WO2004063214A2 - Antibodies that specifically recognize sumo-conjugated proteins - Google Patents

Abstract

The invention provides branched peptides containing 11 or more amino acid residues matching the structure at the conjugation point of conjugates of SUMO proteins, such as SUMO-1, to proteins that are targets for conjugation by the SUMO proteins. The peptides can be used to generate antibodies that specifically react with the SUMO protein-target protein conjugates, and not with the unconjugated SUMO protein or unconjugated target protein. The antibodies can be used to detect diseases and to screen for effectors that activate or inhibit conjugation or deconjugation.

Description

ANTIBODIES THAT SPECIFICALLY RECOGNIZE SUMO- CONJUGATED PROTEINS

GOVERNMENT FUNDING The invention described herein was developed with support from the

National Institutes of Health, under project number 1-ZOl-HDOO 1902-09 (project name: SUMO family small ubiquitin-like modifiers in higher eukaryotes). The U.S. Government may have certain rights in the invention.

PRIORITY OF INVENTION

This application claims priority from United States Provisional Application Number 60/438,685, filed 8 January 2003, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

The small ubiquitin-related modifiers (SUMO) proteins (also called sentrins) are a group of proteins within the larger family of ubiquitin-like proteins. The SUMO proteins, like ubiquitin, and the other ubiquitin-like proteins, are covalently attached to target proteins resulting in branched isopeptide-linked conjugates. The ubiquitin-like proteins share a highly conserved 3 -dimensional structure, but in many cases share relatively little amino acid sequence homology. The SUMO proteins, however, have a higher amino acid sequence homology among themselves, generally above about 40%. The ubiquitin-like proteins include ubiquitin, Rubl (also called Neddδ), Neddl, and the SUMO proteins. The SUMO proteins include SUMO-1 (also called UBL1, Sentrin, PIC1, GMP1, or SMT3c), SUMO-2, SUMO-3, and the Saccharomyces cerevisiae Smt3 protein (for reviews see Kretz-Remy et al., ^• Biochem. Cell Biol. 77:299 (1999); Muller et ah, Nature Reviews. Molecular Cell Biol. 2:202 (2001); Seeler et al., Oncogene 20:7243 (2001)). The best characterized ubiquitin-like protein is ubiquitin, a small protein of 76 amino acids. Post-translational modification of proteins by ubiquitin is involved in a variety of cellular processes, including regulation of intracelmlar protein breakdown, cell cycle regulation, signal transduction, transcription, and antigen presentation (Hochstrasser, Annu. Rev. Genet 30:405 (1996); Hershko et al., Annu. Rev. Biochem. 67:425 (1998)). The best characterized role of ubiquitin attachment to proteins is in targeting the substrate proteins for delivery to a protease complex called the 26S proteasome (Narshavasky, Trends Biochem. Sci. 22:383 (1997)). The functional significance of SUMO modification is less well characterized than is that of ubiquitin. SUMO proteins have been found in a variety of eukaryotes, including yeast, plants, and humans. SUMO-1 can be conjugated to a variety of cellular proteins, including promyelocytic leukemia protein (PML), Ran-GTPase-activating protein (RanGAPl), inhibitor of ΝF-κB (IκBα), RanBP2, SplOO, HDPK2, ρ53, topoisomerase I, c-Jun, and the Werner's Syndrome gene product (Seeler et al., Oncogene 20:7243 (2001); Mahajan et al., J. Cell Biol.140.259 (1998); Muller et al., EMBO J. 17:61 (1998); Desterro et al.. J. Biol. Chem. 274:10618 (1999)).

Certain disease states may be linked to SUMO conjugation. In particular, acute promyelocytic leukemia, which is characterized by an increase in nuclear bodies in leukocytes. The formation of nuclear bodies has been linked to SUMO-1 modification of PML.

A need exists for methods to identify, quantitate, and localize conjugates of SUMO proteins to their target proteins. Tools are needed that will assist in the study of the biological role of particular SUMO proteins. Methods to diagnose disease states linked to a rise or fall in the amount of a SUMO protein conjugate or linked to a particular localization of a SUMO protein conjugate are also needed. Further, methods are needed to assay for effector molecules that inhibit or activate conjugation of a particular SUMO protein or all SUMO proteins, or otherwise alter the amount of a SUMO protein conjugate in a cell.

SUMMARY OF THE INVENTION

It has been surprisingly discovered that branched peptides of 11 residues or more spanning the branch points of SUMO protein-target protein conjugates can be used to generate antibodies that specifically recognize the SUMO protein- target protein conjugates, i.e., that recognize the conjugates and do not recognize the unconjugated SUMO protein or unconjugated target protein.

It has also been discovered that branched peptides of 10 or fewer residues spanning the conjugation point sometimes do not generate antibodies that recognize the SUMO protein conjugate or either of the separate proteins. Specifically a 10-residue branched peptide of the SUMO-1-RanGAPl conjugate failed to generate antibodies that recognized the conjugate or either of the separate proteins. The invention provides a branched peptide containing a first peptide fragment of 4-48 amino acid residues containing the sequence Gln-Xaa^Gly-Gly at the carboxy terminus of the first peptide fragment, wherein Xaa¹ is any amino acid. The branched peptides further contain a second peptide fragment of 2-46 amino acid residues containing at least one lysine residue. In the branched peptide, the first peptide fragment is covalently linked to the second peptide fragment by an isopeptide bond between the α-carboxy of the carboxy terminal glycine of the first peptide fragment and the side chain ε-amine of one of the at least one lysine residues of the second peptide fragment. The first and second peptide fragments collectively contain 11-50 amino acid residues. The second peptide fragment is optionally modified at its carboxy terminus with (Q-

C )alkoxy, (Cι-C )alkylamido, (Cι-C )alkylcarbonyloxy, a saccharide, an amino acid, or a peptide of 2-3 residues, with each group optionally substituted on carbon with mercapto, amino, hydroxy, or carboxy. The first and second peptide fragments are also independently optionally modified at their amino termini with (Cι-C₄)alkyl, (Cι-C₄)alkylcarbonyl, a saccharide, an amino acid, or a peptide of 2-3 amino acid residues, with each group optionally substituted on carbon with mercapto, amino, hydroxy, or carboxy.

The invention provides a compound of formula (I)

(I)

In formula (I) NH-X'-X^X-'-CO is a first peptide fragment containing 2-48 amino acid residues that is a carboxy terminal fragment of a SUMO protein. X³- CO is the carboxy terminal amino acid residue of the SUMO protein, NH-X¹ is an internal amino acid residue of the SUMO protein 1-47 amino acid residues from the carboxy terminal residue in the sequence of the SUMO protein, and X² is the 0-46 amino acid residues between NH-X¹ and X³-CO in the sequence of the SUMO protein.

NH

I

(CH₂)₄

X⁴— HN— C— C— X⁵

H II O

(II)

Formula (II) is a second peptide fragment containing 2-48 amino acid residues including the lysine residue depicted by its structure. This second peptide fragment is a fragment of a target protein, where the lysine residue of the second peptide fragment is a site of conjugation with the SUMO protein. X⁴ and X⁵ are each 0-47 amino acid residues. Y¹ and Y² are each independently H, (Ci- C₄)alkyl, (Cι~C₄)alkylcarbonyl, a saccharide, an amino acid, or a peptide of 2-3 amino acid residues; each optionally substituted on carbon with mercapto, amino, hydroxy, or carboxy. Y³ is hydroxy, (Cι-C )alkoxy, (Cι-C₄)alkylamido, (Cι-C₄)alkylcarbonyloxy, a saccharide an amino acid, or a peptide of 2-3 residues; each optionally substituted on carbon with mercapto, amino, hydroxy, or carboxy. NH-X¹-X²-X³-CO, X⁴, and X⁵ collectively contain from 10 to 49 amino acid residues. The compounds of formula (I) are branched peptides of 11 to 50 amino acid residues matching the amino acid sequence of a SUMO- protein-target protein conjugate at the area surrounding the branch point of the conjugate, with the amino and carboxy termini of the branched peptides optionally appended with the Y groups discussed above. The branch point of the branched peptide is an isopeptide linkage to the ε-amino group of the side chain of a lysine residue on the target protein. The branched peptide optionally has groups including alkyl, alkylcarbonyl, a saccharide, an amino acid, or a short peptide, all optionally substituted with mercapto, hydroxy, amino, or carboxy groups, appended to one or more of the amino or carboxy termini of the peptides. Another embodiment of the invention is an in vitro synthesized protein- peptide conjugate. The protein-peptide conjugate contains (a) a branched peptide containing: a first peptide fragment of 4-48 amino acid residues having a carboxy terminus and an amino terminus, the residues at the carboxy terminus of the first peptide fragment having a sequence GlnrXaa¹ -Gly-Gly, Xaa¹ being any amino acid, and the remaining residues have any sequence; and a second peptide fragment of 2-46 amino acid residues having a carboxy terminus and an amino terminus, and at least one residue of the second peptide fragment being a lysine residue. The first peptide fragment is covalently linked to the second peptide fragment by an isopeptide bond between the α-carboxy group of the carboxy terminal glycine of the first peptide fragment and the side chain ε-amino group of one of the at least one lysine residues of the second peptide fragment. The first and second peptide fragments collectively have 11-50 amino acid residues. The first and second peptide fragments are unmodified at their amino termini, or one or both of the first and second peptide fragments is modified at its amino terminus with (Cι-C₄)alkyl, (Cι-C₄)alkyl having at least one substituent on carbon, (Cι-C )alkylcarbonyl, (Cι-C₄)alkylcarbonyl having at least one substituent on carbon, a saccharide, or a saccharide having at least one substituent on carbon, wherein each substituent is independently mercapto, amino, hydroxy, or carboxy. The second peptide fragment is unmodified at its carboxy terminus or is modified at its carboxy terminus with (Cι-C₄)alkoxy, (Cι-C₄)alkoxy having at least one substituent on carbon, (Cι-C₄)alkylamido, (d- C₄)alkylamido having at least one substituent on carbon, (d- C₄)alkylcarbonyloxy, (Cι-C )alkylcarbonyloxy having at least one substituent on carbon, a saccharide, or a saccharide having at least one substituent on carbon, wherein each substituent is independently mercapto, amino, hydroxy, or carboxy. The branched peptide is covalently linked to (b) a protein.

Another embodiment of the invention is an in vitro synthesized conjugate of (a) a branched peptide as described above, preferably a compound of formula (I), covalently linked to (b) a protein. This also is referred to herein as a protein- peptide conjugate.

Another embodiment of the invention is a method of generating an antibody involving administering to a vertebrate a composition containing a branched peptide as described above, preferably a compound of formula (I). This method can generate antibodies that are specific for the SUMO protein- target protein conjugate whose sequence the branched peptide matches.

Another embodiment of the invention is an antibody that specifically recognizes a conjugate of a SUMO protein with a target protein. Another embodiment of the invention is a method of diagnosing a disease characterized by an altered presence, amount, or physiological location of a SUMO protein-target protein conjugate in a mammal. The method involves contacting a first physiological sample obtained from the mammal with an antibody that specifically recognizes a SUMO protein-target protein conjugate to detect the conjugate, and comparing the detecting of the conjugate with a standard in order to diagnose the disease.

Another embodiment of the invention is a method of screening for effectors that alter the level of a SUMO protein-target protein conjugate in a sample. The method involves acquiring a first sample comprising a test compound and (1) a SUMO protein, a target protein, and at least one enzyme that catalyzes the conjugation of the SUMO protein to the target protein to form the conjugate, and/or (2) the conjugate and at least one enzyme that catalyzes the dissociation of the conjugate into the SUMO protein and the target protein. The method also involves contacting the first sample with an antibody that specifically recognizes the conjugate to detect the conjugate, and comparing the detecting of the conjugate with a standard in order to determine whether the test compound is an effector.

In a preferred embodiment of the invention, the branched peptides are isolated and/or purified branched peptides.

DETAILED DESCRIPTION OF THE INVENTION Definitions

"Target protein" refers to a protein that is a natural target for conjugation in a living organism by a SUMO protein and that may be found in a conjugated state.

