ZA200007766B - Disintegrin homologs. - Google Patents
Disintegrin homologs. Download PDFInfo
- Publication number
- ZA200007766B ZA200007766B ZA200007766A ZA200007766A ZA200007766B ZA 200007766 B ZA200007766 B ZA 200007766B ZA 200007766 A ZA200007766 A ZA 200007766A ZA 200007766 A ZA200007766 A ZA 200007766A ZA 200007766 B ZA200007766 B ZA 200007766B
- Authority
- ZA
- South Africa
- Prior art keywords
- seq
- polypeptide
- molecule
- residues
- polypeptide molecule
- Prior art date
Links
- 101800001224 Disintegrin Proteins 0.000 title claims description 47
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 420
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 378
- 229920001184 polypeptide Polymers 0.000 claims description 344
- 210000004027 cell Anatomy 0.000 claims description 209
- 238000000034 method Methods 0.000 claims description 106
- 102000040430 polynucleotide Human genes 0.000 claims description 86
- 108091033319 polynucleotide Proteins 0.000 claims description 86
- 239000002157 polynucleotide Substances 0.000 claims description 86
- 150000001413 amino acids Chemical class 0.000 claims description 59
- 210000001519 tissue Anatomy 0.000 claims description 51
- 108091005804 Peptidases Proteins 0.000 claims description 36
- 239000004365 Protease Substances 0.000 claims description 36
- 230000000295 complement effect Effects 0.000 claims description 35
- 102000035195 Peptidases Human genes 0.000 claims description 31
- 210000004556 brain Anatomy 0.000 claims description 26
- 125000000539 amino acid group Chemical group 0.000 claims description 24
- 238000001727 in vivo Methods 0.000 claims description 22
- 210000002216 heart Anatomy 0.000 claims description 21
- 239000002773 nucleotide Substances 0.000 claims description 19
- 125000003729 nucleotide group Chemical group 0.000 claims description 19
- 108020001507 fusion proteins Proteins 0.000 claims description 18
- 102000037865 fusion proteins Human genes 0.000 claims description 18
- 210000000278 spinal cord Anatomy 0.000 claims description 16
- 239000013604 expression vector Substances 0.000 claims description 15
- 238000000338 in vitro Methods 0.000 claims description 15
- 210000002027 skeletal muscle Anatomy 0.000 claims description 15
- 230000008611 intercellular interaction Effects 0.000 claims description 13
- 238000013518 transcription Methods 0.000 claims description 12
- 230000035897 transcription Effects 0.000 claims description 12
- 238000012258 culturing Methods 0.000 claims description 9
- 210000004748 cultured cell Anatomy 0.000 claims description 6
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 4
- 108090000623 proteins and genes Proteins 0.000 description 149
- 102000004169 proteins and genes Human genes 0.000 description 88
- 235000018102 proteins Nutrition 0.000 description 87
- 235000001014 amino acid Nutrition 0.000 description 64
- 229940024606 amino acid Drugs 0.000 description 51
- 108020004414 DNA Proteins 0.000 description 50
- 230000027455 binding Effects 0.000 description 46
- 239000012634 fragment Substances 0.000 description 37
- 238000003556 assay Methods 0.000 description 34
- 102000006495 integrins Human genes 0.000 description 33
- 108010044426 integrins Proteins 0.000 description 33
- 125000003275 alpha amino acid group Chemical group 0.000 description 31
- 230000000694 effects Effects 0.000 description 30
- 102000005962 receptors Human genes 0.000 description 30
- 108020003175 receptors Proteins 0.000 description 30
- 230000004927 fusion Effects 0.000 description 28
- 239000013598 vector Substances 0.000 description 28
- -1 MDCs Proteins 0.000 description 26
- 230000014509 gene expression Effects 0.000 description 25
- 108020004635 Complementary DNA Proteins 0.000 description 24
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 21
- 238000010804 cDNA synthesis Methods 0.000 description 21
- 239000002299 complementary DNA Substances 0.000 description 21
- 239000011347 resin Substances 0.000 description 21
- 229920005989 resin Polymers 0.000 description 21
- 230000003248 secreting effect Effects 0.000 description 21
- 238000006467 substitution reaction Methods 0.000 description 20
- 108020004705 Codon Proteins 0.000 description 19
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 18
- 125000006239 protecting group Chemical group 0.000 description 18
- 238000000746 purification Methods 0.000 description 18
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 17
- 238000004519 manufacturing process Methods 0.000 description 17
- 108091022885 ADAM Proteins 0.000 description 16
- 102000029791 ADAM Human genes 0.000 description 16
- 239000000243 solution Substances 0.000 description 16
- 241000894007 species Species 0.000 description 16
- 238000003786 synthesis reaction Methods 0.000 description 16
- 108091028043 Nucleic acid sequence Proteins 0.000 description 15
- 241001452677 Ogataea methanolica Species 0.000 description 15
- 230000015572 biosynthetic process Effects 0.000 description 15
- 230000008859 change Effects 0.000 description 15
- 239000000499 gel Substances 0.000 description 15
- 239000000203 mixture Substances 0.000 description 15
- 239000000523 sample Substances 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 14
- 150000001875 compounds Chemical class 0.000 description 14
- 238000005859 coupling reaction Methods 0.000 description 14
- 235000018417 cysteine Nutrition 0.000 description 14
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 14
- 241000588724 Escherichia coli Species 0.000 description 13
- 101710112987 Zinc metalloproteinase-disintegrin-like crotastatin Proteins 0.000 description 13
- 230000004913 activation Effects 0.000 description 13
- 239000000556 agonist Substances 0.000 description 13
- 230000004069 differentiation Effects 0.000 description 13
- 230000005764 inhibitory process Effects 0.000 description 13
- 239000005557 antagonist Substances 0.000 description 12
- 239000003446 ligand Substances 0.000 description 12
- 241000701161 unidentified adenovirus Species 0.000 description 12
- 108700021041 Disintegrin Proteins 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- 239000000463 material Substances 0.000 description 11
- 239000012528 membrane Substances 0.000 description 11
- 108020004999 messenger RNA Proteins 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 230000035755 proliferation Effects 0.000 description 11
- 238000011282 treatment Methods 0.000 description 11
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 10
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 10
- 108010076504 Protein Sorting Signals Proteins 0.000 description 10
- 241000700605 Viruses Species 0.000 description 10
- 229940072056 alginate Drugs 0.000 description 10
- 229920000615 alginic acid Polymers 0.000 description 10
- 235000010443 alginic acid Nutrition 0.000 description 10
- 230000003171 anti-complementary effect Effects 0.000 description 10
- 230000008878 coupling Effects 0.000 description 10
- 238000010168 coupling process Methods 0.000 description 10
- 239000003814 drug Substances 0.000 description 10
- 239000003550 marker Substances 0.000 description 10
- 238000012216 screening Methods 0.000 description 10
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 9
- 230000036755 cellular response Effects 0.000 description 9
- 239000003153 chemical reaction reagent Substances 0.000 description 9
- 238000003776 cleavage reaction Methods 0.000 description 9
- 210000002950 fibroblast Anatomy 0.000 description 9
- 230000002068 genetic effect Effects 0.000 description 9
- 239000013615 primer Substances 0.000 description 9
- 208000037803 restenosis Diseases 0.000 description 9
- 230000007017 scission Effects 0.000 description 9
- 230000008685 targeting Effects 0.000 description 9
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 8
- 102000005741 Metalloproteases Human genes 0.000 description 8
- 108010006035 Metalloproteases Proteins 0.000 description 8
- 206010028980 Neoplasm Diseases 0.000 description 8
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 8
- 230000002391 anti-complement effect Effects 0.000 description 8
- 108010008730 anticomplement Proteins 0.000 description 8
- 210000001772 blood platelet Anatomy 0.000 description 8
- 239000003795 chemical substances by application Substances 0.000 description 8
- 231100000433 cytotoxic Toxicity 0.000 description 8
- 230000001472 cytotoxic effect Effects 0.000 description 8
- 230000002950 deficient Effects 0.000 description 8
- 229940079593 drug Drugs 0.000 description 8
- 210000004962 mammalian cell Anatomy 0.000 description 8
- 239000011159 matrix material Substances 0.000 description 8
- 230000037361 pathway Effects 0.000 description 8
- 230000009805 platelet accumulation Effects 0.000 description 8
- 230000014616 translation Effects 0.000 description 8
- 241000701447 unidentified baculovirus Species 0.000 description 8
- 230000002792 vascular Effects 0.000 description 8
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 7
- 101710121366 Disintegrin and metalloproteinase domain-containing protein 11 Proteins 0.000 description 7
- 102100031107 Disintegrin and metalloproteinase domain-containing protein 11 Human genes 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 7
- 238000000636 Northern blotting Methods 0.000 description 7
- 108091027981 Response element Proteins 0.000 description 7
- 239000003636 conditioned culture medium Substances 0.000 description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 210000002889 endothelial cell Anatomy 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 210000003527 eukaryotic cell Anatomy 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 239000003112 inhibitor Substances 0.000 description 7
- 230000003993 interaction Effects 0.000 description 7
- 238000013507 mapping Methods 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 210000000130 stem cell Anatomy 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 101800000620 Disintegrin-like Proteins 0.000 description 6
- 108091060211 Expressed sequence tag Proteins 0.000 description 6
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 6
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 229920002684 Sepharose Polymers 0.000 description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 6
- 239000004473 Threonine Substances 0.000 description 6
- 230000006907 apoptotic process Effects 0.000 description 6
- 239000011324 bead Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 210000004413 cardiac myocyte Anatomy 0.000 description 6
- 210000000349 chromosome Anatomy 0.000 description 6
- 230000006539 extracellular acidification Effects 0.000 description 6
- 230000002163 immunogen Effects 0.000 description 6
- 238000010348 incorporation Methods 0.000 description 6
- 230000005855 radiation Effects 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 235000004400 serine Nutrition 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 235000008521 threonine Nutrition 0.000 description 6
- 239000003053 toxin Substances 0.000 description 6
- 238000012546 transfer Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 5
- 102000014914 Carrier Proteins Human genes 0.000 description 5
- 101710201511 Coagulation factor X-activating enzyme heavy chain Proteins 0.000 description 5
- 206010053567 Coagulopathies Diseases 0.000 description 5
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 108091029865 Exogenous DNA Proteins 0.000 description 5
