CN1421456A - Human apolipoprotein polypeptide and its use in resisting vascular proliferation diseases - Google Patents

Abstract

The present invention is huamn apolipoprotein polypeptide and its use in resisting vascular proliferation diseases. Abnormal proliferation of vessel may cause malignant tumor and the vessel change in retina of later stage diabetics may cause blindness. Vascular endothelial cell proliferation resisting therapy is one new way in treating such diseases. Apolipoprotein is one giant glucoprotein in human blood with 38 placentiform structure bodies. The end placentiform structure body (AK38) has vascular endothelial cell proliferation resisting function. Based on the amino acid sequence structure of AK38, polypeptide P5 and its compound P51-P57 with vascular endothelial cell proliferation resisting activity are synthesized. They may be produced in industry.

Description

The polypeptide of human apolipoprotein and the purposes of anti-angiogenic proliferative disease thereof

Technical field

The present invention relates to a peptide species, especially the polypeptide P5 that includes of people's lipophorin and synthetics thereof function with anti-angiogenic proliferative disease.

Background technology

Lipophorin [Apo (a)] is a huge glycoprotein that is present in the human blood.Its physiological function is not separated bright (1) as yet.But its DNA and aminoacid sequence have been verified (2).Contain 38 pie structures in its huge protein molecular structure.These 38 pie structures become the series connection form closely to combine.

The round pie structure is meant the tectosome of the cake shape with dicyclo that contains 3 pairs of inner disulfide linkage formation.It approximately contains the amino acid about 90, extensively is present in the different protein moleculars.This structure is found in hemoglutinin the earliest.The basic function that the round pie structure is taken on is the interaction between molecule and the molecule.For example tissue plasminogen activator (TPA) contains two round pie structures.A pie structure nearest apart from the C-terminal of TPA has identification and combined function to thrombus, and bootable TPA brings into play directed SL thrombus effect.

The aminoacid sequence and the composition of 38 pie structures of human apolipoprotein are different, can be divided into 11 types.Be positioned at the least significant end of pie structure concatermer, i.e. the 38th pie structure, and be named as AK38, and having anti-vascular endothelial cell growth function, the registration number of this structure in gene pool Gene Bank is AY039748.

Summary of the invention

The life that the human normal cell is supported in blood circulation exists.Blood circulation is made of blood vessel, and minimum blood vessel is called capillary vessel, and the whole body that gathers forms network, and oxygen and nutrient delivery are arrived intravital each corner.Adult blood vessel is relatively stable under normal physiological conditions, no longer infiltration and development.So healthy people's blood circulation network range is changeless basically.But when the blood circulation network has undesired plus-minus,, all may cause illness as extending or short circuit.The morbid state that blood vessel blockage forms local cutout and short circuit shows as diseases such as big cerebral apoplexy and myocardial infarction.And the unusual extension of blood circulation network can cause the malignant development of tumour.

Do not have in tumor tissues inside under the situation of blood vessel existence, tumour can not constitute fatal threat to human body.The malignancy of tumor tissues results from and produces new capillary vessel from the tumor tissues blood vessel that had existed already on every side.Capillary endothelial cell around tumor tissues is subjected to the stimulation of cancer cells secretory substance to begin division, and they pass basilar membrane and remove to invade contiguous cancer cell tissue and generate new capillary vessel in cancer cell tissue inside with the cancer cells of a matter as invasion.Cancer cells is growth fast under the supply that obtains new vessel conveying nutriment.Because of its mass consumption oxygen of growing fast, form the inner partial spatial anoxic condition of cancer cell tissue.But and anoxic condition irritation cancer emiocytosis vascular endothelial growth factor (VEGF) and Basic Fibroblast Growth Factor (bFGF) etc.These somatomedins excite the blood vessel of a new round to increase.These newborn capillary vesseies provide necessary oxygen of growth and nutriment to inner intrusion of tumor tissues and extension to tumour cell, cause the vicious cycle development of tumor tissues.The paraplasm of capillary vessel also can cause the damage and the synovitis of cartilaginous tissue.Old spot skiascope disease blind and the later stage diabetic subject also is the direct result of blood vessel hyperplasia.Anti-angiogenic prolotherapy is considered to cure the new effective way of these diseases.