"SUMO protein" refers to a protein having at least about 30% amino acid sequence identity to human SUMO-1 and that in vivo is conjugated to target proteins by an isopeptide linkage between the carboxy terminus of the SUMO protein and a lysine side chain of the target protein. Amino acid sequence identity can be calculated with BLAST 2.0 using the default parameters, as available at www.ncbi.nlm.nih.gov. The amino acid sequence of mature human SUMO-1 is shown below. Mature SUMO-1 sequence:

1 MSDQEAKPST ED GDKKEGE YIKLKVIGQD SSEIHF VKM TTHLKKLKES YCQRQGVP 61 SLRFLFEGQR IADNHTPKEL GMEEEDVIEV YQEQTGG {SEQ ID NO:l)

The amino acid sequences of human SUMO-2, human SUMO-3, and S. cerevisiae Smt3 are disclosed in Figure 1 of Muller, et al. In a particular embodiment, the mature SUMO protein has the sequence Gln-Xaa¹ -Gly-Gly at its carboxy terminus, where Xaa¹ is any amino acid.

"Amino acid residue" as used herein refers to the structure -HN-CH(R)- CO- , including amino terminal or carboxy terminal residues, except when it is clear from the context that the term "amino acid residue" when applied to an amino terminal residue refers to the structure H₂N-CH(R)-CO- and when applied to a carboxy terminal residue refers to the structure -HN-CH(R)-COOH.

"Effector" as used herein includes enzyme inhibitors and activators and compounds that by any mechanism alter the amount of a SUMO-target protein conjugate in a physiological sample.

"Branched peptide" as used herein includes compounds of formula (I), including those with non-amino acid groups appended to one or more amino or carboxy termini of the first and second peptide fragments of the branched peptide structure. "Isopeptide linkage" as used herein refers to any aminoacyl bond between two amino acids that is not between the α-amino and α-carboxyl groups of the two amino acids. Generally, as used herein it refers to a linkage between the α-carboxyl of the carboxy terminal amino acid of one peptide fragment and the side chain ε-amino group of a lysine residue of another peptide fragment. As used herein "matching" when referring to amino acid sequences or chemical structures, means the sequences or structures are identical.

As used herein, "carboxy terminal residue of a SUMO protein" refers to the residue that is at the carboxy terminus of the mature SUMO protein as it exists in nature after proteolytic processing of one or more residues from the C- terminus of the SUMO protein precursor by a specific protease. As used herein, "an antibody that specifically recognizes a conjugate of a SUMO protein with a target protein" refers to an antibody that recognizes the conjugate and does not recognize either the unconjugated SUMO protein or the unconjugated target protein, i.e., has a substantially higher binding affinity for the conjugate than for either of the unconjugated proteins.

Description

One embodiment of the present invention provides branched peptides of 11-50 amino acid residues having an amino acid sequence that matches the sequence at the branch point of a SUMO protein-target protein conjugate. These peptides can be used to raise antibodies that are specific for the SUMO-target protein conjugates to which they correspond, i.e., the antibodies recognize only the conjugate and not the unconjugated SUMO protein or the unconjugated target protein. hi one embodiment, the SUMO protein and the target protein of the conjugate are both human proteins.

One embodiment of the invention provides branched peptides containing a first peptide fragment of 4-48 amino acid residues and a second peptide fragment of 2-46 amino acid residues including a lysine residue. The first peptide fragment is linked to the second peptide fragment by an isopeptide bond between the carboxy terminal α-carboxy group of the first peptide fragment and the side chain ε-amino group of the lysine residue of the second peptide fragment.

In a particular embodiment of the branched peptides of the invention, the branched peptide contains 11-50 amino acid residues. hi one embodiment of the branched peptides of the invention, the amino acid sequence of the first peptide fragment is identical to the amino acid sequence at the carboxy terminus of a SUMO protein.

In another embodiment of the invention, the second peptide fragment of the branched peptide contains the amino acid sequence Xaa¹⁰-Lys-Xaaⁿ-Xaa¹²; wherein Xaa¹⁰ is an aliphatic amino' acid, Xaa¹¹ is any amino acid, and Xaa¹² is Glu or Pro. The side chain of the lysine residue of the sequence is linked by the isopeptide bond to the first peptide fragment. hi one embodiment, the amino acid sequence of the second peptide fragment is identical to a portion of the amino acid sequence of a target protein that is a target for conjugation with a SUMO protein. In the target protein, the lysine residue of the second peptide fragment is a site of conjugation of the target protein with the SUMO protein.

In a specific embodiment, the SUMO protein is SUMO-1. hi a particular embodiment, the SUMO-1 is human SUMO-1. In other specific embodiments, the SUMO protein is SUMO-2, SUMO-3, or Smt3.

In a specific embodiment, the SUMO proteins have at least about 40% amino acid sequence identity to human SUMO- 1 , preferably 50%, more preferably 65%, more preferably 80%, most preferably 90% sequence identity. These identities are especially preferred when the sequence at the branch point region matches the branch point sequence region of SUMO-1 protein. In a particular embodiment, the mature SUMO proteins have a glycine as the carboxy terminal residue, hi another specific embodiment, the mature SUMO proteins have an amino acid sequence of Gly-Gly as the last two residues at the carboxy terminal. In another specific embodiment, the mature SUMO proteins having any of the sequence identity percentages given above have an amino acid sequence of Gln-Xaa¹ -Gly-Gly as the sequence of the last four residues at the carboxy terminal, where Xaa¹ can be any amino acid. In a specific embodiment, Xaa¹ is threonine (SEQ ID NO:2).

In a specific embodiment of the branched peptides of the invention, the first peptide fragment contains at its carboxy terminus the amino acid sequence Gln-Xaa¹ -Gly-Gly, where Xaa¹ is Thr or He (SEQ ID NO:3). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Xaa²-Gln-Xaa^! -Gly-Gly, where Xaa² is Glu or Gin (SEQ ID NO:4). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Xaa³ -Xaa²-Gln-Xaa¹ -Gly-Gly, where Xaa³ is Gin or Arg (SEQ ID NO:5). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Xaa⁴-Xaa³-Xaa²-Gln-Xaa^] -Gly-Gly, where Xaa⁴ is Tyr, Phe, or His (SEQ ID NO:6). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Xaa⁵-Xaa⁴-Xaa³-Xaa²-Gln-Xaa¹-Gly- Gly, where Xaa⁵ is Nal or Ala (SEQ ID ΝO:7). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Xaa⁶-Xaa⁵-Xaa⁴-Xaa³-Xaa²-Gln-Xaa¹ -Gly-Gly, where Xaa⁶ is Asp or Glu (SEQ ID NO:8). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Ile-Xaa⁶-Xaa -Xaa - Xaa³-Xaa²-Gln-Xaa¹ -Gly-Gly (SEQ LD NO:9). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Xaa⁷-Ile-Xaa⁶-Xaa⁵-Xaa⁴-Xaa³-Xaa²-Gln-Xaa¹-Gly-Gly, where Xaa⁷ is Val, Thr, or He (SEQ LD NO: 10). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Asp- Xaa⁷-Ile-Xaa⁶-Xaa⁵-Xaa⁴-Xaa³-Xaa²-Gln-Xaa¹-Gly-Gly (SEQ ID NO: 11). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Xaa⁸-Asp-Xaa⁷-Ile-Xaa⁶-Xaa⁵-Xaa⁴-Xaa³- Xaa²-Gln-Xaa¹-Gly-Gly, where Xaa⁸ is Glu or Asn (SEQ ID NO: 12). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Xaa⁹-Xaa⁸-Asp-Xaa⁷-Ile-Xaa⁶-Xaa⁵-Xaa⁴-Xaa³-Xaa²- Gln-Xaa¹ -Gly-Gly, where Xaa⁹ is Asp or Glu (SEQ ID NO: 13). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Glu-Xaa⁹-Xaa⁸-Asp-Xaa⁷-Ile-Xaa⁶-Xaa⁵-Xaa⁴-Xaa³- Xaa²-Gln-Xaa^! -Gly-Gly (SEQ LD NO: 14). In another specific embodiment, the first peptide fragment contains at its carboxy terminus the amino acid sequence Met-Glu-Xaa⁹-Xaa⁸-Asp-Xaa⁷-Ile-Xaa⁶-Xaa⁵-Xaa⁴-Xaa³-Xaa²-Gln-Xaa¹-Gly- Gly (SEQ ID NO: 15).

In a specific embodiment of the branched peptides of the invention, the second peptide fragment contains the amino acid sequence Xaa¹⁰-Lys-Xaa^π- Xaa¹², wherein Xaa¹⁰ is Ala, Val, Leu, or He, Xaa¹¹ is any amino acid, and Xaa¹² is Glu or Pro.

In specific embodiments, the target protein is RanGAPl (SEQ ID NO:39)

1 ASEDIAKLA ETLAKTQVAG GQI-SFKGKS KLNTAEDAKD VIKEIEDFDS EALR EGNT 61 VGVEAARVIA KA--EKKSELK RCH SDMFTG RLRTEIPPAL ISLGEG ITA GAQLVELDI-S 121 DNAFGPDGVQ GFEA KSSA CFT QE KLN NCGMGIGGGK ILAAALTECH R SSAQG PL 181 A KVFVAGRN RLENDGATAL AEAFRVIGTL EEVHMPQNGI NHPGITALAQ AFAV PLLRV 241 INLNDNTFTE KGAVAMAETL KT RQVEVIN FGDC VRSKG AVAIADAIRG GLPKLKEI-NL 301 SFCEIKRDAA LAVAEAMADK AELEKLDI-NG NTLGEEGCEQ LQEVLEGFNM AKVI-AS SDD 361 EDEEEEEEGE EEEEEAEEEE EEDEEEEEEE EEEEEEEPQQ RGQGEKSATP SRKI DPNTG 421 EPAPV SSPP PADVSTFLAF PSPEKI-LRLG PKSSV IAQQ TDTSDPEKW SAFLKVSSVF 481 KDEATVRMAV QDAVDAL QK AFNSSSFNSN TFLTR I-VHM G L SED VK AIA LYGPLM 541 A NHMVQQDY FPKA-jAPI-LI- AFVTKPNSAL ESCSFARHS QTLYKV or PML (SEQ ID NO:40)

₁ MEPAPARSPR PQQDPARPQE PTMPPPETPS EGRQPSPSPS PTERAPASEE EFQF RCQQC

6₁ Q_AE_AKCPKLL PCLHTLCSGC LEASGMQCPI CQAP PLGAD TPA DNVFFE SLQRRLSVYR

121 QIVDAQAVCT RCKESADF C FECEQ LCAK CFEAHQ FLK HEARPLAE R NQSVREFLDG 181 TRKT NIFCS NP HRTPT T SIYCRGCSKP LCCSCALLDS SHSELKCDIS AEIQQRQEE

241 DAMTQALQEQ DSAFGAVHAQ MHAAVGQLGR ARAETEELIR ERVRQWAHV RAQERELLEA

30₁ VDARYQRDYE EMASRLGRLD AV QRIRTGS ALVQRMKCYA SDQEVI-D HG FLRQALCRLR

361 QEEPQS QAA VRTDGFDEFK VRLQDLSSCI TQGKDAAVSK KASPEAASTP RDPIDVDLPE

421 EAERVKAQVQ ALGLAEAQPM AWQSVPGAH PVPVYAFSIK GPSYGEDVSN TTTAQKRKCS 481 QTQCPRKVI MESEEGKEAR LARSSPEQPR PSTSKAVSPP HI-DGPPSPRS PVIGSEVFLP

