AU688310B2

AU688310B2 - Recombinant alpha-N-acetylgalactosaminidase enzyme and cDNA encoding said enzyme

Info

Publication number: AU688310B2
Application number: AU64175/94A
Authority: AU
Inventors: Jack Goldstein; Alex Zhu
Original assignee: New York Blood Center Inc
Current assignee: New York Blood Center Inc
Priority date: 1993-03-26
Filing date: 1994-03-28
Publication date: 1998-03-12
Anticipated expiration: 2014-03-28
Also published as: CA2159083A1; CA2159083C; WO1994023070A1; EP0694081A1; EP0694081A4; AU6417594A; JPH08508406A

Description

I b r~rl 'WO 94123070 PCTUS94/03338 RECOMBINANT a-N-ACETYLGALACTOSAMINIDASE ENZYME AND cDNA ENCODING SAID ENZYME Statement of Government Interest This invention was made with government support under NMRDC Grant Number N0014-90-J-1638. As such, the government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS This Application is a Continuation-In-Part of Application Serial No. 07/964,756 filed October 22, 1992, entitled PREPARATION OF ENZYME FOR CONVERSION OF SUB-TYPE A AND AB ERYTHROCYTES.

FIELD OF THE INVENTION This invention relates to a recombinant enzyme for use in the removal of type A antigens from the surface of cells in blood products, thereby converting certain sub-type A blood products to type 0 blood products and certain type AB blood products to type B blood products. This invention further relates to methods of cloning and expressing said recombinant enzyme. More particularly, this invention is directed to a recombinant chicken liver a-N-acetylgalactosaminidase enzyme, methods of cloning and expressing said recombinant m-N-acetylgalactosaminidase enzyme, and a method of removing type A antigens from the surface of cells in type A and AB ~L~Llb~ I IB~II~AIBRII~AIC C I II III~- I *WO 94123070 PCT/US94/03338 2 blood products using said recombinant a-N-acetylgalactosaminidase enzyme by contacting said enzyme with blood products so as to remove the terminal moiety of the A-antigenic determinant from the surface of cells (for example, erythrocytes) in said blood products, while allowing the structure and function of the cells in the blood products to remain intact. The recombinant a-N-acetylgalactosaminidase enzyme of this invention provides a readily available and cost-efficient enzyme which can be used in the removal of type A antigens from the surface of cells in type A and AB blood products. Treatment of certain sub-type A blood products with the recombinant enzyme of this invention provides a source of cells free of the A antigen, which blood products are thereby rendered useful in transfusion therapy in the same manner of 0 type blood products.

BACKGROUND OF THE INVENTION As used herein, the term "blood products" includes whole blood and cellular components derived from blood, including erythrocytes (red blood cells) and platelets.

There are more than thirty blood group (or type) systems, one of the most important of which is the ABO system.

This system is based on the presence or absence of antigens A and/or B. These antigens are found on the surface of erythrocytes and on the surface of all endothelial and most IIII-11 ~RIADls~ACraB~ -I r- Ir Illlrl WO 94/23070 PCT/US94/03338 3 epithelial cells as well. The major blood product used for transfusion is erythrocytes, which are red blood cells containing hemoglobin, the principal function of which is the transport of oxygen. Blood of group A contains antigen A on its erythrocytes. Similarly, blood of group B contains antigen B on its erythrocytes. Blood of group AB contains both antigens, and blood of group 0 contains neither antigen.

The blood group structures are glycoproteins or glycolipids and considerable work has been done to identify the specific structures making up the A and B determinants or antigens. It has been found that the blood group specificity is determined by the nature and linkage of monosaccharides at the ends of the carbohydrate chains. The carbohydrate chains are attached to a peptide or lipid backbone which is embedded in the lipid bi-layer of the membrane of the cells. The most important (imnauno-dominant or immuno-determinant) sugar has been found to be N-acetylgalactosamine for the type A antigen and galactose for the type B antigen.

There are three recognized major sub-types of blood type A. These sub-types are known as Al, A intermediate (Ain t and A 2 There are both quantitative and qualitative differences which distinguish these three sub-types.

Quantitatively, Al erythrocytes have more antigenic A sites, terminal N-acetylgalactosamine residues, than Aint erythrocytes which in turn have more antigenic A sites than sereaansaa~uMMsM I II_ k WO 94/23070 PCT/US94/03338 4

A

2 erythrocytes. Qualitatively, the transferase enzymes responsible for the formation of A antigens differ biochemically from each other in Al, Ain t and A 2 individuals. Some A antigens found in Al cells contain dual A antigenic sites.

Blood of group A contains antibodies to antigen B.

Conversely, blood of group B contains antibodies to antigen A.

Blood of group AB has neither antibody, and blood group 0 has both. A person whose blood contains either (or both) of the anti-A or anti-B antibodies cannot receive a transfusion of blood containing the corresponding incompatible antigen(s). If a person receives a transfusion of blood of an incompatible group, the blood transfusion recipient's antibodies coat the red blood cells of the transfused incompatible group and cause the transfused red blood cells to agglutinate, or stick together. Transfusion reactions and/or hemolysis (the destruction of red blood cells) may result therefrom.

In order to avoid red blood cell agglutination, transfusion reactions and hemolysis, transfusion blood type is cross-matched against the blood type of the transfusion recipient. For example, a blood type A recipient can be safely transfused with type A blood which contains compatible antigens. Because type 0 blood contains no A or B antigens, it can be transfused into any recipient with any blood type, i.e., recipients with blood types A, B, AB or 0. Thus, type 0 blood I ~a ~lq _P~~b IBII~R~LI Il~ Cr-, 'WO 94123070 PCT/US94/03338 is considered "universal", and may be used for all transfusions. Hence, it is desirable for blood banks to maintain large quantities of type 0 blood. However, there is a paucity of blood type 0 donors, Therefore, it is useful to convert types A, B and AB blood to type 0 blood in order to maintain large quantities of universal blood products.

In an attempt to increase the supply of type 0 blood, methods have been developed for converting certain type A, B and AB blood to type 0 blood. For example, U.S. Patent No.

4,609,627 entitled "Enzymatic Conversion of Certain Sub-Type A and AB Erythrocytes" ("the '627 Patent"), which is incorporated herein by reference, is directed to a process for converting Ain t and A 2 (including A 2 B erythrocytes) to erythrocytes of the H antigen type, as well as to compositions of type B erythrocytes which lack A antigens, which compositions, prior to treatment, contained both A and B antigens on the surface of said erythrocytes. The process for converting Aint and A 2 erythrocytes to erythrocytes of the H antigen type which is described in the '627 Patent includes the steps of equilibrating certain sub-type A or AB erythrocytes, contacting the equilibrated erythrocytes with purified chicken liver a-N-acetylgalactosaminidase enzyme for a period sufficient to convert the A antigen to the H antigen, removing the enzyme from the erythrocytes and re-equilibrating the erythrocytes.

As described in the '627 Patent, a-N-acetylgalactoaminidase clm IF WO 94/23070 PCT/US94/03338 6 obtained from an avian liver (specifically, chicken liver) source was found to have superior activity in respect of enzymatic conversion or cleavage of A antigenic sites.

Prior to the present invention, it was necessary to purify the enzyme from an avian liver source, a process which is time consuming and can be expensive. Hence, a need has arisen to develop an enzyme source which is more readily available. In addition, a need has arisen to develop an enzyme useful in blood product conversion which enzyme is cost-efficient.

A simplified purification process is described in a related application, Serial No. 07/964,756, filed October 22, 1992, entiUled "Preparation of Enzyme for Conversion of Sub-Type A and AB Erythrocytes". This process, as described in the related application, utilizes chicken liver as a source of enzyme and, therefore, requires a number of purification steps. Despite this simplified process, it is still desirable to provide a more readily available and controlled source of enzyme, that being cloned and expressed enzyme. This would provide an enzyme source which is more consistent and which is readily purified at less cost and expense, with a still further reduced number of purification steps. Additionally, a recombinant, cloned enzyme allows for specific protein sequence modifications, which can be introduced to generate an enzyme I~EB~PBBAIIL~ -sl= WO 94/23070 PCT/US94/03338 7 with optimized specific activity, substrate specificity and pH range.

-N-acetylgalactosamnidase enzymes are characterized (and thereby named) by their ability to cleave N-acetylgalactosamine sugar groups. In isolating or identifying these enzymes, their activity is assessed in the laboratory by evaluating cleavage of synthetic substrates which mimic the sugar groups cleaved by the enzymes, with p-nitrophenylglycopyranoside derivatives of the target sugar groups being commonly used. Although very useful in enzyme identification and isolation procedures (the quantitative cleavage of these synthetic substrates can be used to readily distinguish (and thereby identify) enzymes isolated from different sources), these synthetic substrates are simple structurally and small-sized and mimic only a portion of the natural glycoproteins and glycolipid structures which are of primary concern, those being the A antigens on the surface of cells.

A natural glycolipid substrate, originally isolated from sheep erythrocytes, is the Forsmann antigen (globopentaglycosylceramide). The Forsmann antigen substrate appropriately mimics the natural A antigen glycolipid structures and is therefore utilized to predict the activity of a-N-acetylgalactosaminidase enzymes against the A antigen substrate. Isolated Forsmann antigen glycolipids have been qbl IP~III~P~BllsllC qpa ]I~b~rYIIC1~ r~ C 9~ =C~ra 'WO 94/23070 PCT/US94/03338 8 shown to inhibit hemolysis of sheep red cells by immune rabbit anti-A serum in the presence of serum complement.

a-N-acetylgalactosaminindase enzyme has been isolated from a number of sources besides chicken liver (described above), including bacteria, mollusks, earthworms, and human liver. The human -N-acetylgalactosaminidase enzyme has been purified, sequenced, cloned and expressed. For example, in "Human a-N-Acetylgalactosaminidase Molecular Cloning, Nucleotide Sequence and Expression of a Full-length cDNA", by Wang et al., in The Journal of Biological Chemistry, Vol. 265, No. 35, pages 21859-21866 (December 15, 1990), the cDNA encoding human a-N-acetylgalactosaminidase was sequenced. In addition, in "Molecular Cloning of a Full-Length cDNA for Human a-N-Acetylgalactosaminidase (a-Galactosidase by Tsuji et al., in Biochemical And Biophysical Research Communications, Vol. 163, No. 3, pages 1498-1504 (September 29, 1989), the cDNA encoding human a-N-acetylgalactosaminidase was sequenced. Both the nucleotide sequence and the amino acid sequence of human a-N-acetylgalactosaminidase is published therein. Further, PCT Application No. WO 92/07936 discloses the cloning and expression of the cDNA which encodes human a-N-acetylgalactosaminidase.