Purpose of the present invention: the blood vessel tissue is to be made of endotheliocyte.The extension of blood vessel is the result that endotheliocyte is constantly bred expansion.Hit and support the needed neovascularity of the pernicious growth of tumour body, promptly attacking to tumour provides the effective way of the delivery system of growth desired nutritional is the growth of control vascular endothelial cell.So suppress the technical field of vascular endothelial cell growth is the new hope for the treatment of vascular proliferative diseases such as cancer.Biotechnology is widely used in contemporary pharmaceutical industry, and many protein drugs are by large-scale production.But the function that how to concentrate macromolecule protein is promptly carried out the polypeptide autogenic therapy in the smaller polypeptides body, is an important directions of modern biological medicine.The present invention is the combination of polypeptide body technology and anti-angiogenic hyperplasia technology.Several anti-vascular endothelial cell growth promoters or the factor have been developed and have been applied to treat vascular proliferative disease.But the demonstration human toxicity that has is as many Meads (Thalidomide is a kind of tranquilizer, can cause baby's deformity) in wiping.What have has only very weak anti-vascular endothelial cell growth activity, for example Interferon, rabbit and estrogen antagonist.And newfound protein anti-vascular endothelial cell growth factor is difficult for being produced by gene engineering method.So keeping under the prerequisite of pharmaceutical activity, easy and to produce efficiently be the crux problem of anti-angiogene medicine.

With respect to engineered protein, short and small polypeptide body is easier to be synthesized and mass production.Therefore the invention provides polypeptide body anti-angiogene technology.Simultaneously, we know that the polypeptide body of human body homology does not cause the reaction of exempting from service usually.So the present invention will pay attention to screening the human body homology polypeptide body with anti-vascular endothelial cell growth activity.

Technical scheme: polypeptide body and proteinic essentially consist unit all are amino acid.By the carboxyl (COOH) of an amino acid molecular and the amino (NH of another amino acid molecular ₂) phase condensation formation peptide bond.The dipeptides body is to connect two compounds that amino acid molecular forms by peptide bond.The compound that the polypeptide body is formed by plural amino acid molecular condensation.There is the different natural polypeptides body of numerous length and composition in the cell, constitutes various biochemical reaction, embody special cell activities.Long and polypeptide body that D structure arranged is usually by appellation protein.At present, the amino acid of about 80 of automatic producing technology polymerizables that constitutes by program control and biochemical organic synthesis principle.The polypeptide body of here being discussed is meant and contains the following molecule of 28 amino acid.

Round pie structure with human apolipoprotein is polypeptide P5 and the synthetics P51 to P57 thereof that the source screening has the anti-vascular endothelial cell growth activity, studies its aminoacid sequence and anti-angiogenic proliferative effect.

Concrete grammar: the peptide body is purified by anti-phase C18 high pressure liquid chromatography separometer (HPLC) then by Fmoc method synthetic (3,4) more than involved in the present invention.Polypeptide body behind the purifying and process mass analysis collection of illustrative plates (MS) are determined.Then, use the inhibition activity (5) of BCE cell measurement polypeptide body to vascular endothelial cell proliferation.50% o'clock the concentration that the polypeptide body is grown with respect to control group to the inhibition degree of BCE growth is called as IC ₅₀

The polypeptide body is the aminoacid sequence synthetic (Fig. 1) according to above-mentioned AK38 lipophorin.Polypeptide body and amino acid all adopt international conventional IUPAC-IUB name symbolic representation (6).Molecule ends A c and NH ₂Represent acetoxy group and amino respectively, serve as the protection mission of polypeptide body N end and C end.

The molecular composition of the polypeptide body of lipophorin is as follows: (its aminoacid sequence is seen Fig. 1)

P1：

Ac-Ile-Val-Pro-Ser-Leu-Gly-Pro-Pro-Ser-Glu-Gln-Asp-NH ₂

P2：

Ac-Met-Phe-Gly-Gln-Gly-Lys-Tyr-Arg-Gly-Lys-Lys-Ala-Thr-

Thr-Val-Thr-Thr-Val-Thr-Pro-NH ₂

P3：

Ac-Gln-Glu-Trp-Ala-Ala-Gln-Glu-Pro-His-Arg-His-Ser-Thr-

Phe-Ile-Pro-Gly-Thr-Asn-Lys-Trp-Ala-Gly-Leu-Glu-Lys-Asn-

Tyr-NH ₂

P4：

Ac-Arg-Asn-Pro-Asp-Gly-Asp-Ile-Asn-Gly-Pro-Trp-NH ₂

P5：

Ac-Tyr-Thr-Met-Asn-Pro-Arg-Lys-Leu-Phe-Asp-Tyr-NH ₂

P6：

Ac-Asp-Ile-Pro-Leu-NH ₂

P7：

Ac-Ala-Ser-Ser-Ser-Phe-Asp-NH ₂

These polypeptide bodies big or small different in size is not that no regularity is selected.They have individual common ground: promptly they are the aminoacid sequences between per two adjacent halfcystines of round pie structure.So they do not contain halfcystine, can avoid at intermolecular or inner formation disulfide linkage.