541 NSNHVASGAG EAEERWVIS SSEDSDAENS VSSSPQSEVL Y KVHGAHGD RRATVLASP

601 ASPLLASPL LASPVSAEST RSLQPAL HI PPPS ASPPA R.

In other particular embodiments, the target protein is SplOO (Sternsdorf et al, J. Cell Biol. 139:1621 (1997); Sternsdorf et al., J. Biol. Chem.. 274:12555 (1999)), p53 (Gostissa et al, EMBO J., 18:6462 (1999); Rodriguez et al., EMBO J.. 18:6455 (1999); Muller et al, J. Biol. Chem.. 275:13321 (2000)), p73 (Minty et al., J. Biol. Chem.. 275:36316 (2000)), HIPK2 (Kim et al., Proc. Natl. Acad. Sci. USA. 96:12350 (1999)), TEL (Chakrabarti et al., Proc. Natl. Acad. Sci. USA, 97:13281 (2000); Chakrabarti et al., Proc. Natl. Acad. Sci. USA. 96:7467 (1999)), c-Jun (Muller et al., J. Biol. Chem.. 275:13321 (2000)), androgen receptor (Poukka et al, Proc. Natl. Acad. Sci. USA. 97:14145 (2000)), IκBα (Desterro et al., Mol. Cell.. 2:233 (1998)), Mdm2 (Buschmann et al, Cell, 101:753 (2000)), Topo I (Mao et al., Proc. Natl. Acad. Sci. USA. 97:4046 (2000)), Topo II (Mao et al, J. Biol. Chem.. 275:26066 (2000)), WRN (Kawabe et al, J. Biol. Chem.. 275:20963 (2000)), RanBP2 (Saitoh et al., Curr. Biol, 8:121 (1998)), GLUT1 (Giorgino et al., Proc. Natl. Acad. Sci. USA. 97:1125 (2000)), GLUT4 (Giorgino et al, Proc. Natl. Acad. Sci. USA. 97:1125 (2000)), Ttk69 (Lehembre et al., Mol. Cell. Biol.. 20:1072 (2000), Dorsal (Bhaskar et al., J. Biol. Chem.. 275:4033 (2000)), CaMK (Long et al., J. Biol. Chem.. 275:40765 (2000)), a septin (Takahashi et al, Biochem. Biophys. Res. Commun., 259:582 (1999); Johnson et al., J. Cell. Biol. 147:981 (1999)), CMV IE1 (Muller et al., J. Virol. 73:5137 (1999)), CMV IE2 (Hofhiann et al, J. Virol. 74:2510 (2000)), EBV BZLF1 (Adamson et al, J. Virol. 74:1224 (2000)), or HPV/BPV El (Rangasamy et al, J. Biol Chem.. 275:37999 (2000)).

In a specific embodiment, the conjugate is SUMO-1-RanGAPl. In a specific embodiment where the conjugate is SUMO- 1-RanG API, the compound of formula (I) is QTGG (SEQ ID NO: 16) HMGLLK ISE (SEQ ID NO: 17)

In this depiction of the structure of compounds of formula (I), the letters are the single letter amino acid abbreviations; the lines of letters indicate linear peptide fragments with the amino terminus on the left and the carboxy terminus on the right, and the vertical line indicates an isopeptide bond between the α-carboxyl of the C-terminal glycine and the side chain ε-amine of the lysine residue.

In another particular embodiment where the conjugate is SUMO-1- RanGAP 1 , the compound of formula (I) is

QEQTGG ( SEQ ID NO : 18 )

HMGLLKSEDK ( SEQ ID NO : 19 ) hi a particular embodiments where the SUMO protein is SUMO-1, the carboxy terminal fragment of SUMO-1 in the branched peptide is GG, TGG, QTGG (SEQ ED NO: 16), EQTGG (SEQ LD NO:20), QEQTGG (SEQ ID NO: 18), YQEQTGG (SEQ ID NO:21), VYQEVTGG (SEQ ID NO:22), EVYQEQTGG (SEQ ID NO:23), or IEVYQEQTGG (SEQ ID NO:24).

In particular embodiments where the target protein is RanGAP 1, the second peptide fragment (the fragment of the target protein) is GLLKSE (SEQ ID NO:25), HMGLLKSE (SEQ ID NO: 17), GLLKSEDK (the branch point lysine is the first lysine; SEQ ID NO:26), HMGLLKSEDK (SEQ ID NO: 19), LVHMGLLKSE (SEQ ID NO:27), or RLLVHMGLLKSE (SEQ ID NO:28). PML can be conjugated with SUMO-1 at lysine residues 65, 160, and 490 of PML. Thus, in particular embodiments where the target protein is PML, the second peptide fragment includes Lys-65, Lys-160, or Lys-490 of PML.

In particular embodiments where the target protein is PML, the second peptide fragment (the fragment of the target protein) is WFLKHE (SEQ ID NO:29), HQWFLKHE (SEQ ID NO:30), WFLKHEAR (SEQ ID NO:31), WFLKHEARPL (SEQ ID NO:32), LKHEARPL (SEQ LD NO:33),

HQWFLKHEAR (SEQ ID NO:34), EAHQWFLKHE (SEQ ID NO:35), or CFEAHQWFLKHE (SEQ ID NO:36).

In a particular embodiment where the conjugate is SUMO-1 -PML, the compound of formula (I) is QTGG ( SEQ ID NO : 16 )

HQWFLKHE (SEQ ID NO : 30 ) In another particular embodiment where the conjugate is SUMO-1-PML, the compound of formula (I) is

QEQTGG (SEQ ID NO : 18 )

I HQWFLKHEAR (SEQ ID NO: 34)

The branched peptides may be compounds of formula (I). In a specific embodiment, Y¹ and Y² in formula (I) are each hydrogen and Y³ is hydroxy.

In a specific embodiment of the invention, NH-X¹-X²-X³-CO, X⁴, and X⁵ in formula (I) collectively contain 10-39 amino acid residues (i.e., the first and second peptide fragments collectively contain 11 to 40 amino acid residues, including the branch point lysine residue). In other specific embodiments, NH- X¹-X²-X³-CO, X⁴, and X⁵ collectively contain 10-29, 10-24, 10-22, 10-19, 11- 19, 11-22, 11-24, 11-29, 11-39, 12-24, 13-24, 13-29, 13-39, 14-24, 15-29, 15-39, 15-49, 16-24, 17-29, 17-39, or 17-49 residues.

In a particular embodiment of the protein peptide conjugates of the invention, the protein to which the peptide or compound of formula (I) is conjugated is keyhole limpet hemocyanin or ovalbumin.

Another embodiment of the invention is an antibody that specifically recognizes a conjugate of a SUMO protein with a target protein.

In particular embodiments, the SUMO protein is SUMO-1, SUMO-2, SUMO-3 or Smt3. In particular embodiments, the target protein is RanGAP 1 or PML. hi other particular embodiments, the target protein is SplOO, p53, p73, HIPK2, TEL, c-Jun, androgen receptor, I/cBα, Mdm2, Topo I, Topo II, WRN, RanBP2, GLUT1, GLUT4, Werner's syndrome gene product, Ttk 69, Dorsal, CaMK, a septin, CMV IE1, CMV IE2, EBV BZLF1, or HPV/BPV El. In a particular embodiment, the antibody recognizes the SUMO- 1 -

RanGAP 1 conjugate. In a particular embodiment, this antibody also recognizes a branched peptide of the invention containing an amino acid sequence matching the amino acid sequence at the branch point of the SUMO-1 -RanGAP 1 conjugate. In another particular embodiment, the antibody recognizes the SUMO-1- PML conjugate. In a particular embodiment, this antibody also recognizes a branched peptide of the invention containing an amino acid sequence matching the amino acid sequence at the branch point of the SUMO-1-PML conjugate. In a particular embodiment, this antibody recognizes the compound

QTGG ( SEQ ID NO : 16 )

I HQ FLKHE ( SEQ ID NO : 3 0 )

In a particular embodiment, the antibody is monoclonal. In another particular embodiment, the antibody is polyclonal. hi a particular embodiment, the antibody also specifically recognizes a branched peptide of the invention, hi a particular embodiment, the antibody recognizes a branched peptide of the invention whose first and second peptide fragments match the amino acid sequence at the branch point of the SUMO- target protein conjugate recognized by the antibody.

In particular embodiments, the antibody specifically recognizes a denatured conjugate of the SUMO protein with the target protein, the native conjugate of the SUMO protein with the conjugate, or both the native and a denatured conjugate of the SUMO protein with the target protein. In a particular embodiment, the antibody is spin labeled or dye labeled.

Another embodiment of the invention provides a method of diagnosing a disease characterized by an altered presence, amount, or physiological location of a SUMO protein-target protein conjugate in a mammal. The method involves contacting a first physiological sample obtained from the mammal with an antibody that specifically recognizes a SUMO protein-target protein conjugate to detect the conjugate, and comparing the detecting of the conjugate with a standard to diagnose the disease.

In a particular embodiment of the method of diagnosing a disease, the contact step detects the presence of the conjugate in the sample, and the presence of the conjugate is diagnostic of the disease. In another specific embodiment, the contacting detects the absence of the conjugate, and the absence of the conjugate is diagnostic of the disease.

In another specific embodiment, the contact step identifies the quantity of the conjugate in the sample, and the quantity of the conjugate is diagnostic of the disease. In another specific embodiment, the contact step identifies the intracellular location of the conjugate, and the intracellular location of the conjugate is diagnostic of the disease.

In a particular embodiment, the disease diagnosed is acute promyelocytic leukemia. In particular embodiments when the disease is acute promyelocytic leukemia, the contacting detects the intracellular location of the SUMO-1 -PML conjugate. For instance, the contacting may detect an absence or decreased amount of the conjugate in nuclear bodies, or the presence or an increased amount of the conjugate in the cytoplasm. The contacting may also, in particular embodiments where the disease is acute promyelocytic leukemia, detect a lowered quantity of the SUMO-1 -PML conjugate in a sample obtained from a person with the disease compared to the quantity of conjugate in a sample obtained from a person without acute promyelocytic leukemia.

In a specific embodiment, the method of diagnosing a disease further involves contacting a second physiological sample from the mammal with an antibody that specifically recognizes the unconjugated target protein to detect unconjugated target protein. The first and second physiological samples can be the same or separate samples.

Another embodiment of the invention is a method of screening for effectors that alter the level of a SUMO protein-target protein conjugate in a sample. The method involves acquiring a first sample containing a test compound and (1) a SUMO protein, a target protein, and at least one enzyme that catalyzes the conjugation of the SUMO protein to the target protein to form the conjugate, and/or (2) the conjugate and at least one enzyme that catalyzes the dissociation of the conjugate into the SUMO protein and the target protein. Next, the method involves contacting the first sample with an antibody that specifically recognizes the conjugate to detect the conjugate, and comparing the detecting of the conjugate with a standard to determine whether the test compound is an effector. hi specific embodiments of a method of screening for effectors, the first sample is a cell, a tissue, a physiological sample, or an organism.

In a specific embodiment, the first sample is a mammalian sample. In a specific embodiment, the first sample is an in vitro sample. In a specific embodiment, the in vitro sample is purified. In a specific embodiment, the contacting detects the presence of the conjugate in the first sample. In another specific embodiment, the contacting detects the absence of the conjugate in the first sample, h another specific embodiment, the contacting detects the quantity of the conjugate in the first sample.

In another specific embodiment of a method of screening for effectors, the method further involves acquiring a second sample containing (1) a SUMO protein, a target protein, and at least one enzyme that catalyzes the conjugation of the SUMO protein to the target protein to form the conjugate, and/or (2) the conjugate and at least one enzyme that catalyzes the dissociation of the conjugate into the SUMO protein and the target protein, where the second sample does not contain the test compound. This method involves contacting the second sample with an antibody that specifically recognizes the conjugate to detect the conjugate in the second sample; and comparing the detection of the conjugate in the first sample with the detection of the conjugate in the second sample to determine whether the test compound is an effector.

In a particular embodiment of a method of screening for effectors, the first sample contains the SUMO protein, the target protein, and at least one enzyme that catalyzes the conjugation of the SUMO protein to the target protein. In this embodiment, detecting the conjugate in the first sample can be used to determine whether the test compound is an effector that activates or inhibits the conjugation.

In another specific embodiment of a method of screening for effectors, the first sample contains the conjugate and at least one enzyme that catalyzes the dissociation of the conjugate. In this embodiment, the detecting the conjugate in the first sample can be used to determine whether the test compound is an effector that activates or inhibits the dissociation of the conjugate.