Although human a-N-acetylgalactosaminidase has been purified, sequenced, cloned and expressed, it is not appropriate for use in removing A antigens from the surface of I -a ~a olrl~ YRlasl~BIIIIIB~L~B~Is~ r~, 'WO 94/2301070 PCT/US94/03338 9 cells in blood products. In determining whether an enzyme is appropriate for use in removing A antigens from the surface of cells, one must consider the following enzyme characteristics, particularly with respect to the Forsmann antigen substrate: substrate specificity, specific activity or velocity of the substrate cleavage reaction, and pH optimum. Substrate specificity is measured in the Km value, which measures the binding constant or affinity of an enzyme for a particular substrate. The lower a Km value, the more tightly an enzyme binds its substrate. The velocity of an enzyme cleavage reaction is measured in the Vmax, the reaction rate at a saturating concentration of substrate. A higher Vmax indicates a faster cleavage rate. The ratio of these two parameters, Vmax/Km, is a measure of the overall efficiency of an enzyme in reacting with (cleaving) a given substrate. A higher Vmax/Km indicates greater enzyme efficiency. For successful and clinically applicable removal of A antigens from the surface of cells, the enzyme must be sufficiently active at or above a pH at which the cells being treated can be maintained. The procedure described in the '627 patent calls for treatment of cells at or above a pH of 5.6. Therefore, the pH optimum of an appropriate enzyme must still provide reasonable enzyme activity at this pH. These specific characteristics (Vmax/Km, Vmax, Km and pH optimum) are reported for the human a-N-acetylgalactosaminidase enzyme in "Studies on Human I r L~ Rlrmasli~%BBIRBBI~B~Ba~gsB~ L WO 94/23070 PCT/US94/03338 Liver a-galactosidases", by Dean et al. in The Journal of Biological Chemistry, Vol. 254, No. 20, pages 10001-10005 (1979).

The Vmax/Km value for the Forsmann antigen of human a-N-ac.ylgalactosaminidase is 0.46, as compared to a Vmax/Km value of 5.0 for the chicken liver enzyme, indicating an approximately ten-fold difference in efficiency. The Km is lower and the Vmax is higher for the chicken liver enzyme, compared to the human enzyme. Further, human a-N-acetylgalactosaminidase has a pH optimum for the Forsmann antigen of 3.9, compared to 4.7 for chicken liver a-N-acetylgalactosaminidase. By all of these enzyme characteristics, human a-N-acetylgalactosaminidase enzyme is not suitable for NEmoval of A antigens, particularly when compared to the chicken liver enzyme.

As a result, a need still existed to develop an enzyme which is capable of removing A antigens from the surface of cells in blood products, wherein said enzyme is readily available and cost-efficient.

It is therefore an object of this invention to provide a recombinant enzyme for use in the removal of A antigens from the surface of cells in blood products.

It is another object of this invention to provide a recombinant enzyme for use in the removal of A antigens from the surface of cells in blood products wherein said enzyme is 1 I DsoRlassaaa~arasMlsl aa~ II lyl It 'I Ir WO 94/23070 PCT/US94/03338 11 readily available and may be manufactured on a cost-efficient basis.

It is a further object of this invention to provide methods of cloning and expressing a recombinant enzyme useful in the removal of A antigens from the surface of cells in blood products.

It is yet another object of this invention to provide a method of removing A antigens from the surface of cells in blood products using a recombinant enzyme.

BRIEF DESCRIPTION OF THE DRAWINGS The above brief description, as well as further objects and features of the present invention, will be more fully understood by reference to the following detailed description of the presently preferred, albeit illustrative, embodiment of the present invention when taken in conjunction with the accompanying drawing wherein: Figure 1 represents a diagram of the strategy used to clone and sequence the chicken liver m-N-acetylgalactosaminidase cDNA; Figure 2 represents the nucleic acid sequence and the deduced amino acid sequence of the chicken liver m-N-acetylgalactosaminidase cDNA clone; Figure 3 represents the expression of chicken liver a-N-acetylgalactosaminidase in bacteria and rabbit reticulocyte lysate as shown by Western blot; L, LII ~w~ss~IBa~ Ils 3 r e IC 3 r 'WO 94/23070 PCT/US94/03338 12 Figure 4 represents a homology comparison between a-N-acetylgalactosaminidases and a-galactosidases; and Figure 5 represents the expression of chicken liver a-N-acetylgalactosaminidase in yeast as shown by Western blot.

SUMMARY OF THE INVENTION This invention is directed to a recombinant chicken liver a-N-acetylgalactosaminidase enzyme, which enzyme has a molecular weight of about 45 kDa, is immunoreactive with an antibody specific for chicken liver a-N-acetylgalactosaminidase, and also has about 80% amino acid sequence homology with human a-N-acetylgalactosaminidase enzyme. The recombinant chicken liver a-N-acetylgalactosaminidase enzyme of this invention has the amino acid sequence depicted in Figure 2, from amino acid number 1 to amino acid number 406. This invention is further directed to methods of cloning and expressing the recombinant chicken liver a-N-acetylgalactosaminidase enzyme, and to a method of using said enzyme to remove A antigens from the surface of cells in blood products so as to convert said blood products of certain A sub-types to type 0, thereby rendering said blood products universal for use in transfusion therapy.

DETAILED DESCRIPTION OF THE INVENTION This invention is directed to a recombinant enzyme for use in the removal of type A antigens from the surface of cells II -II 'WO 94/23070 WO 9423070PCTlUS94/03338 13 in blood products, thereby converting certain sub-type A blood products to type 0 blood products and certain sub-type AB blood products to type B blood products. The recombinant chicken liver m-N-acetylgalactosaminidase enzyme of this invention has a molecular weight of about 45 kDa and is immunoreactive with an antibody specific for chicken liver a-Nacetylgalactosaminidase. In addition, the recombinant enzyme of this invention -has about 80% amino acid sequence homology with human a-N-acetylgalactosaminidase enzyme. The recombinant chicken liver m-N-acetylgalactosaminidase enzyme of this invention has the following nucleic acid and deduced amino acid sequence: SEQ ID NO 1: ATG CTG GAG MAC GGG CTG GCG CGG ACC CCG CCC ATG GGC TGG TTG GCC Met Leu Glu Asn Gly Leu Ala Arg Thr Pro Pro Met Gly Trp Leu Ala TGG GAG CGC TTC CGC TGC MAC GTG AAC TGC CGG GAG GAC CCC CGC GAG Trp Glu Arg Phe Arg Cys Asn Val Asn Cys Arg Glu Asp Pro Arg Gln TGC ATC ACT GAG ATG CTC TTC ATG GAG ATG GCA GAC CGA ATA GCA GAG Cys Ile Ser Glu Met Leu Phe Met Glu Met Ala Asp Arg Ile Ala Glu GAC CCC TGG AGG GAG CTG CCC TAC MAG TAC ATC MAT ATC CAT GAC TGC Asp Gly Trp Arg Glu Leu Gly Tyr Lys Tyr Ile Asn Ile Asp Asp Cys TGG CCC CCC MAG CAG CGT GAC ACT GAG CCC CCC CTG GTG CCT GAC CCC Trp Ala Ala Lys Cln Arg Asp Thr Glu Gly Arg Leu Val Pro Asp Pro GAG AGG TTC CCC CGG GGC ATT MAG GCC TTG CCT GAC TAC GTT CAT CC Glu Arg Phe Pro Arg Gly Ile Lys Ala Leu Ala Asp Tyr Val His Ala CGA GGC TTC MAG CTC CCC ATT TAT GCC GAC CTG CCC AGA CTC ACC TGT Arg Gly Leu Lys Leu Gly Ile Tyr Cly Asp Leu Cly Arg Leu Thr Cys GGA GGC TAG OGA CCC ACC ACG CTC GAC GGT GTG GAG GAG GAC GGA GAG Cly Cly Tyr Pro Cly Thr Thr Leu Asp Arg Val Glu Gln Asp Ala Gln F 7, 1. 4 Ir 0 N 'WO 94/23070 WO 9423070PCTIUS94/03338 ACC TTC GCT GAG TGG GGT GTG GAC ATG Thr

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Arg Leu TCA CCG CAG CTT AAG CAG AGG TCC CAG ATG TCC ACA TCC TGG CCA CTA GTG TGC CTG AGT GAA AAM GCT GAT CTG GCC CTC AGG Pro Leu Leu**

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TGA TCT GAG ATG GMA GCA BII~R~a*IB~s~ r c WO 94/23070 PCTIUS94/03338 16' A DNA vector containing a sequence encoding chicken liver a-N-acetylgalactosaminidase was deposited under the Budapest Treaty with the American Type Culture Collection, Rockville, Maryland, on March 17, 1993, tested and found viable on March 22, 1993 and catalogued as ATCC No. 75434.

The recombinant chicken liver M-Nacetylgalactosaminidase enzyme of this invention can be cloned and expressed so that it is readily available for use in the removal of A antigens from the surface of cells in blood products. The enzyme of this invention can be cloned and expressed by screening a chicken liver cDNA library to obtain the cDNA sequence which encodes the chicken liver a-N-acetylgalactosaminidase, sequencing the encoding cDNA once is determined, cloning the encoding cDNA and expressing a-N-acetylgalactosaminidase from the cloned encoding cDNA.