Find by anti-vascular endothelial cell growth activity analysis these polypeptide bodies: polypeptide body P1, P2, P6 and P7 almost do not have activity; P3 only demonstrates faint activity; P4 has activity; P5 polypeptide body then demonstrates the strongest anti-vascular endothelial cell growth activity (Fig. 2).The IC50 of P5 is 0.1 micro-molar concentration (Fig. 3).

The theoretical molecular of the P5 polypeptide body that activity is the strongest is 1488.66Da.The quality collection of illustrative plates of synthetic P5 polypeptide body shows that its quality is 1489.41Da (Fig. 4).The accurate scope of actual measured value meets the molecular weight of Theoretical Calculation.

P5 polypeptide body contains 11 amino acid.In order to dwindle the molecular weight of polypeptide body littlelyr, on the basis of P5 polypeptide body, synthesized polypeptide body P51 to P57.Their molecular composition dependency as shown in Figure 5.In these polypeptide bodies, the molecular structure that has has kept the anti-vascular endothelial cell activity (Fig. 6) of P5 polypeptide body.

Description of drawings

The amino acid sequence of polypeptide figure of Fig. 1 .AK38 lipophorin

AK38 gene cDNA sequence and corresponding amino acid sequence thereof are as shown in the figure.The pairing position of line part displayed polypeptides body.6 halfcystines are the dividing point of 7 (P1-P7) polypeptide bodies.

Fig. 2. the active synoptic diagram of the anti-vascular endothelial cell of polypeptide body

X-axis is polypeptide (0.75 micro-molar concentration) among the figure

Y-axis is endotheliocyte (BCE) propagation (%)

Shown in the figure the different restraining effect of 7 polypeptide bodies to the growth of BCE cell.The concentration of each polypeptide body is 0.75 micro-molar concentration.The result is the mean value of three groups of tests.

Fig. 3. the concentration of polypeptide body P5 and the synoptic diagram of activity relationship

X-axis is the micro-molar concentration of polypeptide among the figure

Y-axis is endotheliocyte (BCE) propagation (%)

The biological activity of the polypeptide body P5 of different concns shown in the figure.The polypeptide body is represented with its per-cent with respect to the control group extent of growth the inhibition degree of BCE growth.

Fig. 4. the molecular mass collection of illustrative plates of polypeptide body P5

X-axis is quality (m/z), and Y-axis is count value (counts).Acceleration voltage: 20 kilovolts.The crest of quality 1489.41Da is the monomer of polypeptide body F5.

Fig. 5. the aminoacid sequence figure of polypeptide P5 and P51 to P57

P51 is the N end parts of P5, and P52 is the replacement of P51, and P53 to P56 is the displacement of P51 and adds and subtract mutant that P57 is the C end parts of P5.

The active comparison diagram of Fig. 6 .P5 and P51 to P57 polypeptide body

X-axis is polypeptide (0.75 micro-molar concentration) among the figure

Y-axis is endotheliocyte (BCE) propagation (%)

P5 shown in the figure and displacement thereof and adding subtracts the inhibition activity of mutant to the growth of BCE cell.The concentration of each polypeptide body is 0.75 micro-molar concentration (three groups of test mean value).