Peptide Epitopes of the Invention The branched peptides of the invention include epitopes that are recognized by antibodies that specifically recognize SUMO-target protein conjugates. The epitopes can be all or only a portion of the branched peptides of the invention. In some embodiments of the invention, substantially all of the antibodies raised against the branched peptides of the invention specifically recognize a SUMO-target protein conjugate. For example, in a monoclonal antibody preparation, at least 80%, at least 90%, at least 95%, or at least 99°/ the antibody molecules may specifically recognize the SUMO-target protein conjugate. For another example, in a polyclonal antiserum, at least 80%, at 1 90%, at least 95%, or at least 99% of the antibody molecules that recognize 1 SUMO-target protein conjugate may specifically recognize the SUMO-targe protein conjugate (Le., not recognize the unconjugated SUMO protein or the unconjugated target protein). In other embodiments, only a portion of the antibodies raised against the branched peptides of the invention specifically recognize a SUMO-target protein conjugate.

Peptide Variants and Derivatives

The invention is also directed to variants and derivatives of the isolat peptide epitopes that can generate SUMO-specific antibodies. Amino acid residues of the isolated peptides and peptide variants can genetically encoded L-amino acids, naturally occurring non-genetically enco L-amino acids, synthetic L-amino acids or D-enantiomers of any of the abov The amino acid notations used herein for the twenty genetically encoded L- amino acids and common non-encoded amino acids are conventional and are shown in Table 1.

Table 1

Branched peptides that are encompassed within the scope of the invention can have one or more amino acids substituted with an amino acid of similar or different chemical and/or physical properties, so long as these variant and derivative peptides retain the ability to generate antibodies that specifically recognize the SUMO-target protein conjugates.

When generating a variant or derivative peptide, amino acids that reside within similar classes or subclasses can be substituted for amino acids in a reference peptide or amino acid sequence. As known to one of skill in the art, amino acids can be placed into three main classes: hydrophilic amino acids, hydrophobic amino acids and cysteine-like amino acids, depending primarily on the characteristics of the amino acid side chain. These main classes maybe further divided into subclasses. Hydrophilic amino acids include amino acids having acidic, basic or polar side chains and hydrophobic amino acids include amino acids having aromatic or apolar side chains. Apolar amino acids may be further subdivided to include, among others, aliphatic amino acids. The definitions of the classes of amino acids as used herein are as follows: "Hydrophobic Amino Acid" refers to an amino acid having a side chain that is uncharged at physiological pH and that is repelled by aqueous solution. Examples of genetically encoded hydrophobic amino acids include He, Leu and Val. Examples of non-genetically encoded hydrophobic amino acids include t- BuA. "Aromatic Amino Acid" refers to a hydrophobic amino acid having a side chain containing at least one ring having a conjugated π-electron system (aromatic group). The aromatic group may be further substituted with substituent groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfonyl, nitro and amino groups, as well as others. Examples of genetically encoded aromatic amino acids include phenylalanine, tyrosine and tryptophan. Commonly encountered non-genetically encoded aromatic amino acids include phenylglycine, 2-naphthylalanine, j3-2-thienylalanine, 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid, 4-chlorophenylalanine, 2- fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine. "Apolar Amino Acid" refers to a hydrophobic amino acid having a side chain that is generally uncharged at physiological pH and that is not polar. Examples of genetically encoded apolar amino acids include glycine, proline and methionine. Examples of non-encoded apolar amino acids include Cha. "Aliphatic Amino Acid" refers to an apolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain. Examples of genetically encoded aliphatic amino acids include Ala, Leu, Val and He. Examples of non-encoded aliphatic amino acids include Nle. "Hydrophilic Amino Acid" refers to an amino acid having a side chain that is attracted by aqueous solution. Examples of genetically encoded hydrophilic amino acids include Ser and Lys. Examples of non-encoded hydrophilic amino acids include Cit and hCys.

"Acidic Amino Acid" refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).

"Basic Amino Acid" refers to a hydrophilic amino acid having a side chain pK value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Examples of genetically encoded basic amino acids include arginine, lysine and histidine. Examples of non-genetically encoded basic amino acids include the non-cyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.

"Polar Amino Acid" refers to a hydrophilic amino acid having a side chain that is uncharged at physiological pH, but where a bond in the side chain has a pair of electrons that are held more closely by one of the atoms involved in the bond. Examples of genetically encoded polar amino acids include asparagine and glutamine. Examples of non-genetically encoded polar amino acids include citmlline, N-acetyl lysine and methionine sulfoxide.

"Cysteine-Like Amino Acid" refers to an amino acid having a side chain capable of forming a covalent linkage with a side chain of another amino acid residue, such as a disulfide linkage. Typically, cysteine-like amino acids generally have a side chain containing at least one thiol (SH) group. An example of a genetically encoded cysteine-like amino acid is cysteine. Examples of non- genetically encoded cysteine-like amino acids include homocysteine and penicillamine. As will be appreciated by those having skill in the art, the above classifications are not absolute. Several amino acids exhibit more than one characteristic property, and can therefore be included in more than one category. For example, tyrosine has both an aromatic ring and a polar hydroxyl group. Thus, tyrosine has dual properties and can be included in both the aromatic and polar categories. Similarly, in addition to being able to form disulfide linkages, cysteine also has an apolar character. Thus, while not strictly classified as a hydrophobic or an apolar amino acid, in many instances cysteine can be used to confer hydrophobicity to a peptide. Certain commonly encountered amino acids that are not genetically encoded and that can be present, or substituted for an amino acid, in the peptides, peptide variants and peptide derivatives of the invention include, but are not limited to, /3-alanine (b-Ala) and other omega-amino acids such as 3- aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr), 4-aminobutyric acid and so forth; c--aminoisobutyric acid (Aib); e-aminohexanoic acid (Aha); δ- amino valeric acid (Ava); N-methylglycine (MeGly); ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Melle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); 2- naphthylalanine (2-Nal); 4-chlorophenylalanine (Phe(4-Cl)); 2- fluorophenylalanine (Phe(2-F)); 3-fluorophenylalanine (Phe(3-F)); 4- fluorophenylalanine (Phe(4-F)); penicillamine (Pen); 1,2,3,4- tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (liArg); N-acetyl lysine (AcLys); 2,3-diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu); p- aminophenylalanine (Phe(pNH₂)); N-methyl valine (MeVal); homocysteine (hCys) and homoserine (hSer). These amino acids also fall into the categories defined above.

The classifications of the above-described genetically encoded and non- encoded amino acids are summarized in Table 2, below. It is to be understood that Table 2 is for illustrative purposes only and does not purport to be an exhaustive list of amino acid residues that may comprise the peptides, variants and derivatives described herein. Other amino acid residues that are useful for making the peptides, peptide variants and peptide derivatives described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and the references cited therein. Amino acids not specifically mentioned herein can be conveniently classified into the above-described categories on the basis of known behavior and/or their characteristic chemical and/or physical properties as compared with amino acids specifically identified.

TABLE 2

Peptides of the invention can have any amino acid substituted by any similarly classified amino acid to create a variant or derivative peptide, so long as the peptide variant or derivative retains an ability to bind to the biomolecule or tissue to which the unaltered or reference peptide bound.

Synthesis of branched peptides

Branched peptides of the invention can be purchased from commercial suppliers of custom peptides, such as AnaSpec, Inc. (San Jose, California). Peptides can also be synthesized by methods known in the art, such as those described in Plaue et al, and references cited therein. For instance, the peptide

QTGG ( SEQ ID NO : 16 )

I HMGLLKSE ( SEQ ID NO : 17 ) is synthesized using terbutyloxycarbonyl (Boc) as the amino group protecting group starting from Boc-Glu (OcHx) Pam resin prepared as described in Plaue,

S. et al, (Tetrahedron Lett. 28:1401 (1987)), with sequential addition then of the

S, K, L, L, G, M, and H residues from the C-terminal to amino terminal direction to form a linear peptide. For trifunctional amino acids, side chain protecting groups should be used. Among suitable protecting groups are cyclohexyl for Glu, benzyl for Tlir, 2-chlorobenzyloxycarbonyl for Lys, Tosyl for Arg, paramethylbenzyl for Cys, 2,6-dichlorobenzyl for Tyr, and fluorenylmethyloxycarbonyl (Fmoc) for the branch point Lys.

The Boc groups are removed using 65% trifluoroacetic acid in dichloromethane for 13 minutes. Coupling reactions are performed in dimethylformamide (DMF) with a threefold excess of hydroxybenzotriazol active ester prepared just before use. After deprotection of the last residue H in the linear peptide HMGLLKSE (SEQ ID NO: 17), the amino terminus is acetylated using a 10 molar excess of acetic anhydride in the presence of the same amount of diisopropylethylamine for 10 minutes. The Fmoc group of the branch point lysine is then deprotected by two treatments of 50% piperidine in DMP (2 minutes each). The residues G, G, T, and Q are then assembled sequentially onto the lysine side chain as described above. The peptide is cleaved from the solid support using hydrogen fluoride in the presence of 10% (vol/vol) of p-cresol as scavenger for 45 minutes at 0°C.

The same procedure can be used to synthesize other branched peptides. Other methods of manual and automated peptide sequencing are also known in the art, and can be used to synthesize the branched peptides of the invention. For instance, suitable methods are disclosed in Bodanszky, M., et al. The Practice of Peptide Synthesis. Springer- Verlag, New York, (1994).

Certain groups such as (Cι-C )alkyl, (Cι-C )alkylcarbonyl, a saccharide, (Cι-C₄)alkoxy, (Cι-C₄)alkylamido, and (Cι-C₄)alkylcarbonyloxy can be appended to the amino or carboxy termini of the branched peptides of the invention by techniques known in the art. For example, suitable reagents and reaction conditions are disclosed, e.g, in Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, Cary and Sundberg (1983); Advanced Organic Chemistry. Reactions, Mechanisms, and Structure, Second Edition, March (1977); Protecting Groups in Organic Synthesis, Second Edition, Greene, T.W., and Wutz, P.G.M., John Wiley & Sons, New York; and Comprehensive Organic Transformations, Larock, R.C., Second Edition, John Wiley & Sons, New York (1999). Raising Antibodies

The invention also provides antibodies made by available procedures that can bind an epitope on a branched peptide of the invention.

To generate antibodies, the branched peptides should be coupled to a carrier protein. Suitable carrier proteins include keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), rabbit serum albumin, and ovalbumin. Methods of coupling to the carrier protein include single-step and two-step glutaraldehyde coupling, coupling with m-maleimoidobenzoyl-N- hydroxysuccinimide ester, carbodiimides, or bis-diazotized benzidine. Protocols for these coupling methods are found in Harlow, Ed et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988).

The immunogen made up of the branched peptides coupled to a carrier protein is used to immunize a vertebrate animal in order to induce the vertebrate to generate antibodies to the branched peptide. Preferably the immunogen is injected along with an adjuvant, such as Freund's adjuvant, to enhance the immune response. Suitable vertebrates include rabbits, mice, rats, hamsters, and chickens.

Hybridomas to synthesize monoclonal antibodies can be prepared by methods known in the art. See, for instance, Wang, H., et al, Antibody Expression and Engineering, Am. Chem. Soc, Washington, DC (1995).

Polyclonal and monoclonal antibodies can be isolated by methods known in the art. See, for instance, id. and Harlow et al.