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 5
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 5
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 5
- 241000235648 Pichia Species 0.000 description 5
- 108010029485 Protein Isoforms Proteins 0.000 description 5
- 102000001708 Protein Isoforms Human genes 0.000 description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 5
- 101710154810 Snake venom metalloproteinase Proteins 0.000 description 5
- 101710144366 Snake venom metalloproteinase acutolysin-C Proteins 0.000 description 5
- 101710177459 Snake venom metalloproteinase fibrolase Proteins 0.000 description 5
- 101710197154 Zinc metalloproteinase-disintegrin VMP-II Proteins 0.000 description 5
- 101710112278 Zinc metalloproteinase-disintegrin-like Proteins 0.000 description 5
- 101710200352 Zinc metalloproteinase-disintegrin-like VMP-III Proteins 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 239000000427 antigen Substances 0.000 description 5
- 230000000890 antigenic effect Effects 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 210000001367 artery Anatomy 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 5
- 108091008324 binding proteins Proteins 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 230000000747 cardiac effect Effects 0.000 description 5
- 230000007910 cell fusion Effects 0.000 description 5
- 230000035602 clotting Effects 0.000 description 5
- 230000001086 cytosolic effect Effects 0.000 description 5
- 238000012217 deletion Methods 0.000 description 5
- 230000037430 deletion Effects 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 210000002458 fetal heart Anatomy 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 239000002502 liposome Substances 0.000 description 5
- 229920002521 macromolecule Polymers 0.000 description 5
- 230000001404 mediated effect Effects 0.000 description 5
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- 230000004766 neurogenesis Effects 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000002265 prevention Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 238000007634 remodeling Methods 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- 230000001131 transforming effect Effects 0.000 description 5
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 5
- 235000002374 tyrosine Nutrition 0.000 description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 4
- ORILYTVJVMAKLC-UHFFFAOYSA-N Adamantane Natural products C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 108091026890 Coding region Proteins 0.000 description 4
- 229920002307 Dextran Polymers 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 4
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 4
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 4
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 4
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- 108700008625 Reporter Genes Proteins 0.000 description 4
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 208000024248 Vascular System injury Diseases 0.000 description 4
- 208000012339 Vascular injury Diseases 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 4
- 230000000692 anti-sense effect Effects 0.000 description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 4
- 230000000975 bioactive effect Effects 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 230000002759 chromosomal effect Effects 0.000 description 4
- 230000014107 chromosome localization Effects 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 238000004520 electroporation Methods 0.000 description 4
- 238000010828 elution Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000001415 gene therapy Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 239000003102 growth factor Substances 0.000 description 4
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 4
- 235000014304 histidine Nutrition 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 206010020718 hyperplasia Diseases 0.000 description 4
- 230000001965 increasing effect Effects 0.000 description 4
- 230000002401 inhibitory effect Effects 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 229960003136 leucine Drugs 0.000 description 4
- 210000004185 liver Anatomy 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 4
- 238000002703 mutagenesis Methods 0.000 description 4
- 231100000350 mutagenesis Toxicity 0.000 description 4
- 210000000107 myocyte Anatomy 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 210000000056 organ Anatomy 0.000 description 4
- 230000036961 partial effect Effects 0.000 description 4
- 238000010647 peptide synthesis reaction Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000017854 proteolysis Effects 0.000 description 4
- 230000022379 skeletal muscle tissue development Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 4
- 125000003396 thiol group Chemical group [H]S* 0.000 description 4
- 239000013603 viral vector Substances 0.000 description 4
- BDNKZNFMNDZQMI-UHFFFAOYSA-N 1,3-diisopropylcarbodiimide Chemical compound CC(C)N=C=NC(C)C BDNKZNFMNDZQMI-UHFFFAOYSA-N 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 102100035882 Catalase Human genes 0.000 description 3
- 108010053835 Catalase Proteins 0.000 description 3
- 108010048623 Collagen Receptors Proteins 0.000 description 3
- 241000699802 Cricetulus griseus Species 0.000 description 3
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 3
- 241000702421 Dependoparvovirus Species 0.000 description 3
- QOSSAOTZNIDXMA-UHFFFAOYSA-N Dicylcohexylcarbodiimide Chemical compound C1CCCCC1N=C=NC1CCCCC1 QOSSAOTZNIDXMA-UHFFFAOYSA-N 0.000 description 3
- QMMFVYPAHWMCMS-UHFFFAOYSA-N Dimethyl sulfide Chemical compound CSC QMMFVYPAHWMCMS-UHFFFAOYSA-N 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- KOSRFJWDECSPRO-WDSKDSINSA-N Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(O)=O KOSRFJWDECSPRO-WDSKDSINSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 102100025305 Integrin alpha-2 Human genes 0.000 description 3
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 3
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 3
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 3
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 3
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- 108700025832 Serum Response Element Proteins 0.000 description 3
- 241000256251 Spodoptera frugiperda Species 0.000 description 3
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 210000001789 adipocyte Anatomy 0.000 description 3
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 3
- 150000001408 amides Chemical class 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 238000002399 angioplasty Methods 0.000 description 3
- 210000003433 aortic smooth muscle cell Anatomy 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 206010003246 arthritis Diseases 0.000 description 3
- 125000003118 aryl group Chemical group 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 235000003704 aspartic acid Nutrition 0.000 description 3
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 3
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 3
- 229960000074 biopharmaceutical Drugs 0.000 description 3
- 229960002685 biotin Drugs 0.000 description 3
- 235000020958 biotin Nutrition 0.000 description 3
- 239000011616 biotin Substances 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 230000022159 cartilage development Effects 0.000 description 3
- 230000003197 catalytic effect Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000001684 chronic effect Effects 0.000 description 3
- 229940095074 cyclic amp Drugs 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 239000012636 effector Substances 0.000 description 3
- 235000020776 essential amino acid Nutrition 0.000 description 3
- 239000003797 essential amino acid Substances 0.000 description 3
- 230000007717 exclusion Effects 0.000 description 3
- 210000002744 extracellular matrix Anatomy 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 125000002485 formyl group Chemical group [H]C(*)=O 0.000 description 3
- 235000013922 glutamic acid Nutrition 0.000 description 3
- 239000004220 glutamic acid Substances 0.000 description 3
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 210000002064 heart cell Anatomy 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 3
- 230000028993 immune response Effects 0.000 description 3
- 208000014674 injury Diseases 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 229910021645 metal ion Inorganic materials 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 3
- 229920002401 polyacrylamide Polymers 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 239000002243 precursor Substances 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 150000003235 pyrrolidines Chemical class 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 210000002363 skeletal muscle cell Anatomy 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000010532 solid phase synthesis reaction Methods 0.000 description 3
- 230000009870 specific binding Effects 0.000 description 3
- 231100000765 toxin Toxicity 0.000 description 3
- 108700012359 toxins Proteins 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 3
- 230000005760 tumorsuppression Effects 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 2
- PDRJLZDUOULRHE-ZETCQYMHSA-N (2s)-2-amino-3-pyridin-2-ylpropanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=N1 PDRJLZDUOULRHE-ZETCQYMHSA-N 0.000 description 2
- DFZVZEMNPGABKO-ZETCQYMHSA-N (2s)-2-amino-3-pyridin-3-ylpropanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=CN=C1 DFZVZEMNPGABKO-ZETCQYMHSA-N 0.000 description 2
- FQFVANSXYKWQOT-ZETCQYMHSA-N (2s)-2-azaniumyl-3-pyridin-4-ylpropanoate Chemical compound OC(=O)[C@@H](N)CC1=CC=NC=C1 FQFVANSXYKWQOT-ZETCQYMHSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 2
- JMTMSDXUXJISAY-UHFFFAOYSA-N 2H-benzotriazol-4-ol Chemical compound OC1=CC=CC2=C1N=NN2 JMTMSDXUXJISAY-UHFFFAOYSA-N 0.000 description 2
- HJBLUNHMOKFZQX-UHFFFAOYSA-N 3-hydroxy-1,2,3-benzotriazin-4-one Chemical compound C1=CC=C2C(=O)N(O)N=NC2=C1 HJBLUNHMOKFZQX-UHFFFAOYSA-N 0.000 description 2
- XWHHYOYVRVGJJY-QMMMGPOBSA-N 4-fluoro-L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(F)C=C1 XWHHYOYVRVGJJY-QMMMGPOBSA-N 0.000 description 2
- 108091007507 ADAM12 Proteins 0.000 description 2
- WRDABNWSWOHGMS-UHFFFAOYSA-N AEBSF hydrochloride Chemical compound Cl.NCCC1=CC=C(S(F)(=O)=O)C=C1 WRDABNWSWOHGMS-UHFFFAOYSA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 241000201370 Autographa californica nucleopolyhedrovirus Species 0.000 description 2
- 241000271566 Aves Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 241000699800 Cricetinae Species 0.000 description 2
- 108050006400 Cyclin Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 102100031112 Disintegrin and metalloproteinase domain-containing protein 12 Human genes 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 2
- 108010074860 Factor Xa Proteins 0.000 description 2
- 102000001133 Fertilins Human genes 0.000 description 2
- 108010069446 Fertilins Proteins 0.000 description 2
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 2
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 2
- 241000700588 Human alphaherpesvirus 1 Species 0.000 description 2
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 2
- 108060003951 Immunoglobulin Proteins 0.000 description 2