This may be performed by obtaining an amplified human m-N-acetylgalactosaminidase fragment capable of use as a screening probe, screening a chicken liver cDNA library, such as the one described hereinabove, using the amplified human a-N-acetylgalactosaminidase fragment as a probe so as to obtain the cDNA sequence of the chicken liver cDNA library which encodes chicken liver m-N-acetylgalactosaminidase, sequencing the encoding DNA, cloning the encoding DNA and expressing chicken liver a-N-acetylgalactosaminidase enzyme from the cloned encoding cDNA. Alternatively, screening can be ~lslP91111~ "BssIPP 1~BC~r~an~ Llr I I rr ~I 'WO 94/23070 PCT/US94/03338 17 performed using antibodies which recognize chicken liver a-N-acetylgalactosaminidase.

Methods which are well known to those skilled in the art can be used to construct expression vectors containing the chicken liver a-N-acetylgalactosaminidase coding sequence, with appropriate transcriptional/translational signals for expression of the enzyme in the corresponding expression systems. Appropriate organisms, cell types and expression systems include: cell-free systems such as a rabbit reticulocyte lysate system, prokaryotic bacteria, such as E. coli, eukaryotic cells, such as yeast, insect cells, mammalian cells (including human hepatocytes or Chinese hamster ovary (CHO) cells), plant cells or systems, and animal systems including oocytes and transgenic animals, The entire chicken liver a-N-acetylgalactosaminidase coding sequence or functional fragments of functional equivalents thereof may be used to construct the above expression vectors for production of functionally active enzyme in the corresponding expression system. Due to the degeneracy of the DNA code, it is anticipated that other DNA sequences which encode substantially the same amino acid sequence may be used. Additionally, changes to the DNA coding sequence which alter the amino acid sequence of the chicken liver a-N-acetylgalactosaminidase enzyme may be introduced which result in the expression of functionally active enzyme. In L I I I ~s~~l~L~pq IDBWg1~9*9~1 L, 'WO 94/23070 PCTIUS94/03338 18 particular, amino acid substitutions may be introduced which are based on similarity to the replaced amino acids, particularly with regard to the charge, polarity, hydrophobicity, hydrophilicity, and size of the side chains of the amino acids.

Once a recombinant chicken liver m-N-acetylgalactosaminidase enzyme is cloned and expressed, said enzyme can be used to remove A antigens from the surface of cells in blood products. Methods of utilizing chicken liver a-N-acetylgalactosaminidase to remove A antigens from the surface of erythrocytes can be found in U.S. Patent No.

4,609,627 issued September 2, 1986 to Goldstein, entitled "Enzymatic Conversion of Certain Sub-type A and AB Erythrocytes", which is incorporated herein by reference.

Sub-type A antigens can be removed from the surface of erythrocytes by contacting the erythrocytes with the recombinant chicken liver a-N-acetylgalactosaminidase enzyme of this invention for a period of time sufficient to remove the A antigens from the surface of the erythrocytes.

EXAMPLE

Isolation and Characterization of the Chicken Liver cDNA Clone Chicken liver a-N-acetylgalactosaminidase was purified to homogeneity. The enzyme was a glycoprotein with a molecular weight of 80 kDa, and was dissociated into two ~-39311sll~eslll~P qqqblpll IC ~WORi~e~BII~- WO 94/23070 PCT/US94/03338 19 identical subunits at pH 7.5. Its optimal pH for cleavage of the synthetic p-nitrophenyl-a-N-acetylgalactosaminylpyranoside substrate was 3.65 and the activity dropped sharply when the pH was raised above 7. The N-terminal sequence obtained from the purified chicken liver a-N-acetylgalactosaminidase showed a strong homology with the corresponding sequence deduced from the human a-N-acetylgalactosaminidase cDNA clone described in Tsuji et al., and Wang et al.

In order to isolate and characterize the cDNA clone for chicken liver a-N-acetylgalactosaminidase, two oligonucleotides, corresponding to nucleotides 688 to 705 and 1219 to 1236 of the human a-N-acetylgalactosaminidase sequence published by Wang, et al. were synthesized. Using human placental mRNA (Clontech) as a template, the specific cDNA was made from the downstream (C-terminal) oligonucleotide. Next, a DNA fragment corresponding to human a-N-acetylgalactosaminidase residues from 688 to 1236 was amplified from the cDNA by the hot-start PCR technique. The PCR reaction mixture was preheated at 95°C for 5 minutes and maintained at 80 0 C while Taq DNA polymerase (Promega) was added to reduce the possible non-specific annealing at lower temperature. 35 cycles of amplification was then carried out as follows: 940C for 1 minute, 50 0 C for 2 minutes and 72*C for 3 minutes. The same conditions for PCR were applied in all of the following experiments.

I, II 'F II Fu-~s~naa~e~--P 'd WO 94/23070 PCT/US94/03338 The PCR-amplified fragment was then used as a radioactively-labeled probe in the screening of a chicken liver cDNA library (Stratagene) based on homology hybridization. The filters containing the library were hybridized with the probe overnight at 42 0 C in a solution of 50% formamide, 0.1% SDS and 0.1 mg/ml salmon sperm DNA. The filters were then washed as follows: 1. 3 X SSC 0.1% SDS, 20 min. room temperature 2. 2 X SSC 0.1% SDS, 20 min. room temperature 3. 1 X SSC 0.1% SDS, 20 min. 56°C 4. 1 X SSC 0.1% SDS, 20 min. 560C The filters were autoradiographed overnight a+ -70 0

C.

The positive clones were picked up for the second-round screening following the same procedure. In total, three consecutive screenings were carried out in order to obtain a well-isolated positive clone.

From approximately one million plaques screened, one positive clone was successfully isolated. The sequencing data indicated that the clone consists of a 1.2 kb 3'-untranslated region and a 0.7 kb coding region which is highly homologous to human a-N-acetylgalactosaminidase. In order to obtain the missing coding sequence, the library was rescreened by using the 1.9 kb cDNA clone as a probe. However, no positive clone was identified by this approach.

I~

~BE~I~R"P~sL C~ u-- SWO 94/23070 PCT/US94/03338 21 The upstream cDNA sequence was then obtained by applying multiple amplification (the nested PCR technique) of a second chicken liver cDNA library (Clontech). Figure 1 represents a diagram of the strategy used to clone and sequence the chicken liver a-N-acetylgalactosaminidase cDNA. The cDNA encoding chicken liver m-N-acetylgalactosaminidase contained a 1.2 kb coding region (slashed area) and a 1.2 kb 3' untranslated region. The arrows at the bottom of the diagram indicate the sequencing strategy. CL1, CL2 and CL3 are oligonucleotides used as primers for the nested PCR. CL1 and CL2 are located at position 924-941 nt and 736-753 nt, respectively (see Figure According to the N-terminal sequence of native chicken liver enzyme, the oligonucleotide CL3 [5'-CTGGAGAAC(T)GGA(GC)CTGGCT(CA)CG] was designed taking into account chicken codon usage and "best guess".

In the first-round PCR amplification, the whole cDNA library was used as a template in the presence of one specific primer (CL1) (see Figure 1) and one universal primer derived from the library vector (5'-CTGGTAATGGTAGCGACC). A small aliquot from the above reaction was directly taken for the second-round amplification with a different set of primers.

The primer CL2 had the sequence located upstream of CL1 (Figure 1) and the second primer, CL3, was designed based on the N-terminal amino acid sequence from purified chicken liver a-N-acetylgalactosaminidase (see Figure A 750 bp srPllsllsl C PI~ C1PqPIPPIII11IPsq~ IIP~ I~aaumaraolmwa~R4~rrs~i Is~ II ~C SWO 94/23070 PCT/US94/03338 22 fragment was sequenced to eliminate any possible PCR artifacts. Since the 750 bp fragment overlapped with the 1.9 kb clone isolated by the library-screening, the two fragments were linked together by PCR to reconstitute the cDNA encoding chicken liver a-N-acetylgalactosaminidase (Figure The DNA sequencing was performed according to standard procedure, and the coding region was sequenced in both orientations.

The Cloned DNA Encodes Chicken Liver e-N-Acetylgalactosaminidase The authenticity of the cDNA clone was established by co-linearity of deduced amino acid sequences with N-terminal and CNBr-digested peptide sequences from purified chicken liver a-N-acetylgalactosaminidase. Figure 2 represents the nucleic acid sequence and deduced amino acid sequence of the chicken liver a-N-acetylgalactosaminidase cDNA clone. The underlined regions in Figure 2 match sequences obtained from the N-terminus and CNBr-derived fragments of enzyme purified from chicken liver. The first 3 nucleotides, ATG, were added during subcloning to serve as the translational initiation codon for protein expression. The polyadenylation signal (AATAAA) at positions 2299-2304 nt is double-underlined. The boxed sequence indicates potential sites for N-glycosylation.

According to the cDNA, the mature protein of 405 amino acids has a molecular mass of about 45 kDa, consistent with that of ~~1111 ~L tr- l I I~ PIR~I WO 94/23070 PCT/US94/03338 23 the purified enzyme estimated by SDS-PAGE. Due to the cloning approach applied, the sequence at the 5' end of the cDNA corresponded to the N-terminal sequence of the mature enzyme isolated from chicken liver.

In order to express the chicken liver m-N-acetylgalactosaminidase in a rabbit reticulocyte lysate, the sequence from 1 to 1260 nucleotides which contained the coding region for chicken liver a-N-acetylgalactosaminidase was subcloned into the vector PCR-II (Invitrogen) in such an orientation that the T7 promoter was located upstream of the insert. Since the N-terminus of the mature protein started with leucine, a translational initiation codon, ATG, was added during the subcloning construction. The construct was then used as a template in a transcription-translation coupled system, TNT system (Promega), for protein expression according to the procedure recommended by the manufacturer.