Be described further below in conjunction with embodiment:

Embodiment

Embodiment 1

Use polypeptide body synthesizer (applied biochemistry: Applied Biosystems 433A).(A) installation contains the synthetic peptide synthesis garden of Asp polypeptide post, and 25 micromoles synthesize by follow procedure then.(B) flushing: with dimethyl formamide DMF (N, Ndimethylformamide) 5 minutes; (C) remove protecting group: the DMF solution 15 minutes that contains 20% piperidines (piperidine); (D) flushing: DMF 5 minutes; (E) sensitization is used: HBTU (O-benzo tripyrrole base-N, N, N ', N '-tetramethyl-alditol hexafluorophosphate)/HBTU (O-benzotriazolyl-N, N, N ', N '-tetramethyl-uronium hexafluorophosphat, 25 micromoles)/HOBT (1-hydroxy benzo tripyrrole HOBT (1-hydroxybenzo-triazole, 25 micromoles)/DIEA (diisopropylethylamine)/DIEA (diisopropylethylamine, 50 micromoles) 30 minutes; (F) combination: 0.1 mmole Gln was at DMF30 minute; (G) flushing: DMF5 minute; (H) repeating step (E)-(G).Order: Glu below step (F) is pressed in conjunction with amino acid, Ser, Pro, Pro, Gly, Leu, Ser, Pro, Val, last Ile.Use THF tetrahydroxy furans (tetrahydrofuran) flushing 5 minutes afterwards, dry 10 minutes.Free polypeptide body: contain 50 microlitre thioanisoles (thioanisole) with fresh solution, 25 microlitre distilled water, 25 microlitre dithioglycols (ethanedithiol) and 0.9 microlitre TFA tetrahydrofuran (THF) (tetrahydrofuran), 0 ℃ 15 minutes, 23C °, 2 hours.

Embodiment 2

Following steps are different with embodiment 1: step (A) is used and is contained the synthetic post of Pro.Step (F) is by following order: Thr, Gly, Thr, Val, Thr, Thr, Ala, Lys, Lys, Gly, Arg, Tyr, Gly, Lys, Gly, Asn, Gly, Phe and Met.

Embodiment 3

Following steps are different with embodiment 1: step (A) is used the synthetic post that contains Tyr.Step (F) is by following order: Asn, Lys, Glu, Leu, Gly, Ala, Trp, Lys, Asn, Thr, Gly, Pro, Ile, Phe, Thr, Ser, His, Arg, His, Pro, Glu, Gln, Ala, Ala, Trp, Glu and Gln.

Embodiment 4

Following steps are different with embodiment 1: step (A) is used the synthetic post that contains Pro.Step (F) is by following order: Pro, Gly, Asn, Ile, Asp, Gly, Asp, Pro, Asn and Arg.

Embodiment 5

Following steps are different with embodiment 1: step (A) is used the synthetic post that contains Tyr.Step (F) is by following order: Asp, Phe, Leu, Lys, Arg, Pro, Asn, Met, Thr and Tyr.

Embodiment 6

Following steps are different with embodiment 1: step (A) is used the synthetic post that contains Leu.Step (F) is by following order: Pro, Ile and Asp.

Embodiment 7

Following steps are different with embodiment 1: step (A) is used the synthetic post that contains Asp.Step (F) is by following order: Phe, Ser, Ser, Ser and Ala.

Embodiment 8

Use anti-phase C18 high pressure liquid chromatography separometer purification aforementioned polypeptides body.The specification of separator column and analytical column is respectively 20 millimeters * 250 millimeters and 4.6 millimeters * 250 millimeters.Buffered soln is A (0.1%TFA+99.9% distilled water) and B (0.01%TFA+99.99% acetonitrile acetonitrile); Gradient: buffered soln A (100%)/B (0%) is to A (0%)/B (100%).The flow velocity of separator column and analytical column is respectively 15 ml/min and 1 ml/min, and the flow process time was respectively 45 minutes and 20 minutes.

Embodiment 9

Measure the polypeptide body inhibition activity of vascular endothelial cell proliferation has been used BCE cell (5).The nutrient solution of BCE cell contains 90%DMEM, the Basic Fibroblast Growth Factor (bFGF) of 10% serum (FBS) and 0.002 mcg/ml.Use the dull and stereotyped cultivation in 24 holes.Grow into 8,000 to 10,000 cells and 0.5 milliliter of nutrient solution in every hole.The test site changes the nutrient solution that contains the polypeptide body into after 12 hours, continues cultivation at 37 ℃ and counts after 3-5 days.

The above polypeptide that includes according to lipophorin [Apo (a)] has the aminoacid sequence of the pie structure (AK38) of anti-vascular endothelial cell growth function, has synthesized a series of polypeptide bodies.Each polypeptide body composition and structure are made up of the aminoacid sequence between two adjacent halfcystines of AK38 round pie structure respectively.Find that from these polypeptide bodies P5 has the anti-vascular endothelial cell growth activity.This polypeptide body contains 11 amino acid: Tyr-Thr-Met-Asn-Pro-Arg-Lys-Leu-Phe-Asp-Tyr.On the basis of P5 polypeptide body, synthesized littler polypeptide body P51 to P57.P5 polypeptides derived body only contains 7-9 amino acid, the union body of described polypeptide N-terminal and C-terminal, and the union body of the single repetition of described polypeptide or a plurality of not homopolypeptide bodies has all been inherited the biological activity of P5 polypeptide body.