- 206010061216 Infarction Diseases 0.000 description 2
- 241000235058 Komagataella pastoris Species 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 2
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 2
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 102000018697 Membrane Proteins Human genes 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- 108091092878 Microsatellite Proteins 0.000 description 2
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Substances CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 2
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 2
- 108091061960 Naked DNA Proteins 0.000 description 2
- 108020004485 Nonsense Codon Proteins 0.000 description 2
- 108091060545 Nonsense suppressor Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 241001504519 Papio ursinus Species 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 102100027330 Phosphoribosylaminoimidazole carboxylase Human genes 0.000 description 2
- 239000004793 Polystyrene Substances 0.000 description 2
- 102000009339 Proliferating Cell Nuclear Antigen Human genes 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 101000780280 Rattus norvegicus Disintegrin and metalloproteinase domain-containing protein 1 Proteins 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 108050008861 SH3 domains Proteins 0.000 description 2
- 102000000395 SH3 domains Human genes 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241000700584 Simplexvirus Species 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 108010000499 Thromboplastin Proteins 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 102100030859 Tissue factor Human genes 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 108020005202 Viral DNA Proteins 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000007792 addition Methods 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000001261 affinity purification Methods 0.000 description 2
- 230000004075 alteration Effects 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 230000000181 anti-adherent effect Effects 0.000 description 2
- 210000002403 aortic endothelial cell Anatomy 0.000 description 2
- 230000002238 attenuated effect Effects 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 230000017531 blood circulation Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007975 buffered saline Substances 0.000 description 2
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 239000001506 calcium phosphate Substances 0.000 description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 description 2
- 235000011010 calcium phosphates Nutrition 0.000 description 2
- BPKIGYQJPYCAOW-FFJTTWKXSA-I calcium;potassium;disodium;(2s)-2-hydroxypropanoate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].C[C@H](O)C([O-])=O BPKIGYQJPYCAOW-FFJTTWKXSA-I 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 125000000837 carbohydrate group Chemical group 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- PFKFTWBEEFSNDU-UHFFFAOYSA-N carbonyldiimidazole Chemical compound C1=CN=CN1C(=O)N1C=CN=C1 PFKFTWBEEFSNDU-UHFFFAOYSA-N 0.000 description 2
- 230000015556 catabolic process Effects 0.000 description 2
- 238000005277 cation exchange chromatography Methods 0.000 description 2
- 230000021164 cell adhesion Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000004640 cellular pathway Effects 0.000 description 2
- 238000007385 chemical modification Methods 0.000 description 2
- 230000003399 chemotactic effect Effects 0.000 description 2
- 125000004218 chloromethyl group Chemical group [H]C([H])(Cl)* 0.000 description 2
- 239000012501 chromatography medium Substances 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- PMMYEEVYMWASQN-IMJSIDKUSA-N cis-4-Hydroxy-L-proline Chemical compound O[C@@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-IMJSIDKUSA-N 0.000 description 2
- 208000018631 connective tissue disease Diseases 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 210000004351 coronary vessel Anatomy 0.000 description 2
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000010511 deprotection reaction Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 238000000502 dialysis Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 230000000447 dimerizing effect Effects 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 2
- 238000005538 encapsulation Methods 0.000 description 2
- 238000013171 endarterectomy Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 150000002118 epoxides Chemical class 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 235000004554 glutamine Nutrition 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 229960004198 guanidine Drugs 0.000 description 2
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 2
- 210000005003 heart tissue Anatomy 0.000 description 2
- 108010044853 histidine-rich proteins Proteins 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 102000018358 immunoglobulin Human genes 0.000 description 2
- 230000007574 infarction Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 230000000302 ischemic effect Effects 0.000 description 2
- 108010045069 keyhole-limpet hemocyanin Proteins 0.000 description 2
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 2
- 108010052968 leupeptin Proteins 0.000 description 2
- 108020001756 ligand binding domains Proteins 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 239000006249 magnetic particle Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 2
- CWWARWOPSKGELM-SARDKLJWSA-N methyl (2s)-2-[[(2s)-2-[[2-[[(2s)-2-[[(2s)-2-[[(2s)-5-amino-2-[[(2s)-5-amino-2-[[(2s)-1-[(2s)-6-amino-2-[[(2s)-1-[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]hexanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-5 Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)OC)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCCN)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CCCN=C(N)N)C1=CC=CC=C1 CWWARWOPSKGELM-SARDKLJWSA-N 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 238000000520 microinjection Methods 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 210000000663 muscle cell Anatomy 0.000 description 2
- 210000003098 myoblast Anatomy 0.000 description 2
- 210000001087 myotubule Anatomy 0.000 description 2
- 230000009707 neogenesis Effects 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 230000037434 nonsense mutation Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000000963 osteoblast Anatomy 0.000 description 2
- NAHBVNMACPIHAH-HLICZWCASA-N p-ii Chemical compound C([C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H]2CSSC[C@H](NC(=O)[C@H](CC=3C=CC=CC=3)NC(=O)CNC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC2=O)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CSSC[C@@H](C(=O)N1)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)=O)C(C)C)C1=CC=CC=C1 NAHBVNMACPIHAH-HLICZWCASA-N 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 2
- 229950000964 pepstatin Drugs 0.000 description 2
- 108010091212 pepstatin Proteins 0.000 description 2
- FAXGPCHRFPCXOO-LXTPJMTPSA-N pepstatin A Chemical compound OC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C[C@H](O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)NC(=O)CC(C)C FAXGPCHRFPCXOO-LXTPJMTPSA-N 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 230000007030 peptide scission Effects 0.000 description 2
- 210000001322 periplasm Anatomy 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- 108010035774 phosphoribosylaminoimidazole carboxylase Proteins 0.000 description 2
- 230000026731 phosphorylation Effects 0.000 description 2
- 238000006366 phosphorylation reaction Methods 0.000 description 2
- 210000001778 pluripotent stem cell Anatomy 0.000 description 2
- 229920002704 polyhistidine Polymers 0.000 description 2
- 229920002223 polystyrene Polymers 0.000 description 2
- 238000011176 pooling Methods 0.000 description 2
- 239000002987 primer (paints) Substances 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000010410 reperfusion Effects 0.000 description 2
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 238000004007 reversed phase HPLC Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 238000013391 scatchard analysis Methods 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 238000002741 site-directed mutagenesis Methods 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 2
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 230000005740 tumor formation Effects 0.000 description 2
- 210000003606 umbilical vein Anatomy 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- 229910001868 water Inorganic materials 0.000 description 2
- UJXJZOCXEZPHIE-YFKPBYRVSA-N (2s)-2-(2-hydroxyethylamino)-4-sulfanylbutanoic acid Chemical compound OCCN[C@H](C(O)=O)CCS UJXJZOCXEZPHIE-YFKPBYRVSA-N 0.000 description 1
- KUHSEZKIEJYEHN-BXRBKJIMSA-N (2s)-2-amino-3-hydroxypropanoic acid;(2s)-2-aminopropanoic acid Chemical compound C[C@H](N)C(O)=O.OC[C@H](N)C(O)=O KUHSEZKIEJYEHN-BXRBKJIMSA-N 0.000 description 1
- JQFLYFRHDIHZFZ-RXMQYKEDSA-N (2s)-3,3-dimethylpyrrolidine-2-carboxylic acid Chemical compound CC1(C)CCN[C@@H]1C(O)=O JQFLYFRHDIHZFZ-RXMQYKEDSA-N 0.000 description 1
- CNPSFBUUYIVHAP-AKGZTFGVSA-N (2s)-3-methylpyrrolidine-2-carboxylic acid Chemical compound CC1CCN[C@@H]1C(O)=O CNPSFBUUYIVHAP-AKGZTFGVSA-N 0.000 description 1
- IZKGGDFLLNVXNZ-KRWDZBQOSA-N (2s)-5-amino-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)N)C(O)=O)C3=CC=CC=C3C2=C1 IZKGGDFLLNVXNZ-KRWDZBQOSA-N 0.000 description 1
- FXGZFWDCXQRZKI-VKHMYHEASA-N (2s)-5-amino-2-nitramido-5-oxopentanoic acid Chemical compound NC(=O)CC[C@@H](C(O)=O)N[N+]([O-])=O FXGZFWDCXQRZKI-VKHMYHEASA-N 0.000 description 1
- CCAIIPMIAFGKSI-DMTCNVIQSA-N (2s,3r)-3-hydroxy-2-(methylazaniumyl)butanoate Chemical compound CN[C@@H]([C@@H](C)O)C(O)=O CCAIIPMIAFGKSI-DMTCNVIQSA-N 0.000 description 1
- CNPSFBUUYIVHAP-WHFBIAKZSA-N (2s,3s)-3-methylpyrrolidin-1-ium-2-carboxylate Chemical compound C[C@H]1CCN[C@@H]1C(O)=O CNPSFBUUYIVHAP-WHFBIAKZSA-N 0.000 description 1
- UHPQFNXOFFPHJW-UHFFFAOYSA-N (4-methylphenyl)-phenylmethanamine Chemical compound C1=CC(C)=CC=C1C(N)C1=CC=CC=C1 UHPQFNXOFFPHJW-UHFFFAOYSA-N 0.000 description 1
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- PNDPGZBMCMUPRI-HVTJNCQCSA-N 10043-66-0 Chemical compound [131I][131I] PNDPGZBMCMUPRI-HVTJNCQCSA-N 0.000 description 1
- WUAPFZMCVAUBPE-NJFSPNSNSA-N 188Re Chemical compound [188Re] WUAPFZMCVAUBPE-NJFSPNSNSA-N 0.000 description 1
- MXHRCPNRJAMMIM-SHYZEUOFSA-N 2'-deoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-SHYZEUOFSA-N 0.000 description 1
- OMGHIGVFLOPEHJ-UHFFFAOYSA-N 2,5-dihydro-1h-pyrrol-1-ium-2-carboxylate Chemical compound OC(=O)C1NCC=C1 OMGHIGVFLOPEHJ-UHFFFAOYSA-N 0.000 description 1
- OTLLEIBWKHEHGU-UHFFFAOYSA-N 2-[5-[[5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy]-3,4-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-3,5-dihydroxy-4-phosphonooxyhexanedioic acid Chemical compound C1=NC=2C(N)=NC=NC=2N1C(C(C1O)O)OC1COC1C(CO)OC(OC(C(O)C(OP(O)(O)=O)C(O)C(O)=O)C(O)=O)C(O)C1O OTLLEIBWKHEHGU-UHFFFAOYSA-N 0.000 description 1
- 125000006280 2-bromobenzyl group Chemical group [H]C1=C([H])C(Br)=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- XEVFXAFXZZYFSX-UHFFFAOYSA-N 3-azabicyclo[2.1.1]hexane-4-carboxylic acid Chemical compound C1C2CC1(C(=O)O)NC2 XEVFXAFXZZYFSX-UHFFFAOYSA-N 0.000 description 1
- GUPXYSSGJWIURR-UHFFFAOYSA-N 3-octoxypropane-1,2-diol Chemical compound CCCCCCCCOCC(O)CO GUPXYSSGJWIURR-UHFFFAOYSA-N 0.000 description 1