In order to produce the recombinant a-N-acetylgalactosaminidase in large quantities in bacteria and purify the enzyme in a single-step fashion, the cDNA was subcloned into the EcoRI site of the pTrcHis vector (Invitrogen) for expression in E. coli. Because of the sequence in the vector, the expressed enzyme contained a polyhistidine-tag in its N-terminus, which permitted one step purification by affinity chromatography from crude cell lysates.

~sslrs~sr~e r 19ep g -~PICC ~L~s$ ~aranra~ ara~8asa~ar~ e~ I F 'WO 94/23070 PCT/US94/03338 24 Figure 3 represents the expression of chicken liver a-N-acetylgalactosaminidase in bacteria and rabbit reticulocyte lysate as shown by Western blotting. Lane 1 through lane 4 demonstrate the results of expression in a rabbit reticulocyte lysate. The expression was carried out in lysate in the presence of 3 5 S-methionine with (lane 1) or without (lane 2) the expression plasmid. Next, 5 il of the reaction sample was loaded to a 12% SDS-PAGE. The gel was dried and autoradiographed for 2 hours and a band of an apparent molecular weight of about 45KDa was visualized with the expression plasmid (lane 1, Figure In order to confirm the authenticity of the expressed protein, a Western blot was performed using a polyclonal antibody raised against a-N-acetylgalactosaminidase purified from chicken liver.

Using non-labelled methionine instead, the same expression reaction was performed for a Western blot (Promega) as shown in lnes 3 and 4, with and without the expression plasmid, respectively. As indicated in Figure 3, the antibody specifically recognized a band from the reaction with expression plasmid (lane but not in the control (lane 4).

Lane 5 shows the protein expressed in bacteria and recognized by the same antibody on Western blot. Lane 6 shows the a-N-acetylgalactosaminidase purified from chicken liver as a positive control. Molecular weight size marker is indicated on the left, Hence, it was confirmed that the -L I~ ~1 Iba~rrsearm~s~ ss~auIsl L I 'WO 94/23070 PCT/US941/03338 isolated cDNA clone codes for the chicken liver a-N-acetylgalactosaminidase.

Comparison of the Cloned Chicken Liver Sequence with other Enzyme Sequences The chicken liver a-N-acetylgalactosaminidase sequence was compared with published sequences of other a-N-acetylgalactosaminidases and a-galactosidases which cleave a-galactose sugar groups. Figure 4 shows a homology comparison between various a-N-acetylgalactosaminidases and a-galactosidases. Alignment was carried out using both the computer program PROSIS (Hitachi Software Engineering Corp., Ltd.) and manual arrangement. Tho amino acid sequences were deduced from cDNAs. Sequences I and II are of a-N-acetylgalactosaminidases from chicken liver and human placenta, respectively. Sequences III, IV, V and VI represent m-galactosidase from human, yeast, Cyamopsis tetraqonoloba and Aspergillus niger, respectively. Sequences IV and VI are truncated at the C-terminus, as indicated by Identical or conservatively substituted amino acid residues (five out of six or more) among the aligned protein sequences are boxed. The numbers above the sequences indicate the relative position of each peptide sequence.

The deduced amino acid sequence from chicken liver a-N-acetylgalactosaminidase cDNA shows approximately homology with the human a-N-acetylgalactosaminidase as ~slrv -I rl I ~RII~B~aa~ I rr r 9e WO 94/23070 PCT/US94/03338 26 determined by PROSIS. This homology indicates the relatedness of the human and chicken liver enzymes, despite the differences in the specific characteristics of the enzymes, particularly with regard to cleavage of the Forsmann antigen, as has already been described. Also, polyclonal antibodies raised against chicken liver a-N-acetylgalactosaminidase enzyme do not cross react with the human enzyme. The specific amino acids responsible for these differences remain to be elucidated.

Yamachi et al. (1990) reported that a human a-N-acetylgalactosaminidase cDNA with an insertion of 70bp at the position corresponding to number 376 in Figure 4 was not enzymatically active in a transient expression study in COS cells. The data suggests that the open reading frame shift caused by this insertion in the C-terminal portion of the molecule is responsible for the loss of enzymatic activity, indicating that amino acids in the C-terminal region may be essential for a-N-acetylgalactosaminidase enzyme activity.

By sequence similarity searching (BLAST) (Altschul et al. 1990) of available protein databases followed by sequence alignment using the PROSIS computer program and manual arrangement, it was found that a-N-acetylgalactosaminidase is highly homologous to a-galactosidases from human, yeast, cyamopsis tetragonoloba and aspergillus niger (ranging from to 68% at the amino acid level). The extent of the amino acid sequence homology, as shown in Figure 4, suggests that these ~I ICIC~-- II 'WO 94/23070 PCT/US94/03338 27 two functionally specific glycosidases might have evolved from a common ancestral gene. Considering the high degree of similarities and the nature of their substrates it is possible that the two exoglycosidases share a similar catalytic mechanism and the critical amino acid residues involved in both active sites are well conserved. The addition of chicken liver a-N-acetylgalactosaminidase cDNA to the family provides further insight into regions of the molecule which are important for the substrate binding specificity and enzymatic activity. Given the availability of cloned enzymes from a number of sources, the active site and catalytic mechanisms of a-N-acetylgalactosaminidase and a-galactosidase enzymes may now be studied by means of cDNA deletion and site-directed mutagenesis.

Expression of Active Chicken Liver a-N-acetylgalactosaminidase in Yeast The first 48 nucleotides of human a-N-acetylgalactosaminidase cDNA (Wang, et al. 1990) which correspond to the signal peptide sequence, were linked to the cloned chicken liver a-N-acetylgalactosaminidase coding region by PCR. The PCR amplified product was subcloned directly into the vector PCR-II (Invitrogen). Two EcoRl sites flanking the insert were used to subclone the entire a-N-acetylgalactosaminidase cDNA into the yeast expression vector pYES2 (Invitrogen) in such an orientation that the GAL 1 ~Psslllsl~-Cr -r I _I eprra~i l~asla~ I WO 94/23070 PCT/US94/03338 28 promoter was located upstream of the insert. The GAL 1 promoter provides expression of the inserted cDNA clone under galactose inducing growth conditions in yeast.

The yeast vector constructs were transformed into the yeast strain, INVSCI (Invitrogen) using standard procedures.

To confirm the expression of the chicken liver a-N-acetylgalactosaminidase in yeast, the total proteins from cell extract and culture supernatant were prepared and separated by 12% SDS-PAGE and a Western blot performed (by standard conditions) using the polyclonal antibody raised against purified chicken liver a-N-acetylgalactosaminidase.

The transformed yeast cells were grown in medium without uracil (Bio 101, Inc.). After 0.2% galactose induction, the cells were centrifuged and protein extracts were prepared using glass bead disruption. The secreted proteins in the culture supernatant were concentrated with a Centricon-30 (Amicon Division, W.R. Grace The Western blot results are depicted in Figure Lanes 1 and 8 of Figure 5 show the a-N-acetylgalactosaminidase purified from chicken liver.

Lane 2 through lane 4 are cell extracts; from the yeast transformed with three different pYES2 constructs: the vector alone (lane chicken liver a-N-acetylgalactosaminidase cDNA coding region (lane and the coding region plus signal sequence (lane Lane 5 is the culture supernatant from _L1- -~~1IIII~IICI~ lesnm~ara~re~- WO 94/23070 PCT/US94/03338 29 transformed yeast used in Lane 4. Lane 7 shows the molecular weight standard. As shown in Figure 5, while the protein without signal peptide was expressed within yeast cells (lane the protein with a signal peptide sequence was predominantly secreted into the media (lane The larger molecular weight of the secreted protein observed on the Western blot was presumably caused by overglycosylation, as was observed for the expression of guar a-galactosidase in yeast (Fellinger, et al. 1991).

To purify the expressed a-N-acetylgalactosaminidase, concentrated culture supernatant was applied to an affinity column containing aminocaproylgalactosylamine agarose. After washing the column, the bound fraction was eluted with buffer containing 50mM N-acetylgalactosamine. This eluate contains expressed a-N-acetylgalactosaminidase of similar molecular weight to that of the enzyme purified from chicken liver, as indicated in lane 6 in Figure The expressed enzyme eluted from the column demonstrates activity toward the synthetic substrate p-nitrophenyl-a-N-acetylgalactosaminylpyranoside at pH 3.6.

Heavily glycosylated enzyme did not bind to the affinity column and showed no activity against synthetic substrate. All the data taken together demonstrate production, secretion and purification of enzymatically active chicken liver a-N-acetylgalactosaminidase in yeast cells.

LLCM It d ~G~AI ~sa~iiplausl WO 94/23070 PCT/US94/03338 Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention.

-~2~1~CF sg~a~oluP~aanrar~ st SWO 94/23070 PCT/US94/03338 31 SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Alex Zhu Jack Goldstein (ii) TITLE OF INVENTION: Recombinant a-N- Acetylgalactosaminidase Enzyme and cDNA Encoding Said Enzyme (iii) NUMBER OF SEQUENCES: 7 0 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Amster, Rothstein Ebenstein STREET: 90 Park Avenue CITY: New York STATE: New York COUNTRY: U.S.A.

ZIP: 10016 COMPUTER READABLE FORM: MEDIUM TYPE: 3.

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CLASSIFICATION:

5 inch 1.44 Mb storage diskette iM PC Compatible

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rd Processor (ASCII) Not yet assigned Not yet assigned Not yet assigned (vii) PRIOR APPLICATION DATA: None (viii) ATTORNEY/AGENT INFORMATION: NAME: Pasqualini, Patricia A.