Antibodies reacting with the branched peptides of the invention can be isolated by affinity purification by passing a mixture containing the antibodies through a matrix with immobilized branched peptide. To prepare chromatography columns containing immobilized branched peptides, for instance, peptides containing cysteine residues or modified with a thiol- containing group can be conjugated to activated thiol Sepharose 4B as described by the supplier (Pharmacia, Piscataway, NJ). Peptides containing a free amino group, e.g., an amino terminus, can be coupled to resins that contain free amino groups and are preactivated with glutaraldehyde. CDI-activated agarose available from Pierce Biotechnology (Rockford, IL) can also be used to couple peptides containing N nucleophiles. Antibody molecules belong to a family of plasma proteins called immunoglobulins, whose basic building block, the immunoglobulin fold or domain, is used in various forms in many molecules of the immune system and other biological recognition systems. A typical immunoglobulin has four polypeptide chains, containing an antigen binding region known as a variable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Clothia et al, J. Mol. Biol 186:651-66 (1985)); Novotny and Haber, Proc. Natl. Acad. Sci. USA 82:4592-4596 (1985)). Depending on the amino acid sequences of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are at least five (5) major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these may be further divided into subclasses (isotypes), e.g. IgG- 1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The heavy chains constant domains that correspond to the different classes of immunoglobulins are called alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), respectively. The light chains of antibodies can be assigned to one of two clearly distinct types, called kappa (K) and lambda (λ), based on the amino sequences of their constant domain. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

The term "variable" in the context of variable domain of antibodies, refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies. The variable domains are for binding and determine the specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed through the variable domains of antibodies. It is concentrated in three segments called complementarity determining regions (CDRs) also known as hypervariable regions both in the light chain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a β-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the β-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies. The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

An antibody that is contemplated for use in the present invention thus can be in any of a variety of forms, including a whole immunoglobulin, an antibody fragment such as Fv, Fab, and similar fragments, a single chain antibody which includes the variable domain complementarity determining regions (CDR), and the like forms, all of which fall under the broad term "antibody", as used herein. The present invention contemplates the use of any specificity of an antibody, polyclonal or monoclonal, and is not limited to antibodies that recognize and immunoreact with a specific antigen. In specific embodiments, in the context of both the diagnostic and screening methods described below, an antibody or fragment thereof is used that is immunospecific for a branched peptide or epitope of the invention.

The term "antibody fragment" refers to a portion of a full-length antibody, generally the antigen binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments. Papain digestion of antibodies produces two identical antigen binding fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily. Pepsin treatment yields an F(ab') ₂ fragment that has two antigen binding fragments that are capable of cross-linking antigen, and a residual other fragment (which is termed pFc'). Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. As used herein, "functional fragment" with respect to antibodies, refers to Fv, F(ab) and F(ab')₂ fragments. Antibody fragments contemplated by the invention are therefore not full- length antibodies but do have similar or improved immunological properties relative to a full-length antibody. Thus, fragments of full-length antibodies are contemplated by the invention. Such antibody fragments may be as small as about 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 amino acids, about 15 amino acids, about 17 amino acids, about 18 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids or more. In general, an antibody fragment of the invention can have any upper size limit so long as it has similar or immunological properties relative to an antibody that binds with specificity to a conjugate of a SUMO protein with a target protein.

Antibody fragments retain some ability to selectively bind with its antigen or receptor. Some types of antibody fragments are defined as follows:

(1) Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule. A Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.

(2) Fab' is the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule.

Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHI domain including one or more cysteines from the antibody hinge region.

(3) (Fab')₂ is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction.

F(ab')₂ is a dimer of two Fab' fragments held together by two disulfide bonds.

(4) Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non-covalent association (V_H -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH -V _L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although generally at a lower affinity than the complete binding site.

(5) A single chain antibody ("SCA") is defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies are also referred to as "single-chain Fv" or "sFv" antibody fragments. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding. For a review of sFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, N.Y., pp. 269- 315 (1994).

The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/11161, and Hollinger et al, Proc. Natl Acad Sci. USA 90: 6444-6448 (1993). The preparation of polyclonal antibodies is well-known to those skilled in the art. See, for example, Green, et al, Production of Polyclonal Antisera, in: Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press); Coligan, et al, Production of Polyclonal Antisera in Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology, section 2.4.1 (1992), which are herein incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See, for example, Kohler & Milstein, Nature, 256:495 (1975); Coligan, et al, sections 2.5.1-2.6.7; and Harlow, et al, in: Antibodies: A Laboratory Manual, page 726 (Cold Spring Harbor Pub. (1988)), which are herein incorporated by reference. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan, et al, sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al, Purification of

Immunoglobulin G (IgG), in: Methods in Molecular Biology. Vol. 10, pages 79- 104 (Humana Press (1992).

Methods of in vitro and in vivo manipulation of monoclonal antibodies are well known to those skilled in the art. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), or may be made by recombinant methods, e.g., as described in U.S. Pat. No. 4,816,567. The monoclonal antibodies for use with the present invention may also be isolated from phage antibody libraries using the techniques described in Clackson et al. Nature 352: 624-628 (1991), as well as in Marks et al, J. Mol Biol 222: 581-597 (1991). Another method involves humanizing a monoclonal antibody by recombinant means to generate antibodies containing human specific and recognizable sequences. See, for review, Holmes, et al, J. Immunol. 158:2192-2201 (1997) and Vaswani, et al, Annals Allergy. Asthma & Immunol. 81:105-115 (19981

The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In additional to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al. Proc. Natl. Acad Sci. 81, 6851-6855 (1984)). Methods of making antibody fragments are also known in the art (see for example, Harlow and Lane, Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, New York, (1988), incorporated herein by reference). Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') . This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, in US Patents No. 4,036,945 and No. 4,331,647, and references contained therein. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of V_H and V chains. This association may be noncovalent or the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise V_H and V_L chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V_H and V_L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow, et al, Methods: a Companion to Methods in Εnzvmology. Vol. 2, page 97 (1991); Bird, et al, Science 242:423-426 (1988); Ladner, et al, US Patent No. 4,946,778; and Pack, et al, Bio/Technology 11:1271-77 (1993).

Another form¹ of an antibody fragment is a peptide containing a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") are often involved in antigen recognition and binding. CDR peptides can be obtained by cloning or constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick, et al, Methods: a Companion to Methods in Εnzymology, Vol. 2, page 106 (1991).

The invention contemplates human and humanized forms of non-human (e.g. murine) antibodies. Such humanized antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a CDR of the recipient are replaced by residues from a CDR of a nonhuman species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.

In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see: Jones et al, Nature 321, 522-. 525 (1986); Reichmann et al, Nature 332, 323-329 (1988); Presta, Curr. On. Struct. Biol 2, 593-596 (1992); Holmes, et al, J. Immunol. 158:2192-2201 (1997) and Vaswani, et al, Annals Allergy, Asthma & Immunol, 81:105-115 (1998).

Antibodies of the invention can also be mutated to optimize their affinity, selectivity, binding strength or other desirable property. A mutant antibody refers to an amino acid sequence variant of an antibody. In general, one or more of the amino acid residues in the mutant antibody is different from what is present in the reference antibody. Such mutant antibodies necessarily have less than 100% sequence identity or similarity with the reference amino acid sequence, hi general, mutant antibodies have at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the reference antibody. Mutant antibodies may have at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the reference antibody. One method of mutating antibodies involves affinity maturation using phage display.

Affinity maturation using phage display refers to a process described in Lowman et al, Biochemistry 30(45): 10832-10838 (1991), see also Hawkins et al, J. Mol Biol 254: 889-896 (1992). While not strictly limited to the following description, this process can be described briefly as involving mutation of several antibody hypervariable regions in a number of different sites with the goal of generating all possible amino acid substitutions at each site. The antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusion proteins. Fusions are generally made to the gene III product of Ml 3. The phage expressing the various mutants can be cycled tlirough several rounds of selection for the trait of interest, e.g. binding affinity or selectivity. The mutants of interest are isolated and sequenced. Such methods are described in more detail in U.S. Patent 5,750,373, U.S. Patent 6,290,957 and Cunningham, B. C. et al, EMBO J. 13(11), 2508-2515 (1994). Such methods can further include constructing a replicable expression vector containing a nucleic acid encoding an antibody polypeptide (e.g., a complete light chain or heavy chain or a CDR portion or other fragment of a light chain or heavy chain). The nucleic acid can also encode a fusion protein comprising an antibody polypeptide and at least a portion of a natural or wild- type phage coat protein. The expression vector can also have a transcription regulatory element operably linked to the nucleic acids encoding the fusion protein. The vector is mutated at one or more selected positions within the nucleic acid encoding the antibody polypeptide to form a family or "library" of plasmids containing related nucleic acids, each encoding a slightly different antibody polypeptide. Suitable host cells are transformed with the family of plasmids. The transformed host cells are infected with a helper phage having a gene encoding the phage coat protein and the transformed, infected host cells are cultured under conditions suitable for forming recombinant phagemide particles. Each recombinant phagemid displays approximately one copy of the fusion protein on the surface of the phagemid particle. To screen the phagemids, phagemid particles are contacted with an epitope or branched peptide of the invention. Phagemid particles that bind are separated from those that do not bind the epitope or branched peptide. Further rounds of selection may be performed by separately cloning phagemids with acceptable binding properties and re-testing their binding affinity one or more times. The plasmids from phagemid particles that appropriately bind the epitope or branched peptide can also be isolated, cloned and even mutated again to further select for the antibody properties desired, e.g. with good binding affinity. The method is applicable to polypeptide complexes that are composed of more than one subunits of polypeptides. In this case, a nucleic acid encoding each subunit of interest is separately fused to a phage coat protein and separately analyzed for its binding properties.

Any cloning procedure used by one of skill in the art can be employed to make the expression vectors used in such affinity maturation/phage display procedures. For example, one of skill in the art can readily employ known cloning procedures to fuse a nucleic acid encoding an antibody hypervariable region to a nucleic acid encoding a phage coat protein. See, e.g., Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989; Sambrook et al, Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 2001.

The invention is therefore directed to a method for selecting antibodies and/or antibody fragments or polypeptides with desirable properties. Such desirable properties can include increased binding affinity or selectivity for the epitopes of the invention

The antibodies and antibody fragments of the invention include isolated antibodies and antibody fragments. An isolated antibody is one that has been identified and separated and/or recovered from a component of the environment in which it was produced. Contaminant components of its production environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. The term "isolated antibody" also includes antibodies within recombinant cells because at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

If desired, the antibodies of the invention can be purified by any available procedure. For example, the antibodies can be affinity-purified by binding an antibody preparation to a solid support to which the antigen used to raise the antibodies is bound. After washing off contaminants, the antibody can be eluted by known procedures. Those of skill in the art will know of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (see for example, Coligan, et al, Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incorporated herein by reference).

In particular embodiments, the antibody will be purified as measurable by at least three different methods: 1) to greater than 95% by weight of antibody as determined by the Lowry method, and in one embodiment more than 99% by weight; 2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequentator; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomasie blue or silver stain. Determining whether antibodies raised are specific for a SUMO-target protein conjugate

Specificity of antibodies for the SUMO-target protein conjugate is tested by immunoblotting (e.g., dot blotting or Western blotting), or ELISA assays. For dot blotting, for example, the conjugate, unconjugated SUMO protein, and unconjugated target protein, preferably in equal quantities, are spotted separately on a nitrocellulose or nylon membrane. The membrane is contacted with antisera or purified antibodies, and then washed to remove nonspecifically bound antibodies, and then bound antibody is detected and optionally quantitated either by the use of radiolabeled reagents or enzyme-labeled reagents. Procedures for dot blotting and Western blotting are disclosed in Harlow, et al.

In ELISA, the conjugate, unconjugated SUMO protein, and unconjugated target protein are separately coated on wells of microtiter plates. The wells are contacted with test antisera or antibodies and bound antibodies are detected or quantitated by the use of radiolabeled reagents or enzyme-labeled reagents.

Use of antibodies specific for a SUMO-target protein conjugate

Antibodies specific for a SUMO-target protein conjugate can be used to localize the conjugate in particular tissues, cell-types, or intracellular locations. For instance, cells can be contacted with the rabbit antibodies specific for the conjugate, and the rabbit antibodies labeled with commercially available fluorescently labeled goat anti-rabbit antibodies, and the intracellular location of the label determined by confocal microscopy.

The specific antibodies also allow an experimenter to quantitate a SUMO-target protein conjugate. One method involves a two-antibody sandwich assay using an immobilized antibody that binds target or SUMO protein at a point other than the conjugation point, so that it binds both unconjugated and conjugated protein. The bound antigen then can be contacted with antibody that specifically recognizes the conjugate. The amount of the conjugate-specific antibody bound then can be quantified by radioactive or enzymatic labeling procedures, thus quantifying the amount of conjugate. Alternatively, the antibodies that specifically recognize the conjugate can be immobilized to a solid substrate. With the immobilized conjugate-specific antibody, a competition assay is used to quantify the amount of a SUMO-target protein conjugate in a sample. A sample containing an unknown quantity of the SUMO-target protein conjugate of interest is mixed with a known amount of labeled SUMO-target protein conjugate or of labeled branched peptide of the SUMO-target protein conjugate. The mixture is then allowed to bind to a subsaturating amount of the specific antibody bound to a solid phase. From the amount of label bound to the solid phase, the amount of unlabeled SUMO-target protein conjugate in the mixture can be derived. Other immunoassays for detecting or quantitating an antigen are known in the art that can be applied to use with the specific antibodies of the invention to detect or quantify the SUMO-target protein conjugate of interest. For instance, procedures for several types of immunoassays are disclosed in Harlow et al.