- 125000006181 4-methyl benzyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])C([H])([H])* 0.000 description 1
- WOVKYSAHUYNSMH-RRKCRQDMSA-N 5-bromodeoxyuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(Br)=C1 WOVKYSAHUYNSMH-RRKCRQDMSA-N 0.000 description 1
- 108091007504 ADAM10 Proteins 0.000 description 1
- 101150096273 ADE2 gene Proteins 0.000 description 1
- 108010066676 Abrin Proteins 0.000 description 1
- 241000228431 Acremonium chrysogenum Species 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 208000004476 Acute Coronary Syndrome Diseases 0.000 description 1
- 241000589156 Agrobacterium rhizogenes Species 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010021809 Alcohol dehydrogenase Proteins 0.000 description 1
- 102100024321 Alkaline phosphatase, placental type Human genes 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 1
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 1
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 1
- 101500021165 Aplysia californica Myomodulin-A Proteins 0.000 description 1
- 101100437118 Arabidopsis thaliana AUG1 gene Proteins 0.000 description 1
- 206010003178 Arterial thrombosis Diseases 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 231100000699 Bacterial toxin Toxicity 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 102100021935 C-C motif chemokine 26 Human genes 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000222128 Candida maltosa Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 241000218645 Cedrus Species 0.000 description 1
- 208000005243 Chondrosarcoma Diseases 0.000 description 1
- 208000031404 Chromosome Aberrations Diseases 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 241000557626 Corvus corax Species 0.000 description 1
- 241000271532 Crotalus Species 0.000 description 1
- 241000271537 Crotalus atrox Species 0.000 description 1
- 241000701022 Cytomegalovirus Species 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 101710093617 Dihydroxyacetone synthase Proteins 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- 108010053187 Diphtheria Toxin Proteins 0.000 description 1
- 102100039673 Disintegrin and metalloproteinase domain-containing protein 10 Human genes 0.000 description 1
- 101150029662 E1 gene Proteins 0.000 description 1
- 241000393496 Electra Species 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 108010075944 Erythropoietin Receptors Proteins 0.000 description 1
- 102100036509 Erythropoietin receptor Human genes 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 101150096839 Fcmr gene Proteins 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 101710195101 Flagellar filament outer layer protein Proteins 0.000 description 1
- 108010067193 Formaldehyde transketolase Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 102000005720 Glutathione transferase Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010092372 Granulocyte-Macrophage Colony-Stimulating Factor Receptors Proteins 0.000 description 1
- 102000016355 Granulocyte-Macrophage Colony-Stimulating Factor Receptors Human genes 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 102100020948 Growth hormone receptor Human genes 0.000 description 1
- 239000007821 HATU Substances 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000690301 Homo sapiens Aldo-keto reductase family 1 member C4 Proteins 0.000 description 1
- 101000897493 Homo sapiens C-C motif chemokine 26 Proteins 0.000 description 1
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 1
- 101001116548 Homo sapiens Protein CBFA2T1 Proteins 0.000 description 1
- 101000716102 Homo sapiens T-cell surface glycoprotein CD4 Proteins 0.000 description 1
- 101000946843 Homo sapiens T-cell surface glycoprotein CD8 alpha chain Proteins 0.000 description 1
- YZJSUQQZGCHHNQ-UHFFFAOYSA-N Homoglutamine Chemical compound OC(=O)C(N)CCCC(N)=O YZJSUQQZGCHHNQ-UHFFFAOYSA-N 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 235000003332 Ilex aquifolium Nutrition 0.000 description 1
- 241000209027 Ilex aquifolium Species 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 102000006496 Immunoglobulin Heavy Chains Human genes 0.000 description 1
- 108010019476 Immunoglobulin Heavy Chains Proteins 0.000 description 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 description 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 1
- 101710149643 Integrin alpha-IIb Proteins 0.000 description 1
- 108010042918 Integrin alpha5beta1 Proteins 0.000 description 1
- 102100025304 Integrin beta-1 Human genes 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 108010038452 Interleukin-3 Receptors Proteins 0.000 description 1
- 102000010790 Interleukin-3 Receptors Human genes 0.000 description 1
- 102000010781 Interleukin-6 Receptors Human genes 0.000 description 1
- 108010038501 Interleukin-6 Receptors Proteins 0.000 description 1
- 244000285963 Kluyveromyces fragilis Species 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 241001138401 Kluyveromyces lactis Species 0.000 description 1
- SNDPXSYFESPGGJ-BYPYZUCNSA-N L-2-aminopentanoic acid Chemical compound CCC[C@H](N)C(O)=O SNDPXSYFESPGGJ-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 239000004395 L-leucine Substances 0.000 description 1
- 235000019454 L-leucine Nutrition 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- SNDPXSYFESPGGJ-UHFFFAOYSA-N L-norVal-OH Natural products CCCC(N)C(O)=O SNDPXSYFESPGGJ-UHFFFAOYSA-N 0.000 description 1
- HXEACLLIILLPRG-YFKPBYRVSA-N L-pipecolic acid Chemical compound [O-]C(=O)[C@@H]1CCCC[NH2+]1 HXEACLLIILLPRG-YFKPBYRVSA-N 0.000 description 1
- DZLNHFMRPBPULJ-VKHMYHEASA-N L-thioproline Chemical compound OC(=O)[C@@H]1CSCN1 DZLNHFMRPBPULJ-VKHMYHEASA-N 0.000 description 1
- KKJQZEWNZXRJFG-UHFFFAOYSA-N L-trans-4-Methyl-2-pyrrolidinecarboxylic acid Chemical compound CC1CNC(C(O)=O)C1 KKJQZEWNZXRJFG-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 208000007177 Left Ventricular Hypertrophy Diseases 0.000 description 1
- 241000713666 Lentivirus Species 0.000 description 1
- 241000270322 Lepidosauria Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 108090000157 Metallothionein Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100243377 Mus musculus Pepd gene Proteins 0.000 description 1
- 102000047918 Myelin Basic Human genes 0.000 description 1
- 108700028031 Myelin Basic Proteins 0.000 description 1
- 102100030856 Myoglobin Human genes 0.000 description 1
- 108010062374 Myoglobin Proteins 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 229920002274 Nalgene Polymers 0.000 description 1
- BDFNAGOUUFOPSP-UHFFFAOYSA-N Nasvin Natural products O1C2=C(Cl)C(O)=C(Cl)C(C)=C2C(=O)OC2=C1C(C(C)=CC)=C(Cl)C(O)=C2CCCC BDFNAGOUUFOPSP-UHFFFAOYSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 241000221960 Neurospora Species 0.000 description 1
- 101100384865 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cot-1 gene Proteins 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 241000320412 Ogataea angusta Species 0.000 description 1
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 1
- 102000016979 Other receptors Human genes 0.000 description 1
- 108091008606 PDGF receptors Proteins 0.000 description 1
- 101150029183 PEP4 gene Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 241000282520 Papio Species 0.000 description 1
- 241000282516 Papio anubis Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 108010067902 Peptide Library Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 108010001441 Phosphopeptides Proteins 0.000 description 1
- 231100000742 Plant toxin Toxicity 0.000 description 1
- 102000011653 Platelet-Derived Growth Factor Receptors Human genes 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 108091028664 Ribonucleotide Proteins 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 241000235347 Schizosaccharomyces pombe Species 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 108010068542 Somatotropin Receptors Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 1
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 1
- 239000012317 TBTU Substances 0.000 description 1
- 108700012920 TNF Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108010022394 Threonine synthase Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108090000253 Thyrotropin Receptors Proteins 0.000 description 1
- 102100029337 Thyrotropin receptor Human genes 0.000 description 1
- 208000026062 Tissue disease Diseases 0.000 description 1
- 108020004566 Transfer RNA Proteins 0.000 description 1
- 241000255993 Trichoplusia ni Species 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 244000301083 Ustilago maydis Species 0.000 description 1
- 235000015919 Ustilago maydis Nutrition 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 229930003448 Vitamin K Natural products 0.000 description 1
- 241000289690 Xenarthra Species 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- VWQVUPCCIRVNHF-OUBTZVSYSA-N Yttrium-90 Chemical compound [90Y] VWQVUPCCIRVNHF-OUBTZVSYSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- PCBMGUSDYHYVBQ-SOOFDHNKSA-N [4-amino-2-[(3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-imidazol-5-yl]phosphonic acid Chemical compound P(=O)(O)(O)C=1N=C(NC1N)C1[C@H](O)[C@H](O)[C@H](O1)CO PCBMGUSDYHYVBQ-SOOFDHNKSA-N 0.000 description 1
- CLZISMQKJZCZDN-UHFFFAOYSA-N [benzotriazol-1-yloxy(dimethylamino)methylidene]-dimethylazanium Chemical compound C1=CC=C2N(OC(N(C)C)=[N+](C)C)N=NC2=C1 CLZISMQKJZCZDN-UHFFFAOYSA-N 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 108020002494 acetyltransferase Proteins 0.000 description 1
- 102000005421 acetyltransferase Human genes 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 208000009956 adenocarcinoma Diseases 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 238000012867 alanine scanning Methods 0.000 description 1
- 108700023471 alginate-polylysine-alginate Proteins 0.000 description 1
- 125000001931 aliphatic group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 230000002152 alkylating effect Effects 0.000 description 1
- 229940037003 alum Drugs 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 238000010976 amide bond formation reaction Methods 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000000689 aminoacylating effect Effects 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 208000036878 aneuploidy Diseases 0.000 description 1
- 231100001075 aneuploidy Toxicity 0.000 description 1
- 230000003527 anti-angiogenesis Effects 0.000 description 1
- 230000002785 anti-thrombosis Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000002257 antimetastatic agent Substances 0.000 description 1
- 210000000709 aorta Anatomy 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 239000000688 bacterial toxin Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 102000012740 beta Adrenergic Receptors Human genes 0.000 description 1
- 108010079452 beta Adrenergic Receptors Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 125000004057 biotinyl group Chemical group [H]N1C(=O)N([H])[C@]2([H])[C@@]([H])(SC([H])([H])[C@]12[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(*)=O 0.000 description 1
- PSHNNUKOUQCMSG-UHFFFAOYSA-K bis[(2,2,2-trifluoroacetyl)oxy]thallanyl 2,2,2-trifluoroacetate Chemical compound [Tl+3].[O-]C(=O)C(F)(F)F.[O-]C(=O)C(F)(F)F.[O-]C(=O)C(F)(F)F PSHNNUKOUQCMSG-UHFFFAOYSA-K 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 201000008275 breast carcinoma Diseases 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000005392 carboxamide group Chemical group NC(=O)* 0.000 description 1