REGISTRATION NUMBER: 34,894 REFERENCE/DOCKET NUMBER: 63475/12 (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: (212) 697-5995 TELEFAX: (212) 286-0854 or 286-0082 TELEX: TWX 710-581-4766 I ~LI WO 94/23070 PCT/US94/03338 32 INFORMATION FOR SEQ ID NO: 1: SEQUENCE CHARACTERISTICS: LENGTH: 2319 TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DESCRIPTION: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes FRAGMENT TYPE: (vi) ORIGINAL SOURCE: ORGANISM: chicken liver

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMEDIATE SOURCE: library (viii) POSITION IN GENOME: unknown CHROMOSOME SEGMENT: MAP POSITION:

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(ix) FEATURE: NAME/KEY: chicken liver c-Nacetylgalactosaminidase

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IDENTIFICATION METHOD: OTHER INFORMATION: PUBLICATION INFORMATION:

AUTHORS:

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JOURNAL:

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PAGES:

DATE:

cd~ 9Ll~t WO 94/23070 WO 9423070PCT/US94/03338 33 DOCUMhENT NUMBER: FILING DATE: PUBLICATION DlATE: RELEVANT RESIDUES, (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: ATG CTG GAG AAC GGG CTG GCG CGG ACC CCG CCC ATG GGC TGG TTG 0CC 48 Met Leu Glu Asn Gly Leo Ala Arg Thr Pro Pro Met Gly Trp Leu Ala TGG GAG CGG TTC CGC TGC AAC GTG AAC TGC COG GAG GAC CCC CGC CAG 96 Trp Glu Arg Phe Arg Cys Asn Val Asn Cys Arg Glu Asp Pro Arg Gin TG ATC AGT GAG ATG CTC TTC ATG GAG ATG GCA GAC CGA ATA GCA GAG 144 Cys Ile Ser Glu Met Leo Phe Met Glu Met Ala Asp Arg Ile Ala Glu GAC GGC TOG AGG GAG CTG GGC TAC AAG TAC ATC AAT ATC OAT GAC TOC 192 Asp Gly Trp Arg GlL Leu Gly Tyr Lys Tyr Ile Asn Ile Asp Asp Cys TGG GCC GCC AAG CAG CGT GAC ACT GAG GOG CGG CTG GTG CCT GAC CCC 240 Trp Ala Ala Lys Gln Arg Asp Thr Glu Gly Arg Leu Val Pro Asp Pro GAG AGG TTC CCC COO GOC ATT AAG GCC TTG GCT GAC TAG GTT CAT GCC 288 Giu Arg Phe Pro Arg Gly Ile Lys Ala Leu Ala Asp Tyr Val His Ala CGA GGC TTG AAG CTG GGC ATT TAT GGC GAC CTG GGC AGA CTC ACC TOT 336 Arg Gly Leo Lys Leo Gly Ile Tyr Gly Asp Leo Gly Arg Leo Thr Cys 2v~ GOA GGC TAC CCA GGC ACC ACG CTG, GAG CGT GTG GAG CAG GACGOCA CAG 384 Gly Gly Tyr Pro Gly Thr Thr Leo Asp Arg Val Glu Gin Asp Ala Gin ACC TTC GCT GAG TGG GGT OTO GAC ATO CTG AAG CTA OAT G TGC TAC 432 Thr Phe Ala Giu Trp Gly Val Asp Met Leu Lys Leo Asp Gly Cys Tyr TGA TCG 000 MOG GAG CAG GCA CAG GOC TAC CCA CAA ATGOCGA AGO 0VCC 480 Ser Ser Oly Lys Glu Gin Ala Gin Oly Tyr Pro Gin Met Ala Arg Ala TTO AAC 0CC ACT GGC CGC CCC ATC GTC TAC TCC TGC AGC TGG CCA GCC 528 Leu Asn Ala Thr Gly Arg Pro Ile Val Tyr Ser Cys Ser Trp Pro Ala TAC CAG GGG GGG CTG CCT CCC MAG GTG MAC TAC ACT CTC CTG GOT GAG 576 Tyr Gin Gly Oly Leo Pro Pro Lys Val Asn Tyr Thr Leo Leo Gly Glu ATC TOC MAC CTG TOG COG MAC TAC GAT GAO ATC CAG GAC TCA TGG GAG 624 Ile Cys Asn Leo Trp Arg Asn Tyr Asp Asp Ile Gin Asp Ser Trp Asp AGO OTO CTT TCC ATC GTO GAC TOG TTC TTC AGA MAC CAG OAT GTG CTG 672 Ser Val Leo Ser Ile Val Asp Trp Phe Phe Thr Asn Gin Asp Val Leo CAG CCG TTT GCT 0CC CCT C C AC TGG MAT GAC CCA GAC ATO CTC ATC 720 Gin Pro Phe Ala Gly Pro Gly His Trp Asn Asp Pro Asp Met Le Ile WO 94/23070 WO9412010PCTIUS94/03338 34 ATT GGA AAT TTC GGT CTC AGC TAT GAG CAG TGA CGT TCC CAA ATG GCC 768 Ile Giy Asn Phe Giy Leu Ser Tyr Giu Gin Ser Arg Ser Gin Met Ala TTG TGG ACC ATT ATG OCA GCT CCA CTC CTC ATG TGG ACC GAC CTG CGC 816 Leu Trp Thr Ile Met Ala Ala Pro Leu Leu Met Ser Thr Asp Leu Arg ACT ATC TCG CCG, AGT GCC AAG AAG ATT CTG GAG AAC. CGC GTG ATG ATC 864 Thr Ile Ser Pro Ser Ala Lys Lys Ile Leu Gin Asn Arq Leu Met Ile GAG ATA AAC GAG GAC CCC TTG GGA ATC CAG GGG CGC AGG ATC ATC AAG 912 Gin Ile Asn Gin Asp Pro Leu Gly Ile Gin Giy Arg Arg Ile Ile Lys GAG GGA TCC CAC ATT GAG GTG TTC CTG CGC CCG GTG TCA GAG GCT GCC 960 lo Giu Gly Ser His Ile Glu Vai Phe Leu Arg Pro Leu Ser Gin Ala Ala AGT GCC CTG GTC TTC TTC AGC CGG AGG AGA GAC ATG Ccc TTC CGC TAC 1008 Ser Ala Leu Val Phe Phe Ser Arg Arg Thr Asp Met Pro Phe Arg Tyr ACC ACC AGT CTT GCC AAG GTT GGC TTC CCC ATG GGA GCT GCA TAT GAG 1056 Thr Thr Ser Leu Ala Lys Leu Gly Phe Pro Met Gly Ala Ala Tyr Giu GTG CAA GAC GTG TAG AGT GGG AAG ATG ATC AGT GGC GTG MAG ACA GGA 1104 Val Gin Asp Vai Tyr Ser Gly Lys Ile Ile Ser Gly Leu Lys Thr Gly GAC MGC TTG ACA GTG ATC ATC MAC CCC TCA GGG GTG GTG ATG TGG TAC 1152 Asp Asn Phe Thr Val Ile Ile Asn Pro Ser Gly Val Val Met Trp Tyr CTG TGT CCC AAA GCA CTG CTC ATC GAG GAG CGM GGT CGT GGG GGG CCC 1200 Leu Cys Pro Lys Ala Leu Leu Ile Gin Gin Gin Ala Pro Gly Gly Pro TCG CGC CTG CCC CTT CTG TGA GGC CGA TGA TTG GGA GCC CTG GGA TAG 1248 Ser Arg Leu Pro Leu Leu ATC TGA CCG CTG CTC MAG TGC CTT CTT CTG GTG TGG CTG GGG GAG GAC 1296 ATG GAG CTT GCT GCT CTG GCA CCA CCT GAT GAT TTC TAC TGA TTC CAC 1344 GTG MAG GAG GAG TTC TTG TTA CTC CCT CCT GAG AGC ATG GMA AGC GCT 1392 CTG AGG TCC TCC TOT GWA AGA GGA GTG TTG CGA GTG ACC ATC CTT TAG 1440 GAG GAG ATG TGG TCA CCT TTT TTC CTT TG TTG GCT TAG GAG MAA GGG 1488 CTG TGG ACA GGC TG ACC GCT CTT CCC AGO GJAG CAT CCC GAG ACC AGO 1536 AGC TGG TGG GGC GAG GCT GTG TCT GTC TGG GAG CG GAT GAG GAG OTA 1584 ACA CG-A CTA GAG TOT AGT CGG CAC ATA ATG AMA AG MAA TCT MAA CMA 1632 MAC GTG TGG GAG TAG TGT ACT GMA CCC OCT CTG OTT ACA GA GAG CAA68 1680 ,WO 94123070 CIS9/33 PCTIUS94/03338 AAC CTG AOT TGT CCA TGC ACA ATC CCA GTA TCC TCA CTG TGG TGT TAG 1728 CAT GAA AAA TTG CAG TCA CAG TGC ATT GTG CAC GAG TGG TOT CTG GAA 1776 GAT GCT GAT GCT TGT TCG TGO TOG TCT TAA GOT GGG AGA TOC TCA TGG 1824 GTG CTG GCC AAG TTG CAT CTC AAT CTT GTG AGO CTG AAC CTT CCA GCA 1872 TTT CTC AGO GMA AOG CTC TTC CTT TTA AAG GCA GCC TOC ACA AAT AGA 1920 AGO GGC TCA GMA GGA CGC ACG AGG AGG GGC TCA GOT GGG CCG TGC TCC 1968 CCT GAC CAC CCC AAG AGG GOT CAA CTA CTC ACC AMA ATC TAC CCC TTT 2016 CMA GGC CAG GTC AGC CCA GGG AGA CGC ACC CMA GOT TMA ACC TCA MAA 2064 CAG GMA ATC ACC CTA TTT TMA ATT AGT GAG AAA TTG MAC TTC CCC ATT 2112 CTA TTC AGA TGA GGG CTA GMA GCC CAC TCT CCT TAG MAG GCA COT GOT 2160 GGA TTC CTG CCC CTT GCA GAG ACA TTG TGG TCT GMA OCA AGA TGC TGA 2208 ATG TGA TCT TTG CAG CGC TOO AMA TGA CAT GTC TOT TTC ATO CTT GTG 2256 TOG GAG ATO OCT TTO TTT TTG TGA TTT TGA CMA TTT MAC TGA MAT AAA 2304 AGO GMA GCA GAG GGG 2319 INFORM4ATION FOR SEQ ID NO: 2: SEQUENCE CHARACTERISTICS: LENGTH: 406 TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DESCRIPTION: cDNA to mRnNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes FRA GZET TYPE: (vi) ORIGINAL SOURCE: ORGANISM: chicken liver