Screening test compounds to identify effectors that inhibit or activate conjugation or deconjugation, or otherwise alter the level of a SUMO-target protein conjugate in vitro or in vivo

The covalent attachment of SUMO proteins to their targets is catalyzed by a specific set of enzymes. A C-terminal hydrolase first cleaves several C- terminal residues from an inactive SUMO precursor protein to form a mature protein with, in the SUMO proteins discovered to date, a GG sequence at the C- terminal of the protein. The mature SUMO protein is activated by an El enzyme to form a SUMO-E1 thioester, which contains a thioester bond between the C- terminal glycine of SUMO and El (Desterro et al. J. Biol. Chem. 274:10618 (1999); Okuma et al, Biochem. Biophys. Res. Commun. 254:693 (1999)). The SUMO protein is then transferred by an E2 enzyme, which forms a thioester linkage with the C-terminus of SUMO through a cysteine residue on E2. The only E2 identified to date involved in SUMO conjugation is Ubc9, which is specific for SUMO proteins and cannot bind ubiquitin (Gong et al, J. Biol. Chem. 272:28198 (1997); Saitoh et al, Curr. Biol 8:121 (1998)). At this point in the ubiquitin pathway, ubiquitin is transferred from E2 to the substrate by an E3 ligase. However, no E3 involved in SUMO conjugation has been identified. In vitro conjugation of SUMO to a target protein has been achieved with El (Aos/Uba2) and E2 (Ubc9) enzymes in the presence of ATP (Desterro et aL. FEBS Lett. 417:297 (1997); Desterro et al, Mol Cell. 2:233 (1998); Okuma et al, Biochem. Biophys. Res. Commun. 254:693 (1999)). However it is difficult to imagine how this simple system could achieve sufficient specificity for particular target proteins in vivo.

The branched peptides of the invention can be used to produce antibodies specific for a SUMO-target protein conjugate. Antibodies specific for a particular conjugate can be used to detect or quantify the conjugate, as discussed above. This allows assays for effectors that activate or inhibit the enzymes involved in conjugation or deconjugation of a SUMO-target protein conjugate of interest. Where an enzyme reaction or enzyme pathway produces a SUMO- target protein conjugate as a product (the conjugation reactions) or consumes one as a reactant (the deconjugation reactions), the specific antibodies can be used to detect enzyme activity or quantify the rate of the enzyme reaction by detecting or quantifying the conjugate as it is produced or consumed.

Effectors that activate enzymes involved in conjugation can be identified by preparing a reaction mixture in which conjugation of the SUMO protein to the target protein occurs. The rate or extent of conjugation is compared in a mixture lacking a test compound versus a mixture containing the test compound to identify test compounds that are effectors that activate or inhibit the conjugation.

Likewise, a reaction mixture can be prepared in which deconjugation of a SUMO-target protein conjugate occurs. The rate or extent of deconjugation is compared in the mixture lacking a test compound versus a comparable mixture containing the test compound to identify effectors that activate or inhibit the deconjugation.

Test compounds can also be tested on in vivo samples to identify effectors that increase or decrease the amount of a SUMO-target protein conjugate in the sample, or alter the tissue, cell type, or intracellular localization of the SUMO-target protein conjugate. For these assays, for instance, the test compounds can be administered to tissue culture samples or whole organisms. Methods of generating and testing large libraries of compounds for biological activity screening are disclosed in Blackwell, H.E., et al, Chemistry and Biology 8:1167 (2001) and demons, P.A., et al, Chemistry and Biology 8:1183 (2001). The following examples are introduced in order that the invention may be more readily understood. They are intended to illustrate the invention, but not limit its scope.

Examples

Example 1

Antibodies are raised against the synthetic branched peptide

QTGG ( SEQ ID NO : 16 ) I

MGLLKSE (SEQ ID NO : 37 ) from the SUMO-1 -RanGAP 1 conjugate. The branched peptide is synthesized by AnaSpec, Inc. (San Jose, CA). Four mg of peptide is conjugated to Keyhole limpet hemocyanin (KLH). Fifty to 500 mg of the immunogen is diluted to one ml with sterile saline and combined with one ml of the appropriate adjuvant. Complete Freund's Adjuvant is used for the initial injection and Incomplete Freund's Adjuvant is used for all subsequent injections of an animal. The antigen and adjuvant are mixed thoroughly to form a stable emulsion, which is injected subcutaneously and provides enhanced immune response from the sustained presence of the immunogen.

The immunogen is injected into a New Zealand White Rabbit. Ten to 14 days after boost injections, blood is collected from the central ear artery with a 19 gauge needle and allowed to clot and retract at 37°C overnight. The clotted blood is then refrigerated for 24 hours before the serum is decanted and clarified by centrifugation at 2500 rpm for 20 minutes. The schedule is as follows. Time 0 Bleed 25 ml (yields 10 ml pre-immune serum.

Immunize with antigen in CFA. Week 3 Immunize with antigen in JFA.

Week 6 Inimunize with antigen in IFA. Week 7 Bleed 50 ml (yields 20 ml serum).

Week 10 Immunize with antigen in IFA.

Week 11 Bleed 50 ml.

Affinity-purified antibodies are prepared from the antiserum by AnaSpec, Inc., by passing the antiserum through a column matrix containing immobilized branched peptide, washing the column, and then eluting bound antibody. Western Blots

Crude HeLa cell extract (10 μg protein) and recombinant RanGAP (10 ng protein) are loaded on separate lanes for SDS-PAGE. The gel is blotted onto a PVDF membrane according to the manufacturer's protocol. The contacting with the antisera and detection of bound antibody is performed as described in any standard Western blot visualization kit. See also Towbin, H, Proc. Natl. Acad. Sci. USA 76:4350-54 (1979).

To block the Western blot filter with branched peptide, the branched peptide is dissolved to 10 mg/ml in DMSO, diluted to 10 ng/ml in PBS plus 0.2% Tween 20® and 5% milk. The 10 ng/ml solution is incubated at room temperature for 10 hours at room temperature with the Western blot filter to block the filter. Results:

The antiserum from the rabbits recognizes a band on SDS-PAGE at approximately 83-90 kDa from HeLa cell extract that corresponds to the expected molecular weight of SUMO- 1 -RanGAP 1 conjugate. The true molecular weight of RanGAP 1 is 63.5 kDa and the true molecular weight of SUMO-1 is 11.1 kDa. The conjugate therefore has a molecular weight of 74.7 kDa. However, the SUMO-1 -RanGAP 1 conjugate runs on SDS-PAGE at approximately 83-90 kDa. The antiserum does not react with unconjugated RanGAP 1 or unconjugated recombinant SUMO-1.

The Western blot filter blocked with the branched peptide is probed with the antiserum, and the antiserum no longer binds to the 83 kDa band. This suggests that the 83 kDa band is the SUMO-1 -RanGAP 1 conjugate, and binding to the conjugate is outcompeted by the excess of branched peptide.

Antibodies that react with the branched peptide are purified by affinity purification. The affinity-purified antibodies react with the 83 kDa band in a Western blot, but not with unconjugated recombinant RanGAP 1 or unconjugated recombinant SUMO-1. The flow-through antiserum from the affinity purification reacts with several bands from the HeLa extract, but not with the 83 kDa band that the affinity-purified antibody reacts with. Conclusions:

The 11-mer SUMO-1 -RanGAP 1 branched peptide used in Example 1 raises antibodies that specifically bind the SUMO-1 -RanGAP 1 conjugate. Example 2

Antibodies are raised against the synthetic branched peptide

QTGG ( SEQ ID NO : 16 ) I

HMGLLKSE ( SEQ ID NO : 17 ) from the SUMO-1 -RanGAP 1 conjugate. The branched peptide is synthesized, the antiserum is prepared, Western blots are performed, blocking of the Western blot with the branched peptide is performed, and affinity purification of antibody that recognizes the branched peptide is performed as described in Example 1. Results:

The antiserum from the rabbits recognizes an approximately 83-90 kDa protein from HeLa cell extract. The antiserum does not react with unconjugated RanGAP 1 or unconjugated recombinant SUMO-1. The Western blot filter is blocked with branched peptide as described in

Example 1. The blocked filter is probed with the antiserum, and the antiserum no longer binds to the 83-90 kDa band. This suggests that the 83-90 kDa band is the SUMO-1 -RanGAP 1 conjugate, and binding to the conjugate is outcompeted by the excess of branched peptide. Antibodies that react with the branched peptide are purified by affinity purification. The affinity-purified antibodies react with the 83-90 kDa band in a Western blot, but not with unconjugated recombinant RanGAP 1 or unconjugated recombinant SUMO-1. The flow-through antiserum from the affinity purification reacts with several bands from the HeLa extract, but not with the 83 kDa band that the affinity-purified antibody reacts with. Conclusions:

The 12-mer SUMO-1 -RanGAP 1 branched peptide used in this Example raises antibodies that specifically bind the SUMO-1 -RanGAP 1 conjugate.

Example 3

Antibodies are raised against the synthetic branched peptide

QEQTGG (SEQ ID NO : 18 )

I MGLLKSEDK (SEQ ID NO : 38 ) from the SUMO-1 -RanGAP 1 conjugate. The branched peptide is synthesized, the antiserum is prepared, Western blots are performed, blocking of the Western blot with the branched peptide is performed, and affinity purification of antibody that recognizes the branched peptide is performed as described in Example 1. Results:

The antiserum from the rabbits recognizes an 83-90 kDa protein from HeLa cell extract. The antiserum does not react with unconjugated RanGAP 1 or unconjugated recombinant SUMO-1.

The Western blot filter is blocked with branched peptide as described in Example 1. The blocked filter is probed with the antiserum, and the antiserum no longer binds to the 83-90 kDa band. This suggests that the 83-90 kDa band is the SUMO-1 -RanGAP 1 conjugate, and binding to the conjugate is outcompeted by the excess of branched peptide.

Antibodies that react with the branched peptide are purified by affinity purification. The affinity-purified antibodies react with the 83-90 kDa band in a Western blot, but not with unconjugated recombinant RanGAP 1 or unconjugated recombinant SUMO-1. The flow-through antiserum from the affinity purification reacts with several bands from the HeLa extract, but not with the 83 kDa band that the affinity-purified antibody reacts with. Conclusions:

The 16-mer SUMO-1 -RanGAP 1 branched peptide used in this Example raises antibodies that specifically bind the SUMO-1 -RanGAP 1 conjugate.

Example 4 Antibodies are raised against the synthetic branched peptide

IEVYQEQTGG (SEQ ID NO: 24)

I HMGLLKSEDK (SEQ ID NO: 19) from the SUMO-1 -RanGAP 1 conjugate. The branched peptide is synthesized, the antiserum is prepared, Western blots are performed, blocking of the Western blot with the branched peptide is performed, and affinity purification of antibody that recognizes the branched peptide is performed as described in Example 1. Results:

The antiserum from the rabbits recognizes an 83-90 kDa protein from HeLa cell extract. The antiserum does not react with unconjugated RanGAP 1 or unconjugated recombinant SUMO-1. The Western blot filter is blocked with branched peptide as described in

Example 1. The blocked filter is probed with the antiserum, and the antiserum no longer binds to the 83 kDa band. This suggests that the 83-90 kDa band is the SUMO-1 -RanGAP 1 conjugate, and binding to the conjugate is outcompeted by the excess of branched peptide. Antibodies that react with the branched peptide are purified by affinity purification. The affinity-purified antibodies react with the 83-90 kDa band in a Western blot, but not with unconjugated recombinant RanGAP 1 or unconjugated recombinant SUMO-1. The flow-through antiserum from the affinity purification reacts with several bands from the HeLa extract, but not with the 83 kDa band that the affinity-purified antibody reacts with. Conclusions:

The 20-mer SUMO-1 -RanGAP 1 branched peptide used in this Example raises antibodies that specifically bind the SUMO-1 -RanGAP 1 conjugate.