- 210000001054 cardiac fibroblast Anatomy 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000008619 cell matrix interaction Effects 0.000 description 1
- 210000004671 cell-free system Anatomy 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- SBVCFQSSHXEIGO-UHFFFAOYSA-N cesium;tetramethylazanium Chemical group [Cs+].C[N+](C)(C)C SBVCFQSSHXEIGO-UHFFFAOYSA-N 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 230000035572 chemosensitivity Effects 0.000 description 1
- 239000005482 chemotactic factor Substances 0.000 description 1
- 239000012829 chemotherapy agent Substances 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 235000013330 chicken meat Nutrition 0.000 description 1
- BFPSDSIWYFKGBC-UHFFFAOYSA-N chlorotrianisene Chemical compound C1=CC(OC)=CC=C1C(Cl)=C(C=1C=CC(OC)=CC=1)C1=CC=C(OC)C=C1 BFPSDSIWYFKGBC-UHFFFAOYSA-N 0.000 description 1
- 231100000005 chromosome aberration Toxicity 0.000 description 1
- 230000015271 coagulation Effects 0.000 description 1
- 238000005345 coagulation Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000032459 dedifferentiation Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 239000003398 denaturant Substances 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000030609 dephosphorylation Effects 0.000 description 1
- 238000006209 dephosphorylation reaction Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- MXHRCPNRJAMMIM-UHFFFAOYSA-N desoxyuridine Natural products C1C(O)C(CO)OC1N1C(=O)NC(=O)C=C1 MXHRCPNRJAMMIM-UHFFFAOYSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 102000004419 dihydrofolate reductase Human genes 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- MGHPNCMVUAKAIE-UHFFFAOYSA-N diphenylmethanamine Chemical compound C=1C=CC=CC=1C(N)C1=CC=CC=C1 MGHPNCMVUAKAIE-UHFFFAOYSA-N 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 229940000406 drug candidate Drugs 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 238000002003 electron diffraction Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 230000003511 endothelial effect Effects 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000020774 essential nutrients Nutrition 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000002095 exotoxin Substances 0.000 description 1
- 231100000776 exotoxin Toxicity 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 230000004545 gene duplication Effects 0.000 description 1
- 238000012254 genetic linkage analysis Methods 0.000 description 1
- 102000018146 globin Human genes 0.000 description 1
- 108060003196 globin Proteins 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 102000035122 glycosylated proteins Human genes 0.000 description 1
- 108091005608 glycosylated proteins Proteins 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000004217 heart function Effects 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 102000054751 human RUNX1T1 Human genes 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- MWFRVMDVLYIXJF-BYPYZUCNSA-N hydroxyethylcysteine Chemical compound OC(=O)[C@@H](N)CSCCO MWFRVMDVLYIXJF-BYPYZUCNSA-N 0.000 description 1
- 125000004029 hydroxymethyl group Chemical group [H]OC([H])([H])* 0.000 description 1
- 230000003463 hyperproliferative effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000000760 immunoelectrophoresis Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 150000004001 inositols Chemical class 0.000 description 1
- 230000006362 insulin response pathway Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000037427 ion transport Effects 0.000 description 1
- GKOZUEZYRPOHIO-IGMARMGPSA-N iridium-192 Chemical compound [192Ir] GKOZUEZYRPOHIO-IGMARMGPSA-N 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 125000005928 isopropyloxycarbonyl group Chemical group [H]C([H])([H])C([H])(OC(*)=O)C([H])([H])[H] 0.000 description 1
- 108090000512 jararhagin Proteins 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- HXEACLLIILLPRG-RXMQYKEDSA-N l-pipecolic acid Natural products OC(=O)[C@H]1CCCCN1 HXEACLLIILLPRG-RXMQYKEDSA-N 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 101150066555 lacZ gene Proteins 0.000 description 1
- 238000002647 laser therapy Methods 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 210000000415 mammalian chromosome Anatomy 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- BRMYZIKAHFEUFJ-UHFFFAOYSA-L mercury diacetate Chemical compound CC(=O)O[Hg]OC(C)=O BRMYZIKAHFEUFJ-UHFFFAOYSA-L 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 208000024191 minimally invasive lung adenocarcinoma Diseases 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 230000009753 muscle formation Effects 0.000 description 1
- 229940100661 nasal inhalant Drugs 0.000 description 1
- 230000006654 negative regulation of apoptotic process Effects 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000007472 neurodevelopment Effects 0.000 description 1
- 239000002858 neurotransmitter agent Substances 0.000 description 1
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000002188 osteogenic effect Effects 0.000 description 1
- YPZRWBKMTBYPTK-UHFFFAOYSA-N oxidized gamma-L-glutamyl-L-cysteinylglycine Natural products OC(=O)C(N)CCC(=O)NC(C(=O)NCC(O)=O)CSSCC(C(=O)NCC(O)=O)NC(=O)CCC(N)C(O)=O YPZRWBKMTBYPTK-UHFFFAOYSA-N 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 210000001539 phagocyte Anatomy 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000008300 phosphoramidites Chemical class 0.000 description 1
- DCWXELXMIBXGTH-UHFFFAOYSA-N phosphotyrosine Chemical compound OC(=O)C(N)CC1=CC=C(OP(O)(O)=O)C=C1 DCWXELXMIBXGTH-UHFFFAOYSA-N 0.000 description 1
- 238000005222 photoaffinity labeling Methods 0.000 description 1
- SHUZOJHMOBOZST-UHFFFAOYSA-N phylloquinone Natural products CC(C)CCCCC(C)CCC(C)CCCC(=CCC1=C(C)C(=O)c2ccccc2C1=O)C SHUZOJHMOBOZST-UHFFFAOYSA-N 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 108010031345 placental alkaline phosphatase Proteins 0.000 description 1
- 239000003123 plant toxin Substances 0.000 description 1
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920002647 polyamide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000037452 priming Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 230000007026 protein scission Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 210000001147 pulmonary artery Anatomy 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 239000013014 purified material Substances 0.000 description 1
- 229950010131 puromycin Drugs 0.000 description 1
- 239000002510 pyrogen Substances 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 238000003127 radioimmunoassay Methods 0.000 description 1
- 238000003156 radioimmunoprecipitation Methods 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 239000001044 red dye Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 238000010845 search algorithm Methods 0.000 description 1
- 238000002805 secondary assay Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000037432 silent mutation Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- PVFDPMYXCZLHKY-MLLWLMKGSA-M sodium [(1R,2R,4aR,8aS)-2-hydroxy-5-[(2E)-2-[(4S)-4-hydroxy-2-oxooxolan-3-ylidene]ethyl]-1,4a,6-trimethyl-2,3,4,7,8,8a-hexahydronaphthalen-1-yl]methyl sulfate Chemical compound [Na+].C([C@@H]1[C@](C)(COS([O-])(=O)=O)[C@H](O)CC[C@]11C)CC(C)=C1C\C=C1/[C@H](O)COC1=O PVFDPMYXCZLHKY-MLLWLMKGSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 108010018381 streptavidin-binding peptide Proteins 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000004114 suspension culture Methods 0.000 description 1
- NPDBDJFLKKQMCM-UHFFFAOYSA-N tert-butylglycine Chemical compound CC(C)(C)C(N)C(O)=O NPDBDJFLKKQMCM-UHFFFAOYSA-N 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 238000011287 therapeutic dose Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 239000003104 tissue culture media Substances 0.000 description 1
- 230000009772 tissue formation Effects 0.000 description 1
- 230000008467 tissue growth Effects 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- VIYXXANHGYSBLY-UHFFFAOYSA-N trimethylsilyl 2,2,2-trifluoroacetate Chemical compound C[Si](C)(C)OC(=O)C(F)(F)F VIYXXANHGYSBLY-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 241001529453 unidentified herpesvirus Species 0.000 description 1
- 125000005500 uronium group Chemical group 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 210000004509 vascular smooth muscle cell Anatomy 0.000 description 1
- 239000002435 venom Substances 0.000 description 1
- 231100000611 venom Toxicity 0.000 description 1
- 210000001048 venom Anatomy 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000019168 vitamin K Nutrition 0.000 description 1
- 239000011712 vitamin K Substances 0.000 description 1
- 150000003721 vitamin K derivatives Chemical class 0.000 description 1
- 229940046010 vitamin k Drugs 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6489—Metalloendopeptidases (3.4.24)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/04—Drugs for skeletal disorders for non-specific disorders of the connective tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/04—Antineoplastic agents specific for metastasis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Description
)
Description . 5 Disintegrin Homologs
Disintegrins have been shown to bind cell surface molecules, including integrins, on the surface of various cells, such as platelets, fibroblasts, tumor, endothelial, muscle, neuronal, bone, and sperm cells.
Disintegrins are unique and potentially useful tools for investigating «cell-matrix and cell-cell interactions.
Additionally, they have been useful in the development of antithrombotic and antimetastatic agents due to their anti-adhesive, anti-migration of certain tumor cells, and antiangiogenesis activities.
Families of proteins which have disintegrin domains include ADAMs (A Metalloprotease and Disintegrin),
MDCs (Metalloprotease/Disintegrin/Cysteine-rich) and SVMPs (Snake Venom Metalloprotease) . . For a review of ADAMs, see Wolfsberg and White,
Developmental Biology, 180:389-401, 1996. ADAMs have " been shown to exist as independent functional units or in conjunction with other members of this family in heterodimeric complexes. Some members of the family exist in multiple isoforms which may have resulted from alternative splicing. ADAMs proteins have been shown to have adhesive as well as anti-adhesive functions. Some members of the ADAMs family have very specific tissue distribution while others are widely distributed. Not all members of this family are capable of manifesting all of the potential functions represented by the domains common to their genetic structure.
The ADAMs are Characterized by having a propeptide domain, a metalloprotease-like domain, a disintegrin-like domain, a cysteine-rich domain, an EGF- ) like domain, and a Cytoplasmic domain.
A prototypical example of this family is ADAM ’ 12. ADAM 12, also known as meltrin a, has a truncated isoform, as well as a full-length isoform, and is involved in muscle cell fusion and differentiation (Gilpin et al.,
J. Biol. Chem. 273:157-166, 1998).
Another prototypical example of this family is
ADAM 1, which forms a heterodimer with ADAM 2 and 1s involved in sperm/egg fusion (Wolfsberg and White, supra) .