STRAIN:

,WO 94/23070 PTU9133 PCT/US94/03338 Met Trp Cys Asp Trp Arg INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(Vii) IMM2EDIATE SOURCE: library (viii) POSITION IN GENOME: unknown CHROMOSOME SEGMENT: MAP POSITION:

UNITS:

(ix) FEATURE: NAME/KE: chicken liver cr-Nacetylgal actosaminidase

LOCATION:

IDENTIFICATION METHOD: OTHER INFORM4ATION: Wx PUBLICATION INFORMATION:

AUTHORS:

TITLE:

JOURNAL:

VOLUME:

PAGES:

DATE:

DOCUMENT NUMB2ER: FILING; DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: eu Glu Asn Gly Leu Ala Arg Thr Pro Pro Met Gly TI iu Arg Phe Arg Cys Asn Val Asn Cys Arg Giu Asp P le Ser Glu Met Leu Phe Met Giu Met Ala Asp Arg I ly Trp Arg Glu Leu Gly Tyr Lys Tyr Ile Asn Ile A la Ala Lys Gin Arg Asp Thr Glu Gly Arg Leu Val P rg Phe Pro Arg Gly Ile Lys Ala Leu Ala Asp Tyr V ly Leu Lys Leu Gly Ile Tyr Gly Asp Leu Gly Arg L

G

I

AG

A:

'rp ro le sp ro al eu Al a Gin Glu Cys Pro Al a Cys WO 94123070 WO 9423070PCT/US94/03338 Gly Gly Tyr Pro Giy Thr Thr Leu Asp Arg Val Giu Gin Asp Ala Gin Thr Ser Leu Tyr Ile Ser Gin Ile Leu Thr Gin Giu Ser Thr Val Asp.

Leu Ser Phe Ser Asn Gin Cys Vai Pro Giy Trp Ile Ilie Gly Aila Thr Gin ALsf Cys A.rg Ala Gly Al a Gly Asn Leu Phe Asn Thr Ser Asn Ser Leu Ser Asp Phe Pro LeuI Glu Lys Thr Gly Leu Ser Al a Phe Ile Pro Gin His Val Leu Vai rhr ?ro Trp Giu Gly Leu Trp Ile Gly Gly Met Ser Asp Ile Phe Ala Tyr Ile Ala Leu Giy Gin Arg Pro Arg Val Pro Leu Ala Ala Pro Giu Phe Lys Ser V'al Leu Val Al a Pro Pro Asn Asp Gly Ser Al a Lys Leu Val Ser Leu Giy Ile Leu Asp Gin Ile Lys Tyr Trp His Tyr Pro Lys Gly Phe Arg Gly Lys Asn Ile Met Giy Val Val1 Asp Phe Trp Giu Leu Ile I.1 e Leu Arg Phe Ile Pro Leu Tyr Tyr Asn Asp Phe Asn Gin Leu Leu Gin Arg Thr Pro Ile Ser Lys Pro Ser Tyr Ile Thr Asp Ser Met Gin Gly Pro Asp Met Ser Giy Leu Gin Cys Thr Gin Asn Pro Arg Ser Asn Arg Leu Met G ly Gly Val Asp Met Ser Leu Asp Gin Asp Ser Thr Arg Arg Ser Pro Ala Leu Val1 Gly Al a Trp Leu Ser Asp Met Gin Asp Leu Ile Gin Phe Ala Lys Met Cys Arg Pro Gly Trp Val1 Leu Met Leu Met Ile Al a Arg Tyr Thr Trp Tyr Ala Ala Glu Asp Leu Ile Ala Arg Ile Lys Ala Tyr Glu Gly Tyr 128 144 160 176 192 208 224 240 256 272 288 304 320 336 352 368 384 400 406 Gin Gin Gin Ala Pro Gly Gly Pro INFORMATION FOR SEQ ID O: 3: SEQUENCE CHARACTERISTI CS: LENGTH: 411 TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DESCRIPTION: cDNA to mRNA VO 94/23070 'WO 9423070 CT/US94103338 (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes FRAGMENT TYPE: (vi) ORIGINAL SOURCE: ORGANISM: human

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPM1'TTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

UNITS:

(ix) FEATURE: NAMFE/KEY: human cz-Nacetylgal actosazninidase

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: PUBLICATION INFORMATION: AUTHORS: Wang et al TITLE: Human a-N-Acetylgalactosaninidase Molecular Cloning, Nucleotide Sequence, and Expression of a Full-Length cDNA JOURNAL: Journal of Biological Chemistry VOLUME: 265 PAGES: 21859-21866 DATE: 1990 DOCUMENT NUMBER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Leu Leu Lys Thr Val Leu Leu Leu Gly His Val Ala Gln Val Leu Met Leu Asp Asn Gly Leu Leu Gln Thr Pro Pro Met Gly Trp Leu Ala 'WO 94/23070 W0 94/3070 'T/US94/03338 Trp Giu Arg Phe Arq Cys Asn Ile Asn Cys Asp Giu Asp Pro Lys Asn Cys Asp Trp Arg Gly Giy Phe Thr Asn Giu Cys Val1 Pro Gly Trp Ile Ilie Lys Aia Ser Gin Asn IlE G l) Ile Phe Leu Tyr Ala Pro Al a Giy Asn Leu Val Asn Thr Ser Asn Ser Leu Ser %Isp ?he Ser Trp Gly Pro Lys Pro G iu Giu Thr Giy Leu Ser Aila Phe Val Ala Gin Leu Val Leu Val Thr Gli, Arg Gly His Leu G iy Trp Giu Gly Lou Trp Ile Gly Giy Lou Gin Asp Ilie Phe 3iy kryr Asp Arg Gly Gly Thr Lys Arg Arg Pro Arg Leu Pro Leu Ala Asn Pro Giu Phe Gin Ser Val Met Asp Sle Sle Thr Val Ala Pro Pro Asn Asn Gly Ser Ala Met Leu Val Ser Lou.

Gly.

Ile Phe Giy Ala Pro Tyr Lou Asp Gin Ile Arg Tyr Trp His Leu Pro Asp Gly Tyr Cys Asn Asp A.sn Met Tyr Ser Phe Ala Asp Met Gly Ala Val1 Asp Phe Trp Giu Lou Ile Ile Met Arg Phe Ile Pro Giu *Thr Giy *Leu Asp Lys Lou Tyr Phe Asn Asp Val1 Asn.

Gin Lou Leu Gin Arg Thr Thr Ile Ser Met Tyr Arg Ala Met Val Lys Pro Ser Tyr Ile Giu Asp Ser Met Gin Gly Pro Asp Gly Ser Gly *Ala Lou Leu Asp Gly Val1 Lou Lys Cys Ser Gin His Pro Arg Ser Asn Arg Lou Met Ser Gly Val1 *Asp *Asn Met Tyr Asn Gin Asp Met Ser Lou Asp Gin Asp Ala Thr Pro Arg Ser I Pro Ile I Leu Arc Ile Pro Val Phe Asp Gly Al a Trp Lou Ser Asp M1et ksp Leu Ile .sn yr lal ~rg Met Asp Asp His Thr Al a Cys Al a Pro Al a Trp Ile Lou Met Lou Met His Lys Arg Tyr' Asp Al a Asp Pro Ser Cys Gin Phe Ala Ala Asp Trp Lou Lou Ala Arg Ile Lys Al a Tyr Giu Glu Gin Cys Lys Leu Met Thr Ser Lou Tyr Ile Ser Gin Ile Lou Thr Lys Giu Ser His Al a Thr 48 64 96 112 128 144 160 176 192 208 224 240 256 272 288 304 320 336 352 368 384 400 411 Val MetTrp Tyr Lou Tyr Pro Ile Lys Asn Lou Giu Met Ser Gin Gin 'WO 94/23070 'WO 9423970 CTIUS94IO3338 INFORMATION FOR SEQ ID NO: 4: SEQUENCE CHARACTERISTICS: LENGTH: 429 TYPE: amino acid STRANVEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DFACRIPTION: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes FRAGMENT TYPE: (vi) ORIGINAL SOURCE: ORGANISM: human

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMThEIATE SOURCE: library (viii) POSITION IN GENOME: unknown CHROMOSOME SEGMENT: MAP POSITION:

UNITS:

(ix) FEATURE: NA1ME/KEY: human a-galactosidase

LOCATION:

IDENTIFICATION METHOD: OHR INFORMATION: Wx PUBLICATION INFORMATION: AUTHORS: Calhoun et al TITLE: Fabry Disease: Isolation of a cDNA Clone Encoding Human a-Galactosidase A JOURNAL: Proceedings of the National Academy of Science USA VOLUMhE: 82 PAGES: 7364-7368 DATE: 1985 ,WO 94123070 PTU9133 PCT/US94/03338 DOCUMhENT NUMBER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: SEQUENCE DESCRIPTION: SEQ ID NO: Met Arg Asp, Arg Ser Trp Ala Phe Leu Phe Asp Leu Arg Trp His Ser Al a Phe Ile pro Gin (xi) Gin Leu Phe Leu Asn Giy Phe Met Giu Lys Lys Asp Pro Gin Pro His Lys Leu Pro Gly Trp Gly Glu Asn Thr Giy Pro Phe Trp Arg Ile Leu Gly Pro Gly Leu Met Ala Gin Ala Asp Pro Arg Al a Leu Cys Leu Al a Arg Gly Gly Ser Val Leu Arg Gin Asn Asp Gly Ser Ala Lys2 Leu Asn Pro Clu Leu His LeU Giy Cys Ala Leu Ala Leu Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Giu Asn Leu Asp Cys Gin Giu Giu Pro Asp Ser Cys Ile Phe Met Giu Met Ala Giu Leu Met Vai Ser Giu Giy Gly Tyr Giu Tyr Leu Cys Ile Asp Asp Cys Trp Met Asp Ser Giu Giy Arg Leu Gin Ala Asp Pro Gin Arg Ile Arg Gin Leu Ala Asn Tyr Val His Ser Lys Giy Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly Phe Gly Tyr Tyr Asp Ile Asp Aia Gin Thr Phe Ala Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser Al.a Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn Ser Ilie Val Tyr Ser Cys Giu Trp Pro Leu Tyr Met Lys Pro Asn Tyr Thr Giu Ile Arg Gin Tyr Cys Asn Phe Ala Asp Ilie Asp Asp Ser Trp Lys Ser Ile Lys Trp Thr Ser Phe Asn Gin Giu Arg Ile Val Asp Val G1y Trp Asn Asp Pro Asp Met Leu Ile Val Gly Asn rrp Asn Gin Gin Val Thr Gin Met Ala Leu Trp Ala ?ro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser kla Leu Leu Gin Asp Lys Asp Ile Val Ala Ile Asn ;ly Lys Gin Gly Tyr Gin Leu Arg Gin Gly Asp Asn 16 32 48 64 96 112 128 144 160 176 192 208 224 240 256 272 288 304 320 336 Phe Giu Val Trp Glu Arg Pro Leu Ser Giy Leu Ala Trp Ala Val Ala VWO 94/23070 VTU9133 IICTIUS94/03338 Met Val Thr Ser Leu Ile Asn Ala Ser Gin Leu Arg Leu Giu Asn Arg Leu Leu Arg Thr Gin Gly Pro Ser Met Glu Ile Lys Gly Val Lys His Ile Gin Met Gly Val Ar g Asn Ser G ly Ala Lys Pro Leu Pro Cys Leu Thr Lys Ar g Asn G iy Gly Asp Tyr Al a Tyr Val1 Leu Thr Ile Ala Cys Phe Ile Giu Trp Thr Leu Leu Gin 368 384 400 416 429 INFORMATION FOR SEQ ID NO: SEQUENCE CHARACTERISTICS: LENGTH: 438 TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DESCRIPTION: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes FR.AGME~NT TYPE: (vi) ORIGINAL SOURCE: ORGANISM: yeast Saccharomyces cerevisiae

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMME~DIATE SOURCE: library (viii) POSITION IN GENOME: unknown CHROMOSOME SEGEN: MAP POSITION:

UNITS:

(ix) FEATURE: NAME/KEY: yeast a-galactosidase (MELl)

LOCATION:

WO 94/23070 PTU9/33 PCT/US94/03338 43 IDENTIFICATION METHOD: OTHER INFORMATION: PUBLICATION INFORMATION: AUTHORS: Liljestrom TITLE: The Nucleotide Sequence of the Yeast MELl Gene JOURNAL: Nucleic Acids Research VOLUME: 13 PAGES: 7257-7268 DATE: 1985 DOCUMNT NUMBER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met Phe Ala Phe Tyr Phe Leu, Thr Ala Cys Ile Ser Leu Lys Gly Val 16 Phe Gly Val Ser Pro Ser Tyr Asn Gly Leu Gly Leu Thr Pro Gln Met 32 Gly Trp Asp Asn Trp Asn Thr Phe Ala Cys Asp Val Ser Glu. Gln Leu 48 Leu Leu Asp Thr Ala Asp Arg Ile Ser Asp Leu. Gly Leu Lys Asp Met 64 Gly Tyr Lys Tyr Ile Ile Leu. Asp Asp Cys Trp Ser Ser Gly Arg Asp Ser Asp Gly Phe Leu Val Ala Asp Glu. Gln Lys Phe Pro Asn Gly Met 96 Gly His Val Ala Asp His Leu. His Asn Asn Ser Phe Leu Phe Gly Met 112 Tyr Ser Ser Ala Gly Glu Tyr Thr Cys Ala Gly Tyr Pro Gly Ser Leu 128 Gly Arg Glu Glu Glu, Asp Ala Gln Phe Phe Ala Asn Asn Arg Val Asp 144 Tyr Leu Lys Tyr Asp Asn Cys Tyr Asn Lys Gly Gln Phe Gly Thr Pro 160 Glu Ile Ser Tyr His Arg Tyr Lys Ala Met Ser Asp Ala Leu Asn Lys 176 Thr Gly Arg Pro Ile Phe Tyr Ser Leu. Cys Asn Trp Gly Gln Asp Leu 192 Thr Phe Tyr Trp Gly Ser Gly Ile Ala Asn Ser Trp Arg Met Ser Gly 208 Asp Val Thr Ala Glu Phe Thr Arg Pro Asp Ser Arg Cys Pro Cys Asp 224 Gly Asp Glu Tyr Asp Cys Lys Tyr Ala Gly Phe His Cys Ser Ile Met 240 Asn Ile Leu, Asn Lys Ala Ala Pro Met Gly Gln Asn Ala Gly Val Gly 256 VO 94/23070 WO 9413070 CT/US94/03338 Gly Trp Asn Asp Leu Asp Asn Leu Glu Val Giy Val Giy Asn Leu Thr Asp Pro Ser Gly Glu Gly Met Lys 210 Val Th-r Asp Leu Ile Ile Tyr Asp Asn Lys Asp Gly G lu Ile Tyr Pro Gly Gin Thr Leu Asn Ile Glu Ile Ser Aila Gin Vai Thr Thr Ser Leu Lys Giy Gin Thr Gly Val1 Leu Ser Thr Tyr Al a Aila Ala Arg Glu Al a Giu Thr Al a Asn His Asn Ser Vali Ile Leu Giu Trp Ser Al a Phe Val1 Ile Trp Gin Leu Ile Asp Al a Thr Ser Asn Val1 Arq Met Asn Phe Ile Ile Glu Met Asn Al a Tyr Trp Gly Phe Tyr Leu Gin Trp Leu Ile Tyr Ser Gly Asp Asp Gly Ser Ala Lys Asn Val1 Giy Ser Ser Leu Arg Tyr Val Ser Asp Asp LeU Ser Leu Ala Lys Asp Lys Ser Ser Thr Asp Arq Gly Asn Thr Gly Ser Tyr Asn Asp Asn Pro Ser Arg Al a Leu 272 288 304 320 336 352 368 384 400 416 432 438 Ser Lys Asn Asp Thr Arg INFORMATION FOR SEQ ID NO: 6: Wi SEQUENCE CHARACTERISTICS: LENGTH: 411 TYPE: amino acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: DESCRIPTION: cDNA to mRNA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes FRAGM'ENT TYPE: (vi) ORIGINAL SOUR.CE: ORGANISM: guar plant Cyamozpsis tetracionoloba

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE:

HAPLOTYPE:

TISSUE TYPE: WO 94/23070 WO 94/3070 CT/US94/03338 Met Leu Arg Giu His Al a Leu Val Tyr CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMMDIATE SOURCE: library (viii) POSITION IN GENOME: unknown CHROMOSOME SEGMENT: MAP POSITION:

UNITS:

(ix) FEATURE-, NAME/KEY: guiar m-galactosidase

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: Wx PUBLICATION INFORMATION: AUTHORS: Overbeeke et al TITLE: Cloning and Nucleotide Sequence of the m-Galactosidase cDNA From Cyaxnopsis tetragonoloba (guar) JOURNAL: Plant Molecular Biology VOLUME: 13 PAGES: 541-550 DATE: 1989 DOCUM~ENT NUMBER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: la Thr His Tyr Ser Ile Ile Gly Gly Met Ile Ile Val Val et Ile Ile Gly Ser Glu Gly Gly Arg Leu Leu Giu Lys Lys ir Ser Ala Glu Ala Glu His Tyr Asn Val Arg Arg Tyr Leu ;n Gly Leu Gly Gin Thr Pro Pro Met Gly Trp Asn Ser Trp ie Gly Cys Asp Ile Asn Glu Asn Val Val Arg Giu Thr Ala ~t Val Ser Thr Gly Leu Ala Ala Leu Gly Tyr Gin Tyr Ile ;p Asp Cys Trp Ala Glu Leu Asn Arg Asp Ser Glu Gly Asn o Asn Ala Ala Ala Phe Pro Ser Gly Ile Lys Ala Leu Ala 1 His Ser Lys Gly Leu Lys Leu Gly Val Tyr Ser Asp Ala

A:

T1 As P1

ME

As Leu Asn Al a Asn Asp Asn Met Asp Gly 16 32 48 64 96 112 128 144 ,WO 94/23070 WO 94/3070 CT/US94/03338 Asn Gin Thr Cys Ser Lys Arg Met Pro Gly Ser Leu Gly His Glu Glu Gin Asp Met Cys Asn Thr Gly Thr Ala His Leu Trp Arg Leu Thr Ala Asp Asn Gly Glu Ser Ser Gly Thr Pro Glu Gly Ala Ser Gin Gin Cys Al a Cys Lys Trp Trp Ile Trp Glu Leu Leu Val G ly Ser Gin Ser Lys Lys Giu Al a Gly Arg Ala Asn Giu Leu Ile Gin Pro Ser Gly Leu Met Thr Asn Leu Trp Thr Asp Asp Tyr Val Ser Gly Leu Arg Thr Val Tyr Phe Leu Leu Giu Thr Ser Pro Arg Gly Asn Lys Ser Ala Thr Ser Val Al a Gly Ser Asp Gly Asn Asp Ser Cys Al a Lys Asp Thr VTal Gly Leu Ser Ile Ser Pro Asp Asp Met His Asp Glu Val Asn Val Asp Glu Thr Trp Ser Gly Gin Ile Lys Leu Phe Ile Ile Lys Lys Thr Al a Ile Pro G ly Val Arg Ile Glu Trp G2.u Ser Arg Val Ser Val Al a Ar g Ser Arg Val1 Lys Pro Trp Asp Al a Val1 Ile Ala Al a Thr Al a Ser Asp Ala Ser Asp Glu Ile Ala Asn Ser Giy Trp Met Val Asn Val1 Trp Lau Glu Tyr Arg Phe Lys Trp Tyr Asn Ala Asp Asn Asp Ile Ser Trp Ile Leu Tyr Phe Ser Asn Ala Gly Leu Asp Gin Leu Leu Asp Giu Asp Lys Pro Ser Ile S er Gly Gly Al a Thr Asp Glu Trp Ile His Ser Tyr Pro Met Gly Met Pro Met Lys Thr Lys Val1 Asn Gly Ser His 160 176 192 208 224 240 256 272 288 304 320 336 352 368 384 400 411 INFORMATION FOR SEQ ID N: 7: SEQUENCE CHARACTERISTICS: LENGTH: 447 TYPE: amino acid STRANDEDNESS: double TOPOLOGY* linear (ii) MOLECULE TYPE: DESCRIPTION: cDNA to mP.NA (iii) HYPOTHETICAL: no (iv) ANTI-SENSE: yes 'WO 94/23070 'wO 9/2300 PI'1US94/03338 47 FRAGMENT TYPE: (vi) ORIGINAL SOURCE: ORGANISM: Aspergillis niger

STRAIN:

INDIVIDUAL ISOLATE: DEVELOPMENTAL STAGE;,

HAPLOTYPE:

TISSUE TYPE; CELL TYPE: CELL LINE:

ORGANELLE:

(vii) IMME'DIATE SOURCE: library (viii) POSITION IN GENOME: unknown CHROMOSOME SEGMEM: MAP POSITION:

UNITS:,

(ix) FEATURE: NAME/KEY:. Aspergillus niger a-galactosidase

LOCATION:

IDENTIFICATION METHOD: OTHER INFORMATION: PUBLICATION INFORMATION: AUTHORS: den Herder et al TITLE: Cloning and Expression of a Member of the Aspergillus niger Gene Family Encoding m-Galactosidase JOURNAL: Molecular and General Genetics VOLUME: 233 PAGES: 404-410 DATE: 1992 DOCUMENT NUMBER: FILING DATE: PUBLICATION DATE: RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Met Ile Gln Gly Leu Glu Ser Ile Met Asn Gln Gly Thr Lys Arg Ile 16 Leu Leu Ala Ala Thr Leu Ala Ala Thr Pro Trp Gin Val Tyr Gly Ser 32 Ile Glu Gln Pro Ser Leu Leu Pro Thr Pro Pro Met Gly Pro Asn Asn 48 Trp Ala Arg Phe Met Cys Asp Leu Asn Glu Thr Leu Phe Thr Glu Thr 64 Ala Asp Thr Met Ala Ala Asn Giy Leu Arg Asp Ala Gly Tyr Asn Arg 'WO 94/,23070 PCT/US94/03338 Ile Asn Leu Asp Asp Cys Trp Met Ala Tyr Gin Arg Ser Asp Asn Gly ser Aila Ser Giu Leu Giu Gin Thr Tyr Aia Leu Leu Phe Ile Leu Ser Asp Asp Thr Ile Leu Let Lys Gi Gin Asp Tyr His Asp Giy Giy Leu Ile Al a Pro Ile Ar g Ar g Ile Al a Lys Pro Gin Trp Tyr Val Asn Met Asp Aia Gly Cys Lys Gin Pro Leu Asn Asn Glu Leu Ser Aia Aia Arg Pro Asp Leu Trp Aia Leu Ala Val Asp Asp Leu Leu Pro Val Glu Asp Ile Glu Gin Giy Asn Lys Thr Asn Asn Arg Ile Thr Al a Trp Tyr His Ala Ser Asn Thr Thr Gin Leu Leu Cys Thr Ala Cys Thr Val1 Tyr Phe Asp Arg Asp Gln Pro Ser Lys Gin Leu Val1 Trp Trp Ala Ser Thr Lys Gly Phe Tyr G iy Ser Trp His Ser Arg Gly Phe Asp Asp Asp Leu Leu Asp Ser Ser Lys Gly Giy Ala Al a His Giu Tyr Ser Ile Pro Leu Ser Glu Pro Ile Asn Gly Gly His Val Phe Phe Tyr Ser Thr Trp Ser Thr Thr Met Gly Thr Ala Ile Leu Leu Lys Leu Lys Ala Val Pro His Pro Trp Gin His Ala Vai Asp Asn Tyr Al a Pro Ala Al a Thr Gly Thr Thr Thr Pro His Phe G ly Gly G ly Gin Pro Met Ile Asn Phe Asp Leu Phe Gin Arg Asn Glu Gin Ala Thr Gly Gly Ser Ile Arg Val1 Ala Asp Leu Tyr Asn Glu Ile Leu Gin Ser Thr Thr Lys Val1 Gly Leu Ile Tyr Asp Thr Leu Tyr Trp Val Asn Asp Lys Ile Ile Al a Leu Thr Asp Ile Phe Lau Pro Tyr Asn Tyr Ser Phe Val Tyr Tyr Pro Arg Ser Asn Thr Al a Val1 cys Ser Arg Val1 Trp Glu His Leu Glu Lys Ala Pro Ser Asn Asp Ser Ala Glu Leu Asn Thr Thr Asp Leu Phe Leu Asp Giu Lys Giu Met Gly Ile Gly Thr Phe His Tyr Al a Al a G iy Ar g T'yr His Se r Asn 96 112 128 144 160 176 192 208 224 240 256 272 288 304 320 336 352 368 384 400 416 432 447 Thr Ala Ser Gly Asn Cys Leu Thr Ala Ala Ser Asn Ser Ser Val PCT/US94/03338 'WO 94123070 49 TNTWfATIONS IMJAnN!G TO A Dzrosr=~ MCROORCANISM (PCk We~ Ina) American Type Culture Collection 17 March 1993 ATCC No. 75434 C. DD~lON~ DDICh2OS ~~un ris inuixaina is w.mad a a, A£W=aI see 0 A DNA vector containing a nucleic acid sequence which encodes chicken liver -N-acetylgalactosaminidase enzyme.

ID. DrixiATED STATOl MR WBJCH WNICATONS AMK MADE ~~iwrlm~~d L. 8'AMTU Gay.MM OF WCATIONS (Iw 759WAR- d100Wwii1to a~kmn W uaiwI uau 'AMiu Howd-i4bramh' P"r 1"riv" CIA" un caW7 IbPbs *wwinw 1"2 Imaifirlspii Far IaWuamdo*I owe"u WoO Th ris aw* sa recre oy to wnuiia.. -a'

Claims

1. A purified and isolated nucleic acid encoding chicken liver a-N-acetylgalactosaminidase enzyme.

2. The nucleic acid of Claim 1 encoding the amino acid sequence contained in Figure 2.

3. The nucleic acid of Claim 1 having the nucleotide sequence contained in Figure 2.

4. The nucleic acid of Claim 1 contained in the vector deposited under ATCC Accession No. 75434. A vector comprising nucleic acid encoding chicken liver a-N-acetylgalactosaminidase enzyme.

6. The vector of Claim 5, wherein the nucleic acid encodes the amino acid sequence contained in Figure 2.

7. The vector of Claim 5, wherein the nucleic acid has the nucleotide sequence contained in Figure 2.

8. The vector of Claim 5, wherein the nucleic acid is contained in the vector deposited under ATCC Accession No.

75434. 9. A cell transformed with a vector comprising nucleic acid encoding chicken liver a-N-acetylgalactosaminidase enzyme. The cell 'of Claim 9, wherein the nucleic acid encodes the amino acid sequence contained in Figure 2. 11. The cell of Claim 9, wherein the nucleic acid has the nucleotide sequence contained in Figure 2. 12. The cell of Claim 9, wherein the nucleic acid is ,contained in the vector deposited under ATCC Accession No. 75434. AMENDED SHEET be~lle I~ ~e r- 51 iA/Us 13. A method for producing recombinant chicken liver a-N-acetylgalactosaminidase comprising culturing a cell transformed with a vector comprising nucleic acid encoding chicken liver a-N-acetylgalactosaminidase enzyme, and recovering a-N- acetylgalactosaminidase from the culture. 14. The method of Claim 13, wherein the nucleic acid encodes the amino acid sequence contained in Figure 2. The method of Claim 13, wherein the nucleic acid has the nucleotide sequence contained in Figure 2. 16. The method of Claim 13, wherein the nucleic acid is contained in the vector deposited under ATCC Accession No. 75434. 17. The method of Claim 13, which further comprises the step of purifying active chicken liver a-N- acetylgalactosaminidase enzyme from said culture by affinity column. 18. The method of Claim 17, wherein the affinity column is aminocaproylgalactosylamine agarose. AM E PL- P'2 1 -r Ir Ir 'WO 94/2.3070 ~U9I33 ,VUS94/03338 52 The method of~ Claim 13 wherein the recombinant chicken liver m-N1-acetylgalactosaxninidase enzyme is immunoreactive with an antibody specific for chicken liver cx-N-acetylgalactosaminidase. 16. The method of Claim 13 wherein the recombinant chicken liver a.-N-acetylgalactosaminidase enzyme has about amino acid sequence homology with humian m-N-acetylgalactosaminidase. 17. The method of Claim 13 wherein the recombinant chicken liver m-N-acetylgalactosaminidase enzyme includes the amino acid sequence depicted in Figure 2, from amino acid number 1 to amino acid number 406.