Application of the present invention: polypeptide of the present invention has the function of anti-vascular endothelial cell growth activity, be applied to treat tumour, hemangiofibroma, granulomatosis, spider shape hemorrhoid, rendu-Osler-Weber disease, von Willebrand disease and prevention and treatment later stage diabetes-induced blind etc. with the blood vessel hyperplasia diseases associated.Described peptide molecule can be made the medical article of the above disease of treatment, is still had the effect of the above-mentioned disease of treatment by the molecule that reduction, increase or displacement produced at the amino acid of described polypeptide.

Reference (1) Mooser V.Seabra MC.Abedin M.LandschulzKT.Marcovina S.Hoobbs H.Apolipoprotein (a) kringle 4-containing fragments in human urine.Journal of Clinic Investigation.1996; 97:858-864. (2) McLean JW.Tomlinson JE.Kuang WJ.Eaton DL.Chen EY.Fless GM.Scanu AM.Lawn RM.CDNA sequence of humanapolipoprotein (a) is homologous to plasminogen.Nature 1987; 330:132-137. (3) Meienhofer J.Hormonal Proteins andPeptides.Academic Press, New York.1973. (4) Schroder G.Lupke K.The Peptides.Acacemic Press, New York.1965. (5) Folkman J.Haudenschild CC.Zetter BR.Long-term culture of capillary endothelialcells.Proceedings of the National Academy ofSciences USA.1979; 76:5217-5221. (6) Anonymous.IUPAC-IUB Joint Commission onBiochemical Nomenclature (JCBN) .Nomenclatureand symbolism for amino acids and peptides.European Journal of Biochemi stry.1984; 138:9-37.

The reference translation:

(1) the garden pie structure 4-of the lipophorin in the Mooser V.Seabra MC.Abedin M.Landschulz KT.Marcovina S.Hoobbs H. people urine suppresses fragment Journal of Clinical Investigation 1996; 97:858-864.

(2) the CDNA sequence of McLean JW.Tomlinson JE.Kuang WJ.Eaton DL.Chen EY.Fless GM.Scanu AM.Lawn RM. human apolipoprotein and profibr(in)olysin are homologous natures 1987; 330:132-137.

(3) Meienhofer J. neurophysin and polypeptide NYAS publication 1973.

(4) Schroder G.Lupke K. polypeptide NYAS publication 1965.

(5) the long-term cultivation NAS journal 1979 of Folkman J.Haudenschild CC.Zetter BR. capillary endothelial cell; 76:5217-5221.

(6) anonymous (collective's works) is used for the name and the biochemical European magazine 1984 of symbol of amino acid and polypeptide about biochemical IUPAC-IUB names joint committee; 138:9-37.

Claims (4)

1, a kind of polypeptide P5 with anti-vascular endothelial cell growth activity is characterized in that described polypeptide P5 and as follows at the amino acid sequence of the synthetic polypeptide P51 to P57 in the basis of polypeptide P5: P5 Tyr Thr Met Asn Pro Arg Lys Leu Phe Asp TyrP51 Tyr Thr Met Asn Pro Arg LysP52 Tyr Thr Thr Asn Pro Arg LysP53 His Val Leu Lys Asn Arg LysP54 Gly Val Ile Thr Asn Pro Arg Ile LysP55 Gly Val Ile Thr Lys Ile LysP56 Gly Val Ile Thr Arg Ile LysP57 Pro Arg Lys Leyu Phe Asp Tyr

2. polypeptide according to claim 1 is characterized in that N-terminal and C-terminal have union body.

3. polypeptide according to claim 1 is characterized in that having the union body of single repetition or a plurality of not homopolypeptide bodies.

4. polypeptide P5 with anti-vascular endothelial cell growth activity, it is characterized in that described polypeptide P5 and synthetic polypeptide P51 to P57 on the basis of polypeptide P5, can be applied to treat the purposes of anti-angiogenic proliferative disease, described peptide molecule can be made the medical article of the above-mentioned disease of treatment.

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