The SVMP family is represented by three classes (P-I, P-11, and P-III). All three classes contain propeptide and metalloprotease domains. The P-II and P-
ITI classes also contain a disintegrin domain, and the P-
ITI class further contains a cysteine-rich domain. These domains are similar in sequence to those found in the
ADAMs. Some members of the SVMP family have a conserved “RGD” amino acid sequence. This tripeptide has been shown to form a hairpin loop whose conformation can disrupt the binding of fibrinogen to activated platelets. This RGD : sequence may be substituted by RSE, MVD, MSE, and KGD in
P-IT SVMPs, and by MSEC, RSEC, IDDC, and RDDC (a ! tripeptide along with a carboxy-terminal cysteine residue) in P-IITI SVMPs. Thus, these sequences may be responsible for integrin binding in the P-II and P-III SVMPs.
A prototypical example of a SVMP is jararhagin, which mediates platelet aggregation by binding to the platelet a; subunit (GPIa) via the disintegrin domain followed by proteolysis of the Bi subunit (GPIIA) (Huang and Liu, J. Toxicol-Toxin Reviews 16: 135-161, 1997).
The proteins of the
Metalloprotease/Disintegrin/Cysteine-rich family are involved in diverse tasks, ranging from roles in fertilization and muscle fusion, TNFa release from plasma
A membranes, intracellular protein cleavage, and essential functions in neuronal development (Blobel, Cell 090:589- - 5 592, 1997). This family is also characterized by the metalloprotease, disintegrin and cysteine-rich domains, as described above.
The present invention provides a novel disintegrin homolog and related compositions whose uses should be apparent to those skilled in the art from the teachings herein.
Within one aspect, the present invention provides an isolated polypeptide molecule comprising a contiguous sequence of 14 amino acids of SEQ ID NO:2.
Within an embodiment the polypeptide molecule comprises residues 437 to 450 of SEQ ID NO:2. Within another embodiment, the polypeptide molecule is between 82 and 232 amino acids in length. Within further embodiments polypeptide molecule is residues 164 to 382 of SEQ ID
Rk NO:2; residues 383 to 464 of SEQ ID NO:2; and/or residues 465 to 696 of SEQ ID NO:2.
Within another aspect, the invention provides an isolated polypeptide molecule selected from the group consisting of: a) a polypeptide molecule comprising residues 164 to 382 of SEQ ID NO:2; b) a polypeptide molecule comprising residues 383 to 464 of SEQ ID NO:2; ¢) a polypeptide molecule comprising residues 465 to 696 of
SEQ ID NO:2; d) a polypeptide molecule comprising residues 438 to 449 of SEQ ID NO:2; e) a polypeptide molecule comprising residues 164 to 464 of SEQ ID NO:2; ff) a polypeptide molecule comprising residues 164 to 696 of SEQ
ID NO:2; g) a polypeptide molecule comprising residues 383 to 696 of SEQ ID NO:2; h) a polypeptide molecule comprising residues 164 to 449 of SEQ ID NO:2; i) a polypeptide molecule comprising residues 438 to 696 of SEQ
ID NO:2; and j) a polypeptide molecule comprising residues } 1 to 696 of SEQ ID NO:2.
Within another aspect is provided an isolated } polynucleotide molecule encoding a polypeptide molecule, wherein the polypeptide molecule comprises a contiguous sequence of 14 amino acids of SEQ ID NO:2. Within an embodiment, the polypeptide molecule comprises residues 437 to 450 of SEQ ID NO:2. Within a further embodiment, the polypeptide molecule is between 82 and 232 amino acids in length. Within further embodiments, the polypeptide molecule is residues 164 to 382 of SEQ ID NO:2; residues 383 to 464 of SEQ ID NO:2; and/or residues 465 to 696 of
SEQ ID NO:2.
Within another aspect, the invention provides an isolated polynucleotide molecule encoding a polypeptide molecule, wherein the polypeptide molecule is selected from the group consisting of: a) a polypeptide molecule comprising residues 164 to 382 of SEQ ID NO:2; Db) a polypeptide molecule comprising residues 383 to 464 of SEQ
ID NO:2; c) a polypeptide molecule comprising residues 465 - to 696 of SEQ ID NO:2; d) a polypeptide molecule comprising residues 438 to 449 of SEQ ID NO:2; ee) a . polypeptide molecule comprising residues 164 to 464 of SEQ
ID NO:2; f) a polypeptide molecule comprising residues 164 to 6%6 of SEQ ID NO:2; gq) a polypeptide molecule comprising residues 383 to 696 of SEQ ID NO:2; h) a polypeptide molecule comprising residues 164 to 449 of SEQ
ID NO:2; i) a polypeptide molecule comprising residues 438 to 696 of SEQ ID NO:2; and Jj) a polypeptide molecule comprising residues 1 to 696 of SEQ ID NO:2.
Within another aspect is provided an isolated polynucleotide encoding a fusion protein having a first segment and a second segment, wherein the first segment comprises a first polypeptide encoding a polypeptide having a protease domain and the second segment comprises
A a second polynucleotide encoding a polypeptide that has a contiguous sequence of 14 amino acids between residues 383 . 5 and 464 of SEQ ID NO:2, and wherein the first segment is positioned amino-terminally to the second segment. Within an embodiment, the protease domain is selected from the group consisting of; a) a protease domain that is a member of the Disintegrin Proteases; and b) a protease domain that is at least 80% identical to amino acid residues 164 to 382 of SEQ ID NO:2.
Within another aspect the invention provides an isolated polynucleotide molecule encoding a polypeptide molecule wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide molecule that encodes a polypeptide molecule that is at least 80 $ identical to residues 383 to 464 of SEQ ID NO:2; and b) a polynucleotide molecule that is complementary to a).
Within an embodiment, the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide molecule that encodes a polypeptide molecule that is at ) least 80 % identical to residues 383 to 696 of SEQ ID
NO:2; and b) a polynucleotide molecule that is ] complementary to a). Within a further embodiment, the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide molecule that encodes a polypeptide molecule that is at least 80 $ identical to residues 1 to 696 of SEQ ID NO:2; and b)} a polynucleotide molecule that is complementary to a).
Within another aspect is provided an expression vector comprising the following operably linked elements: a) a transcription promoter; b) a DNA segment encoding the polypeptide of claim 1; and c) a transcription terminator.
Within an embodiment the DNA segment further encodes ap affinity tag.
Within another aspect, the invention provides a cultured cell into which has been introduced said expression vector, wherein the cell expresses the ) polypeptide encoded by the DNA segment.
Within another aspect, the invention provides a } method of producing a polypeptide comprising culturing the cell expressing the polypeptide encoded by the DNA segment; and recovering the polypeptide.
Within another aspect is provided a method for modulating cell-cell interactions by combining the polypeptide comprising the sequence of 14 contiguous amino acids, with cells in vivo and in vitro. Within an embodiment, the cells are derived from tissues selected from the group consisting of: a) tissues from heart; b) tissues from brain; c) tissues from spinal cord; and d) tissues from skeletal muscle.
Within another aspect, the invention provides an isolated polypeptide molecule comprising a contiguous
Sequence of amino acids, wherein the contiguous sequence of amino acids is selected from the group consisting of: a) SEQ ID NO:7; b) SEQ ID NO:8; c) SEQ ID NO:9; d) SEQ ID
NO:10; and e) SEQ ID NO:11. :
Within another aspect is provide an isolated polynucleotide molecule encoding an isolated polypeptide . molecule, wherein the polypeptide comprises a contiguous sequence of amino acids and is selected from the group consisting of: a) SEQ ID NO:7; b) SEQ ID NO:8; cc) SEQ ID
NO:9; d) SEQ ID NO:10; and e) SEQ ID NO:11.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention and attached drawings.
Figure 1 is a Hopp/Woods hydrophilicity profile of the zdintl protein sequence shown in SEQ ID NO:2. The profile is based on a sliding six-residue window. Buried
G, S, and T residues and exposed H, Y, and W residues were " ignored. These residues are indicated in the figure by lower case letters. - 5 Figure 2 schematically shows a domain level alignment of members of ADAMs, MDCs, and SVMPs.
DISA TRIGA is a SVMP. MS2 HUMAN is an ADAM. HSUTSP1 (TACE) is a MDC. And HSU52370 1 is fertilin-f, ADAM 2. “sig” denotes the secretory signal peptide; “propep” denotes the propeptide domain; “Metal-protease” denotes the metalloprotease domain; “disint” denotes the disintegrin domain; “cys” denotes the cysteine-rich domain; “RGD” denotes a tripeptide, Arginine-Glycine-
Asparagine; and “TMD” denotes the transmembrane domain.
Prior to setting forth the invention in detail, it may be helpful to the understanding thereof to define the following terms:
The term “affinity tag” is used herein to denote a polypeptide segment that can be attached to a second - polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of ] the second polypeptide to a substrate. In principal, any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
Affinity tags include a poly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P, Flag™ peptide (Hopp et al.,
Biotechnology 6:1204-10, 1988), streptavidin binding peptide, or other antigenic epitope or binding domain.
A :
See, in general, Ford et al., Protein Expression and
Purification 2: 95-107, 1991. DNAs encoding affinity tags are available from commercial suppliers (e.g., Pharmacia ’
Bictech, Piscataway, NJ).
The term "allelic variant" is used herein to ’ denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations «can be silent {no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence. The term allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
The terms “amino-terminal” and “carboxyl- terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position.
For example, a certain sequence positioned carboxyl- terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference - sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide. .
The term “complement/anti-complement pair” denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair.
Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of <109 M~1,
A The term “complements of a polynucleotide molecule” is a polynucleotide molecule having a ~ 5 complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
The term “contig” denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to “overlap” a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5'-
ATGGAGCTT-3' are 5’'-AGCTTgagt-3’ and 3’ -tcgacTACC-5".
The term “corresponding to”, when applied to positions of amino acid residues in sequences, means corresponding positions in a plurality of sequences when the sequences are optimally aligned.
The term “degenerate nucleotide sequence” . denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference . polynucleotide molecule that encodes a polypeptide).
Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e.,
GAU and GAC triplets each encode Asp) .
The term "expression vector" is used to denote a
DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription.
Such additional segments include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer,
a polyadenylation signal, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. )
The term “isolated”, when appiied to a polynucleotide, denotes that the polynucleotide has been } removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones. Isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316:774-78, 1985).
An “isolated” polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue. In a preferred form, the isolated : polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is . preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure. When used in this context, the term “isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
The term “operably linked", when referring to
DNA segments, indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
The term “ortholog” denotes a polypeptide or protein obtained from one species that is the functional ° counterpart of a polypeptide or protein from a different
Species. Sequence differences among orthologs are the . 5 result of speciation. “Paralogs” are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, a-globin, b- globin, and myoglobin are paralogs of each other.