Example 5

Antibodies are raised against the synthetic branched peptide

QTGG (SEQ ID NO: 16)

I HQWFLKHE (SEQ ID NO: 30) from the SUMO-1 -PML conjugate. The branched peptide is synthesized, the antiserum is prepared, Western blots are performed, blocking of the Western blot with the branched peptide is performed, and affinity purification of antibody that recognizes the branched peptide is performed as described in Example 1. Results: The antiserum from the rabbits recognizes an 81 kDa protein, corresponding to the expected molecular weight of the SUMO-1 -PML conjugate, from HeLa cell extract. The antiserum does not react with unconjugated recombinant PML or unconjugated recombinant SUMO-1.

The Western blot filter is blocked with branched peptide as described in Example 1. The blocked filter is probed with the antiserum, and the antiserum no longer binds to the 81 kDa band. This suggests that the 81 kDa band is the SUMO-1-PML conjugate, and binding to the conjugate is outcompeted by the excess of branched peptide.

Antibodies that react with the branched peptide are purified by affinity purification. The affinity-purified antibodies react with the 81 kDa band in a Western blot, but not with unconjugated recombinant PML or unconjugated recombinant SUMO-1. The flow-through antiserum from the affinity purification reacts with several bands from the HeLa extract, but not with the 81 kDa band that the affinity-purified antibody reacts with. Conclusions:

The 12-mer SUMO-1 -PML branched peptide used in this Example raises antibodies that specifically bind the SUMO-1 -PML conjugate.

Example 6 Antibodies are raised against the synthetic branched peptide

QEQTGG ( SEQ ID NO : 18 )

I HQ FLKHEAR ( SEQ ID NO : 34 ) from the SUMO-1 -PML conjugate. The branched peptide is synthesized, the antiserum is prepared, Western blots are performed, blocking of the Western blot with the branched peptide is performed, and affinity purification of antibody that recognizes the branched peptide is performed as described in Example 1.

Results:

The antiserum from the rabbits recognizes an 81 kDa protein, corresponding to the expected molecular weight of the SUMO-1 -PML conjugate, from HeLa cell extract. The antiserum does not react with unconjugated recombinant PML or unconjugated recombinant SUMO-1.

The Western blot filter is blocked with branched peptide as described in

Example 1. The blocked filter is probed with the antiserum, and the antiserum no longer binds to the 81 IcDa band. This suggests that the 81 kDa band is the

SUMO-1-PML conjugate, and binding to the conjugate is outcompeted by the excess of branched peptide.

Antibodies that react with the branched peptide are purified by affinity purification. The affinity-purified antibodies react with the 81 kDa band in a Western blot, but not with unconjugated recombinant PML or unconjugated recombinant SUMO-1. The flow-through antiserum from the affinity purification reacts with several bands from the HeLa extract, but not with the 81 kDa band that the affinity-purified antibody reacts with. Conclusions: The 16-mer SUMO- 1 -PML branched peptide used in this Example raises antibodies that specifically bind the SUMO-1 -PML conjugate.

Example 7

Antibodies are raised against the synthetic branched peptide IEVYQEQTGG (SEQ ID NO : 24 )

HQWFLKHEAR ( SEQ ID NO : 34 ) from the SUMO-1-PML conjugate. The branched peptide is synthesized, the antiserum is prepared, Western blots are performed, blocking of the Western blot with the branched peptide is performed, and affinity purification of antibody that recognizes the branched peptide is performed as described in Example 1. Results:

The antiserum from the rabbits recognizes an 81 IcDa protein, corresponding to the expected molecular weight of the SUMO-1 -PML conjugate from HeLa cell extract. The antiserum does not react with unconjugated recombinant PML or unconjugated recombinant SUMO-1.

The Western blot filter is blocked with branched peptide as described in Example 1. The blocked filter is probed with the antiserum, and the antiserum no longer binds to the 81 IcDa band. This suggests that the 81 kDa band is the SUMO-1 -PML conjugate, and binding to the conjugate is outcompeted by the excess of branched peptide.

Antibodies that react with the branched peptide are purified by affinity purification. The affinity-purified antibodies react with the 81 kDa band in a Western blot, but not with unconjugated recombinant PML or unconjugated recombinant SUMO-1. The flow-through antiserum from the affinity purification reacts with several bands from the HeLa extract, but not with the 81 kDa band that the affinity-purified antibody reacts with.

Conclusions:

The 20-mer SUMO-1 -PML branched peptide used in this Example raises antibodies that specifically bind the SUMO- 1 -PML conjugate. Example 8

Antibodies were raised against the synthetic branched peptide

QTGG ( SEQ ID NO : 16 ) I

GLLKSE ( SEQ ID NO : 25 ) from the SUMO-1 -RanGAP 1 conjugate. The branched peptide was synthesized, the antiserum was prepared, Western blots were performed, blocking of the

Western blot with the branched peptide was performed, and affinity purification of antibody that recognizes the branched peptide was performed as described in Example 1.

Western Blots

Crude HeLa cell extract (10 μg protein) and recombinant RanGAP (10 ng protein) were loaded on separate lanes for SDS-PAGE. The gel was blotted onto WESTRAN CLEAR SIGNAL PVDF membrane (Schleicher & Schuell Bioscience, Inc., Keene, New Hampshire) according to the manufacturer's protocol. The contacting with the antisera and detection of bound antibody was performed as described in Towbin, H., Proc. Natl. Acad. Sci. USA 76:4350-54 (1979). Results:

Serum from one of the rabbits recognized a band in the HeLa cell extract of approximately 83 IcDa, which corresponds to the expected molecular weight of SUMO-1 -RanGAP 1. The true molecular weight of RanGAP 1 is 63.5 kDa and the true molecular weight of SUMO-1 is 11.1 kDa. The conjugate therefore has a molecular weight of 74.7 kDa. However, the SUMO-1 -RanGAP 1 conjugate runs on SDS-PAGE at approximately 83-90 kDa. Therefore, the 83 IcDa band may be the SUMO-1 -RanGAP 1 conjugate. This band was not observed with pre-bleed serum obtained from the same rabbit prior to being immunized with the branched peptide. The antiserum from the rabbit did not react with unconjugated RanGAP 1. However, in the HeLa extract lane, many other bands of various molecular weights were detected.

To test for the specificity of the recognition of the 83 kDa band, the blot was blocked with the branched peptide, as described above, to compete for binding to the antibody. The blocked blot was then contacted with the antiserum. If recognition of the band was specific, the band should be lost after blocking with the peptide. The band was still recognized. However, this could be due to insolubility of the peptide causing poor blocking.

The antiserum to the branched peptide was also used for immunoprecipitation with the HeLa extract. The immunoprecipitate was solubihzed with SDS and analyzed by Western blot with antiserum to RanGAP 1, and the 83 kDa protein was detected.

Next, antibodies specific for the branched peptide were purified by affinity purification using a chromatography column with immobilized branched peptide. The flow-through antiserum, depleted of the affinity-purified antibodies was also collected. Western blot of the HeLa extract was perfonned with the affinity-purified antibody and the flow-through antiserum. The 83 kDa band was only observed on the blot using the flow-through antiserum. Conclusion:

These experiments lead to the conclusion that the 10-mer branched peptide from

SUMO-1 -RanGAP 1 does not generate antibodies that recognize either the SUMO-1 -RanGAP 1 conjugate or unconjugated RanGAP 1.

Bibliography Adamson et al, J. Virol. 74: 1224 (2000)

Barnes, et al, Purification of Immunoglobulin G (IgG), in: Methods in

Molecular Biology. Vol. 10, pages 79-104 (Humana Press (1992).

Bhaskar et al, J. Biol Chem.. 275:4033 (2000)

Bird, et al, Science 242:423-426 (1988) Blackwell, H.E., et al, Chemistry and Biology 8:1167 (2001)

Bodanszky, M., et al. The Practice of Peptide Synthesis. Springer-Verlag, New York (1994)

Buschmann et al, Cell, 101:753 (2000)

Cary and Sundberg, Advanced Organic Chemistry, Part B: Reactions and Synthesis, Second Edition, (1983)

Chakrabarti et al, Proc. Natl Acad. Sci. USA. 97:13281 (2000)

Chakrabarti et al, Proc. Natl. Acad. Sci. USA. 96:7467 (1999)

Clackson et al. Nature 352: 624-628 (1991)

Clemons, P.A., et al, Chemistry and Biology 8:1183 (2001) Clothia et al. J. Mol Biol 186:651-66 (1985)

Coligan, et al, Production of Polyclonal Antisera in Rabbits, Rats Mice and

Hamsters, in:Current Protocols in Immunology, section 2.4.1 (1992)

Coligan, et al, Unit 9, Current Protocols in --mmunology. Wiley h terscience, 1991, incorporated by reference).

Cunningham, B. C. et al, EMBO J. 13(11), 2508-2515 (1994)

Desterro et al. J. Biol. Chem. 274:10618 (1999)

Desterro et al, Mol Cell, 2:233 (1998)

Desterro et al. FEBS Lett. 417:297 (1997) Desterro et al. J. Biol Chem. 274:10618 (1999)

Desterro et al, Mol Cell 2:233 (1998)

Fasman, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc. (1989)

Giorgino et al, Proc. Natl Acad. Sci. USA. 97:1125 (2000) Gong et al, J. Biol Chem. 272:28198 (1997)

Gostissa et al, EMBO J.. 18:6462 (1999)

Greene, T.W., and Wutz, P.G.M., Protecting Groups in Organic Synthesis, Second Edition, John Wiley & Sons, New York

Green, et al, Production of Polyclonal Antisera, in: hnmunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press)

Harlow, Ed et al. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988).

Hawkins et al, J. Mol Biol 254: 889-896 (1992)

Hershko et al, Annu. Rev. Biochem. 67:425 (1998) Hochstrasser, Annu. Rev. Genet 30:405 (1996)

Hofmann et al, J. Virol, 74:2510 (2000)

Holmes, et al. J. Immunol. 158:2192-2201 (1997)

Johnson et al, J. Cell. Biol. 147:981 (1999)

Jones et al, Nature 321, 522-525 (1986) Kawabe et al, J. Biol Chem.. 275:20963 (2000)

Kim et al, Proc. Natl. Acad. Sci. USA. 96:12350 (1999)

Larrick, et al, Methods: a Companion to Methods in Enzymology. Vol. 2, page 106 (1991)

Larock, R.C., Comprehensive Organic Transformations, Second Edition, John Wiley & Sons, New York (1999).

Lehembre et al, Mol Cell Biol. 20: 1072 (2000)

Long et al, J. Biol. Chem., 275:40765 (2000)

Lowman et al, Biochemistry 30(45): 10832-10838 (1991) Mahajan et al, J. Cell Biol 140:259 (1998)

Mao et al, Proc. Natl Acad. Sci. USA, 97:4046 (2000)

Mao et al, J. Biol Chem., 275:26066 (2000)

March, Advanced Organic Chemistry, Reactions, Mechanisms, and Structure, Second Edition, (1977) Marks et al, J. Mol Biol. 222: 581-597 (1991)

Milstein, Nature 256, 495 (1975)

Minty et al, J. Biol Chem., 275:36316 (2000)

Morrison et al. Proc. Natl Acad Sci. 81, 6851-6855 (1984)

Muller et al, EMBO J. 17:61 (1998) Muller et al, J. Biol Chem., 275:13321 (2000)

Muller et al, J. Virol, 73:5137 (1999)

Novotny and Haber, Proc. Natl Acad. Sci. USA 82:4592-4596 (1985).

Okuma et al, Biochem. Biophys. Res. Commun. 254:693 (1999)

Pack, et al, Bio/Technology 11:1271-77 (1993) Plaue et al (J. Exp. Med. 169:1607 (1989^{^})

Plaue, S. et al, Tetrahedron Lett. 28:1401 (1987)

Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer- Verlag, N.Y., pp. 269-315 (1994).