A "polynucleotide" is a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vitro, or prepared from a combination of natural and synthetic molecules. Sizes of polynucleotides are expressed as base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases (“kb”). Where the context allows, the latter two terms may describe polynucleotides that are single-stranded or double- stranded. When the term is applied to double-stranded molecules, it is used to denote overall length and will be . understood to be equivalent to the term “base pairs”. It will be recognized by those skilled in the art that the . two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus, all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
A "polypeptide" is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as "peptides.
The term "promoter" is used herein for its art- recognized meaning to denote a portion of a gene containing DNA sequences that provide for the binding of i
RNA polymerase and initiation of transcription. Promoter sequences are commonly, but not always, found in the 5°' ’ non-coding regions of genes.
A "protein" is a macromolecule comprising one or more pclypeptide chains. A protein may also comprise non- peptidic components, such as carbohydrate groups.
Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
The term "receptor" denotes a cell-associated protein that binds to a bioactive molecule (i.e., a ligand) and mediates the effect of the ligand on the cell.
Membrane-bound receptors are characterized by a multi- domain or multi-peptide structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal . transduction. Binding of ligand to receptor results in a conformational change in the receptor that causes an . interaction between the effector domain and other molecule (s) in the cell. This interaction in turn leads to an alteration in the metabolism of the cell. Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids. In general, receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF : receptor, erythropoietin receptor and IL-6 receptor).
The term "secretory signal sequence” denotes a . 5 DNA sequence that encodes a polypeptide (a "secretory peptide”) that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized. The larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
The term “splice variant” is used herein to denote alternative forms of RNA transcribed from a gene.
Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed
RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene. } Molecular weights and lengths of polymers determined by imprecise analytical methods (e.g., gel electrophoresis) will be understood to be approximate values. When such a value is expressed as “about” X or “approximately” X, the stated value of X will be understood to be accurate to +10%.
All references cited herein are incorporated by reference in their entirety.
The present invention is based upon the discovery of a novel cDNA sequence (SEQ ID NO:1) and corresponding polypeptide (SEQ ID NO:2) having homology to disintegrin-like family members (ADAMs, SVMPs and MDCs; referred to herein as Disintegrin Proteases, or “DPs”).
See, for example, Blobel, Cell 90:589-592, 1997, and
Wolfsberg and White, Developmental Biology 180:389-401, 1996. Disintegrins can be involved in, for example, anticoagulation, fertilization, muscle fusion, connective ’ tissue disorders, chondrogenesis, arthritis, metastasis and neurogenesis. i
The secretory signal (also known as a leader sequence, prepro sequence or pre sequence) domain of these polypeptides directs the polypeptide through a secretory pathway of a cell in which it is synthesized. The secretory signal and propeptide domain are cleaved from the full length molecule, resulting in the mature form of the zdintl polypeptide. The protease domain may be active or inactive. Some members of the disintegrin family have “active” zinc catalytic sites which may be regulated by a “cysteine-switch” in the cysteine-rich domain. Examples of family members which have “active” protease domains are
ADAM 1 and ADAM 10, which are involved in sperm/egg fusion and degradation of myelin basic sheath protein, respectively. Other members of this family do not have such a catalytic site and are “inactive”. An example of a family member which contains an inactive protease domain is ADAM 11, which may be involved in tumor suppression. :
Other protein families which are known to have inactive protease domains are the serine proteases. -
The adhesion (disintegrin) domain of this protein binds integrin domains on the surface a multitude of cells, depending on the specificity of the disintegrin.
The predicted binding site within this disintegrin domain is often an amino acid loop comprising about 13 amino acids. The conformation of this sequence upon folding results in a hairpin loop presenting an amino acid
Sequence at its tip. This sequence is often “RGD”, but may be substituted by a variety of other amino acid residues (Wolfsberg and White, supra; and Jia, J. Biol.
Chem. 272:13094-13102 1997). The diversity of these
Sequences may reflect that: 1) not all disintegrin domains serve as ligands for integrins (or other cell surface . receptors); 2) disintegrin domains with different sequences bind to different types of cell surface . 5S receptors; or 3) the important part of the disintegrin structure loop is its structure, not its sequence, and thus, that the receptors for the specific classes of disintegrin domains can recognize a multitude of disintegrin binding loop sequences. Disintegrin domains have been shown to be responsible for cell-cell interactions, including inhibition of platelet aggregation by binding GPIIb/IIIa (fibronectin receptor) and/or
GPIa/IIa (collagen receptor) as well as cell fusion.
Many disintegrin family members have a fusion domain, a relatively hydrophobic domain of about 23 amino acids. This domain is present within some of the ADAM family members, and has been shown to be involved in cell- cell fusion, and particularly in sperm/egg fusion, and muscle fusion.
The cysteine-rich domain varies in the DP family members and is believed to be involved in structurally . presenting the integrin-binding region to integrins.
Many DP family members have a transmembrane } domain, which acts to anchor the polypeptide to the cell membrane.
The signaling domain of disintegrin family members tends to be conserved in length and sites for phosphorylation. However, beyond that they tend to be unique in amino acid composition. Some disintegrin family members may signal by binding to the SH3 domain of Ab],
Src, and/or Src-related SH3 domains.
The zdintl polypeptides of the present invention are a novel member of the DP family. The presence of isoforms of zdintl which also comprise a transmembrane domain would suggest that zdintl will have an alternatively spliced variant with a signaling domain. : The novel zdintl polypeptide-encoding polynuclectides of the present invention were initially identified by performing a Blast similarity search. An i expressed sequence tag corresponding to nucleotides 1097 to 1415 of SEQ ID NO:1 was used to obtain a clone that had been isolated from an infant brain plasmid library.
Examination of the zdintl deduced amino acid sequence (SEQ ID NO:2) permitted identification of the following domains: a propeptide sequence, ending at residue 163 of SEQ ID NO: 2; a protease sequence, residues 164 to 382 of SEQ ID NO: 2; a disintegrin sequence, residues 383 to 464 of SEQ ID NO: 2; and a cysteine-rich sequence, residues 465 to 696 of SEQ ID
NO: 2. Within the disintegrin domain, there is a “disintegrin loop” sequence, residues 438 to 449 of SEQ ID
NO: 2. The amino acid sequence, ECD, which corresponds to residues 443 to 445 of SEQ ID NO: 2, is analogous to the “RGD binding loop” of some other members of the DPs.
Analysis of tissue distribution of zdint1l was performed by the Northern blotting technique using Human -
Multiple Tissue, Master Dot, and human vascular blots.
Strong signals of three transcript sizes, approximately . 3.0 kb, 4.4 kb, and 7.5 kb, were observed in heart on the multiple tissue Northern blots. Faint signals of the same transcript sizes were observed in brain and spinal cord.
Fainter signals of the three transcript sizes were observed in skeletal muscle. The Master Dot Blot showed strong signals in brain, heart, fetal brain, and fetal heart. The human vascular blot showed a strong signal at 3-3.5kb in human aortic endothelial cells and weaker signals in aortic smooth muscle cells and normal human lung fibroblast cells.
The protease domain of zdintl has 49.5% identity to the protease domain of the nearest family - neighbor, ADAM 11, at the polypeptide level, and 58% identity at the polynucleotide level. The disintegrin . S domain of zdintl has 66.7% identity to the disintegrin domain of the nearest family neighbor, ADAM 11, at the polypeptide level, and 64.3% identity at the polynucleotide level. The expression of ADAM 11 has been shown to decrease in breast cancer tissues and, thus, is suggested to act as a tumor suppresser in breast cancer (Emi et al., Nature Gen. 5:151-157, 1993). Additionally,
ADAM 11 has been shown to have multiple isoforms as a result of alternative splicing.
Another protein which is an example of alternative splicing in the DPs is ADAM 12, meltrin a.
The truncated form of this molecule, which lacks the propeptide and metalloprotease domains, is associated with ectopic muscle formation in vivo, but not in vitro, indicating that cells expressing this gene produce a growth factor that acts on neighboring progenitor cells.
Other ADAMs have been considered for treating : angioplasty, acute coronary syndrome, prevention of restenosis on stents, and prevention of excess adhesion following surgical procedures, prevention of metastasis, as well as for degradation of specific proteins, such as, for example, amyloid precursor protein.
POLYNUCLEOTIDES:
The highly conserved amino acids in the disintegrin domain of zdintl can be used as a tool to identify new family members. For instance, reverse transcription-polymerase chain reaction (RT-PCR) can be used to amplify sequences encoding the conserved disintegrin domain from RNA obtained from a variety of tissue sources or cell lines. In particular, highly degenerate primers designed from the zdintl sequences are useful for this purpose. )
The present invention also provides polynucleotide molecules, including DNA and RNA molecules, : that encode the zdintl polypeptides disclosed herein.
Those skilled in the art will readily recognize that, in view of the degeneracy of the genetic code, considerable sequence variation is possible among these polynucleotide molecules. SEQ ID NO:3 is a degenerate DNA sequence that encompasses all DNAs that encode the zdintl polypeptide of
SEQ ID NO:2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO:3 also provides all
RNA sequences encoding SEQ ID NO:2 by substituting U for
T. Thus, zdintl polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 2088 of SEQ ID NO:3 and their RNA equivalents are contemplated by the present invention. Table 1 sets forth the one-letter codes used within SEQ ID NO:3 to denote degenerate nucleotide positions. “Resolutions” are the nucleotides denoted by a code letter. “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y : denotes either C or T, and its complement R denotes A or
G, A being complementary to T, and G being complementary . to C.
TABLE 1 ] —————
Nucleotide Resolution Nucleotide Complement . a ar TT
Cc Cc G G
G G Cc Cc
T T A A
R AG Y CIT
Y clT R AlG
M AlC K GIT
K GIT M A|C
S ClG S ClG
W A|T W A|T
H A|CIT D AlG|T
B CIG|T \% AlCIG \Y AlCI|G B CIGI|T
D AlG|T H AlCIT
N AlICIGIT N AlCIGIT ————
The degenerate codons used in SEQ ID NO: 3, ; 5 encompassing all possible codons for a given amino acid, are set forth in Table 2.