Pouklca et al, Proc. Natl Acad. Sci. USA. 97:14145 (2000) Presta, Curr. Op. Struct. Biol 2, 593-596 (1992);

Rangasamy et al, J. Biol Chem.. 275:37999 (2000)

Reichmann et al, Nature 332, 323-329 (1988)

Rodriguez et al, EMBO J.. 18:6455 (1999)

Saitoh et al, Curr. Biol. 8:121 (1998) Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989;

Sambrook et al, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 2001

Seeler et al, Oncogene 20:7243 (2001) Sternsdorf et al, J. Cell Biol. 139:1621 (1997)

Sternsdorf et al, J. Biol. Chem., 274:12555 (1999)

Takahashi et al, Biochem. Bionhvs. Res. Commun. 259:582 (1999)

Varshavasky, Trends Biochem. Sci. 22:383 (1997) Vaswani, et al, Annals Allergy, Asthma & hnmunol. 81:105-115 (1998)

Wang, H., et al, Antibody Expression and Engineering. Am. Chem. Soc, Washington, DC (1995).

Whitlow, et al, Methods: a Companion to Methods in Enzymology. Vol. 2, page 97 (1991) EP 404,097

WO 93/11161

U.S. Patent No. 4,816,567

U.S. Patent No. 4,816,567

U.S. Patent No. 4,036,945 U.S. Patent No. 4,331,647

U.S. Patent No. 4,946,778

U.S. Patent No. 5,750,373

U.S. Patent No. 6,290,957

All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, is should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

What is Claimed is:

1. A branched peptide comprising: a first peptide fragment of 4-48 amino acid residues having a carboxy terminus and an amino terminus, the residues at the carboxy terminus of the first peptide fragment having a sequence Gln-Xaa¹ -Gly-Gly, Xaa¹ being any amino acid, and the remaining residues of the first peptide fragment having any sequence; a second peptide fragment of 2-46 amino acid residues having a carboxy terminus and an amino terminus, and at least one residue of the second peptide fragment being a lysine residue; wherein the first peptide fragment is covalently linked to the second peptide fragment by an isopeptide bond between the α-carboxy group of the carboxy terminal glycine of the first peptide fragment and the side chain ε-amino group of one of the at least one lysine residue of the second peptide fragment; the first and second peptide fragments collectively have 11-50 amino acid residues; the first and second peptide fragments are unmodified at their amino termini, or one or both of the first and second peptide fragments is modified at its amino terminus with (Cι-C₄)alkyl, (Cι-C )alkyl having at least one substituent on carbon, (Cι-C₄)alkylcarbonyl, (Ci- C )alkylcarbonyl having at least one substituent on carbon, a saccharide, or a saccharide having at least one substituent on carbon, wherein each substituent is independently mercapto, amino, hydroxy, or carboxy; and the second peptide fragment is unmodified at its carboxy terminus or is modified at its carboxy terminus with (Cι-C₄)alkoxy, (Cι-C₄)alkoxy having at least one substituent on carbon, (Cι-C₄)alkylamido, (Ci- C )alkylamido having at least one substituent on carbon, (Q-

C₄)alkylcarbonyloxy, (Cι-C₄)alkylcarbonyloxy having at least one substituent on carbon, a saccharide, or a saccharide having at least one substituent on carbon, wherein each substituent is independently mercapto, amino, hydroxy, or carboxy.

2. A branched peptide of claim 1 wherein the second peptide fragment has 4-46 amine acid residues and four of the residues form the sequence Xaa¹⁰-Lys- Xaaⁿ-Xaa¹²; wherein Xaa¹⁰ is an Ala, Val, Leu, or He, Xaa¹¹ is any amino acid, and Xaa¹² is Glu or Pro; wherein the side chain of the lysine residue of the sequence is linked by the isopeptide bond to the first peptide fragment.

3. A branched peptide of claim 1 wherein the 4-48 amino acid residues of the first peptide fragment form a sequence X-Y₂.₄₆-Z, wherein Z is the carboxy terminal residue of a SUMO protein and X-Y₂.₄₆-Z is the sequence of amino acid residues at the carboxy terminus of the SUMO protein; the 2-46 amino acid residues of the second peptide fragment form a sequence Xb-Ybo-₄ -Zb that is found in a target protein that is a target for conjugation with the SUMO protein; and in the target protein, the lysine residue of the sequence Xb-Ybo-₄₄-Zb that in the second peptide fragment is linked to the first peptide fragment, is a site of conjugation of the target protein with the SUMO protein.

4. A branched peptide of claim 1 covalently linked to a protein.

5. A branched peptide of claim 1 having a structure of a compound of formula (I)

(I)

wherein

NH-X¹-X²-X³-CO is a first peptide fragment having 2-48 amino acid residues; the first peptide fragment is a carboxy terminal fragment of a SUMO protein wherein the carboxy terminal fragment has 2-48 amino acid residues, X - CO is the carboxy terminal amino acid residue of the SUMO protein, NH-X¹ is an internal amino acid residue of the SUMO protein, X² is 0-46 amino acid residues, and NH-X¹-X²-X³-CO is the amino acid sequence of the carboxy terminal fragment of the SUMO protein;

NH I (CH₂)₄

X⁴-HN— C— C— X⁵

H II O is a second peptide fragment having 2-48 amino acid residues including the lysine residue depicted by its structure; the second peptide fragment is a fragment of a target protein; in the target protein, the lysine residue of the second peptide fragment is a site of conjugation of the target protein with the SUMO protein;

X⁴ and X⁵ are each 0-47 amino acid residues;

Y¹ and Y² are each independently H, (Cι-C₄)alkyl, (Cι-C₄)alkyl having at least one substituent on carbon, (Cι-C₄)alkylcarbonyl, (Cι-C₄)alkylcarbonyl having at least one substituent on carbon, a saccharide, a saccharide having at least one substituent on carbon, an amino acid, or a peptide of 2-3 amino acid residues; wherein each substituent is independently mercapto, amino, hydroxy, or carboxy;

Y³ is hydroxy, (C]-C₄)alkoxy, (Cι-C₄)alkoxy having at least one substituent on carbon, (Cι-C )alkylamido, (Cι-C₄)alkylamido having at least one substituent on carbon, (Cι-C₄)alkylcarbonyloxy, (Cι-C₄)alkylcarbonyloxy having at least one substituent on carbon, a saccharide, a saccharide having at least one substituent on carbon, an amino acid, an amino acid having at least one substituent on carbon, a peptide of 2-3 residues, or a peptide of 2-3 residues having at least one substituent on carbon, wherein each substituent is independently mercapto, amino, hydroxy, or carboxy; and

NH-X¹-X²-X³-CO, X⁴, and X⁵ collectively have from 10 to 49 amino acid residues.

6. A branched peptide of claim 5 wherein Y¹ and Y² are each H and Y³ is hydroxy.

7. A branched peptide of claim 5 wherein NH-X^l-X²-X³-CO, X⁴, and X⁵ collectively have from 12 to 24 amino acid residues.

8. A branched peptide of claim 5 wherein the SUMO protein is SUMO- 1 , SUMO-2, SUMO-3, or Smt3.

9. A branched peptide of claim 5 wherein the target protein is RanGAP 1 , PML, SplOO, p53, p73, HIPK2, TEL, c-Jun, androgen receptor, I/ Bc-, Mdm2, Topo I, Topo II, WRN, RanBP2, GLUTl, GLUT4, Werner's syndrome gene product, Ttk 69, Dorsal, CaMK, a septin, CMV IE1, CMV J-E2, EBV BZLFl, or HPV/BPV E1.

10. A branched peptide of claim 5 wherein the SUMO protein is SUMO-1 and the target protein is RanGAP 1.

11. A branched peptide of claim 10 having the structure QTGG (SEQ ID NO : 16)

HMGLLKSE (SEQ ID NO : 17)

12. A branched peptide of claim 5 wherein the SUMO protein is SUMO-1 and the target protein is PML.

13. A branched peptide of claim 12 having the structure

QTGG (SEQ ID NO : 16) HQWFLKHE (SEQ ID NO : 30 )

14. A branched peptide of claim 5 covalently linked to a protein.

15. A method of producing an antibody comprising administering to a vertebrate a branched peptide of claim 5.

16. An antibody produced by the method of claim 15, wherein the antibody specifically recognizes a conjugate of a SUMO protein with a target protein.

17. An antibody of claim 16 wherein the SUMO protein is SUMO- 1 , SUMO-2, SUMO-3, or Smt3.

18. An antibody of claim 16 or 17 wherein the target protein is RanGAP 1 , PML, SplOO, p53, p73, HIPK2, TEL, c-Jun, androgen receptor, IKBO Mdm2, Topo I, Topo II, WRN, RanBP2, GLUTl, GLUT4, Werner's syndrome gene product, Ttk 69, Dorsal, CaMK, a septin, CMV IE1, CMV IE2, EBV BZLFl, or HPV/BPV E1.

19. An antibody of claim 16 wherein the conjugate is a SUMO-1 -RanGAP 1 conjugate or a SUMO-1 -PML conjugate.

20. A method of using an antibody to detect a SUMO protein-target protein conjugate in a sample comprising contacting a sample with an antibody of claim 16 that specifically recognizes a SUMO protein-target protein conjugate to detect the conjugate.

21. A method of diagnosing a disease characterized by an altered presence, amount, or physiological location of a SUMO protein-target protein conjugate in a mammal comprising contacting a first physiological sample obtained from the mammal with an antibody of claim 16 that specifically recognizes a SUMO protein-target protein conjugate to detect the conjugate; and comparing the detecting of the conjugate of the contacting step with a standard to diagnose the disease.

22. A method of claim 21 wherein the contacting detects the presence of the conjugate, the absence of the conjugate, or the quantity of the conjugate in the sample, or identifies the intracellular location of the conjugate, and the presence, absence, or quantity of the conjugate in the sample, or the intracellular location of the conjugate, is diagnostic of the disease.

23. A method of claim 21 wherein the standard is obtained by contacting a second physiological sample from the mammal with an antibody that specifically recognizes unconjugated target protein to detect unconjugated target protein; wherein the first and second physiological samples are the same or separate samples.

24. A method of claim 21 wherein the disease is acute promyelocytic leukemia.

25. A method of screening for effectors that alter the level of a SUMO protein-target protein conjugate in a sample, the method comprising providing a first sample comprising a test compound and (1) a SUMO protein, a target protein, and at least one enzyme that catalyzes the conjugation of the SUMO protein to the target protein to form the conjugate, or (2) the conjugate and at least one enzyme that catalyzes the dissociation of the conjugate into the SUMO protein and the target protein, or (1) and (2); contacting the first sample with an antibody of claim 16 that specifically recognizes the conjugate to detect the conjugate; and comparing the detecting of the conjugate of the contacting step with a standard to determine whether the test compound is an effector.

26. A method of claim 25 wherein the first sample is a cell, tissue, physiological sample, or an organism.

27. A method of claim 25 wherein the contacting detects the presence of the conjugate, the absence of the conjugate, or the quantity of the conjugate in the first sample.

28. A method of claim 25 wherein the standard is obtained by acquiring a second sample not comprising the test compound and comprising (1) a SUMO protein, a target protein, and at least one enzyme that catalyzes the conjugation of the SUMO protein to the target protein to form the conjugate, or (2) the conjugate and at least one enzyme that catalyzes the dissociation of the conjugate into the SUMO protein and the target protein, or (1) and (2); and contacting the second sample with an antibody that specifically recognizes the conjugate to detect the conjugate in the second sample.

29. A method of claim 25 wherein the first sample comprises the SUMO protein, the target protein, and at least one enzyme that catalyzes the conjugation of the SUMO protein to the target protein; wherein the detecting the conjugate in the first sample is used to determine whether the test compound is an effector that activates or inhibits the conjugation.

30. A method of claim 25 wherein the first sample comprises the conjugate and at least one enzyme that catalyzes the dissociation of the conjugate; wherein the detecting the conjugate in the first sample is used to determine whether the test compound is an effector that activates or inhibits the dissociation of the conjugate.