TABLE 2
One
Amino Letter Codons Degenerate )
Acid Code Codon ~~ Cys cc _ T6C TOT Tay :
Ser S AGC AGT TCA TCC TCG TCT WSN
Thr T ACA ACC ACG ACT ACN
Pro P CCA CCC CCG CCT CCN
Ala A GCA GCC GCG GCT GCN
Gly G GGA GGC GGG GGT GGN
Asn N AAC AAT ARDY
Asp D GAC GAT GAY
Glu E GAA GAG GAR
Gln Q CAA CAG CAR
His H CAC CAT CAY
Arg R AGA AGG CGA CGC CGG CGT MGN
Lys K AAA AAG AAR
Met M ATG ATG
Ile I ATA ATC ATT ATH
Leu L CTA CTC CTG CTT TTA TTG YTN
Val \ GTA GTC GTG GTT GTN
Phe F TTC TTT TTY -
Tyr Y TAC TAT TAY
Trp W TGG TGG )
Ter . TAA TAG TGA TRR
Asn|Asp B RAY
Glu|Gln Z SAR
Any X NNN
One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, the degenerate coden for serine (WSN) can, in some circumstances, encode arginine (AGR), and the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY). A . similar relationship exists between codons encoding phenylalanine and leucine. Thus, some polynucleotides . 5 encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2. Variant sequences can be readily tested for functionality as described herein.
One of ordinary skill in the art will also appreciate that different species can exhibit “preferential codon usage.” In general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.
Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm,
Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As used herein, the term “preferential codon usage” or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus ] favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2). For } example, the amino acid Threonine (Thr) may be encoded by
ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential. Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO:3 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, ’ and tested for functionality as disclosed herein.
Within preferred embodiments of the invention the isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:1, or a sequence complementary thereto, under stringent conditions. In general, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tp is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
The isolated polynucleotides of the present invention include DNA and RNA. Methods for preparing DNA and RNA are well known in the art. In general, RNA is isolated from a tissue or cell that produces large amounts of zdintl RNA. Such tissues and cells are identified by
Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA 25..77:5201, 1980), and include heart, brain, skeletal muscle, spinal cord, fetal heart, and fetal brain. Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 18:52-94, 1979). Poly (A)* RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).
Complementary DNA (cDNA) is prepared from poly (A)T RNA using known methods. In the alternative, genomic DNA can be isolated. Polynucleotides encoding zdintl polypeptides are then identified and isolated by, for example, hybridization or PCR. : : A full-length clone encoding zdintl can be obtained by conventional cloning procedures. - 5 Complementary DNA (cDNA) clones are preferred, although for some applications (e.g., expression in transgenic animals) it may be preferable to use a genomic clone, Or to modify a cDNA clone to include at least one genomic intron. Methods for preparing cDNA and genomic clones are well known and within the level of ordinary skill in the art, and include the use of the sequence disclosed herein, or parts thereof, for probing or priming a library.
Expression libraries can be probed with antibodies to zdintl or other specific binding partners. zdintl polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5’ non- coding regions of a zdintl gene. In view of the tissue- specific expression observed for zdintl by Northern blotting, this gene region is expected to provide for heart-, brain-, spinal cord-, and skeletal muscle-specific expression. Promoter elements from a zdintl gene could ] thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. Cloning of 5¢ flanking sequences also facilitates production of zdintl proteins by “gene activation” as disclosed in U.S. Patent
No. 5,641,670, Briefly, expression of an endogenous zdintl gene in a cell is altered by introducing into the zdintl locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting sequence is ga zdintl 5’ non-coding sequence that permits homologous recombination of the construct with the endogenous zdint] locus, whereby the sequences within the construct become operably linked with the endogenous zdintl coding sequence. In this way, an endogenous zdintl promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
The polynucleotides of the present invention can ) also be synthesized using DNA synthesizers. Currently the method of choice is the phosphoramidite method. If chemically synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately. The production of short genes (60 to 80 bp) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them. For the production of longer genes (>300 bp), however, special strategies must be invoked, because the coupling efficiency of each cycle during chemical DNA synthesis is seldom 100%. To overcome this problem, synthetic genes (double-stranded) are assembled in modular form from single-stranded fragments that are from 20 to 100 nucleotides in length. See Glick and Pasternak,
Molecular Biotechnology, Principles and Applications of
Recombinant DNA, (ASM Press, Washington, D.C. 1994); :
Itakura et al., Annu. Rev. Biochem. 53: 323-356 (1984) and
Climie et al., Proc. Natl. Acad. Sci. USA 87:633-637 . (1990).
The present invention further provides counterpart polypeptides and polynucleotides from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. of particular interest are zdintl polypeptides from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate polypeptides. Orthologs of human =zdintl can be cloned using information and compositions provided by the present invention in combination with conventional cloning ) techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses zdintl - 5S as disclosed herein. Such tissue or cell type would include, for example, heart, brain, spinal cord, and skeletal muscle. Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein. A library is then prepared from mRNA of a positive tissue or cell line. A zdintl-encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequences. A cDNA can also be cloned using the polymerase chain reaction, or PCR (Mullis, U.S. Patent No. 4,683,202), using primers designed from the representative human zdintl sequence disclosed herein. Within an additional method, the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to zdintl polypeptide. Similar . techniques can also be applied to the isolation of genomic clones.
Those skilled in the art will recognize that the sequence disclosed in SEQ ID NO:1 represents a single allele of human zdintl and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the DNA sequence shown in SEQ ID NO:1, including those containing silent mutations and those in which mutations result inp amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic wvariants of SEQ ID NO:2. cDNAs generated from alternatively spliced mRNAs, which retain the properties of the zdintl polypeptide are included within the scope of the present invention, as are polypeptides encoded by such
CDNAs and mRNAs. Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic ) libraries from different individuals or tissues according to standard procedures known in the art.
The present invention also provides isolated zdintl polypeptides that are substantially homologous to the polypeptides of SEQ ID NO:2 and their orthologs. The term "substantially homologous" is used herein to denote polypeptides having about 50%, preferably 60% more preferably at least 70%, and even more preferably 80% sequence identity to the sequences shown in SEQ ID NO:2 or their orthologs. Such polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO:2 or its orthologs.) Percent sequence identity is determined by conventional methods.
See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad.
Sci. USA 89:10915-9, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores : using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and .
Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one-letter codes). The percent identity is then calculated as:
Total number of identical matches -_——m x 100 [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences]
= <r > ~ ) = ~ NN mM — = nN NN NN © 1 wn TT +H ™M™ oN N 1 | ) on, ~ =H Hr Mm NN
I | | | I
Ts OW I NN ON = MM ! = nN OO NN = — ~~ oH oo 1 | | I ™ x; NH Mm HO =H Mm NN i I 1 | 1 I J
[0] — fia TN NO MON HN eH
Q ! EE EE
IM]
Ee — TT NM HO MN HM Hm t | | 1 jas @ Mm mM = NN od NN 4 oN NN NN ™M 1 i ) | [ 1 | | | i [G] OW N =r NM MO NO NN Mmm } | i | [ | | 1 [ca] Nn NNO MO mM A NO +H OO +4 ™M oN oN
I | } | I ! | | t
Oo NN NO Mm NN dA OO mM A © A N Hd «oN l | } | [ | | ] i . @] A MT MM HA HM +H NM HoH NN i | | I ] I | ] I } | I | i I [a] OW M OO NN = = Mm IF od MO MM A OO = TT Mm m t I I | | ] | | i l | I 2 CC wr Mm OO OO © + Mm Mm OO NN MM NN oH OO = ~N mM 1 | l ! | | | 1 ] [14 Nn OO NN MO +H OO NN OO Mm NN = MN ~H ~ MOM ~§ mM
I ] | { | t | { | \ i | {
L = NN NN OO + = © NN A =H oH of N +4 4 O Mm NN Oo ] I ] I I I ] 1 I ] 1 f I { £ MZ OU 0m UT mH oa XM 2 boa BHR NS
Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above. ’
Those skilled in the art appreciate that there are many established algorithms available to align two : amino acid sequences. The “FASTA” similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative variant zdintl. The
FASTA algorithm is described by Pearson and Lipman, Proc.
Nat’l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.
Enzymol. 183:63 (1990).
Briefly, FASTA first characterizes sequence similarity by identifying regions shared by the query sequence (e.g., SEQ ID NO:2) and a test sequence that have either the highest density of identities (if the ktup variable is 1) or pairs of identities (if ktup=2), without considering conservative amino acid substitutions, insertions, or deletions. The ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are “trimmed” to include only those residues that . contribute to the highest score. If there are several regions with scores greater than the “cutoff” value (calculated by a predetermined formula based upon the length of the sequence and the ktup value), then the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps. Finally, the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and
Wunsch, J. Mol. Biol. 48:444 (1970); Sellers, SIAM J.
Appl. Math. 26:787 (1974)), which allows for amino acid insertions and deletions. Preferred parameters for FASTA analysis are: ktup=1, gap opening penalty=10, gap : extension penalty=1l, and substitution matrix=BLOSUM62.
These parameters can be introduced into a FASTA program by - S modifying the scoring matrix file (“"SMATRIX"}, as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).
FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above. For nucleotide sequence comparisons, the ktup value can range between one to six, preferably from three to six, most preferably three, with other parameters set as default.
The present invention includes nucleic acid molecules that encode a polypeptide having one or more conservative amino acid changes, compared with the amino acid sequence of SEQ ID NO:2. The BLOSUM62 table 1s an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc.
Nat’l Acad. Sci. USA 89:10915 (1992)). Accordingly, the
BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. As used herein, the language “conservative amino acid substitution” refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.qg., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
Variant zdintl polypeptides or substantially homologous zdintl polypeptides are characterized as having one or more amino acid substitutions, deletions or i additions. These changes are preferably of a minor nature, that 1s conservative amino acid substitutions (see
Table 4) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag. The present invention thus includes polypeptides of from 383 to 464 amino acid residues that comprise a sequence that is at least 50%, preferably at least 60%, and more preferably 80% or more identical to the corresponding region of SEQ ID NO:2. Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the zdintl polypeptide and the affinity tag.
Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites. ’
Table 4 ’ : Conservative amino acid substitutions - 5 Basic: arginine lysine histidine
Acidic: glutamic acid aspartic acid
Polar: glutamine asparagine
Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine tryptophan tyrosine
Small: glycine alanine serine threonine } methionine
The present invention further provides a variety of polypeptide fusions and related multimeric proteins comprising one or more polypeptide fusions. For example, a disintegrin polypeptide domain can be prepared as a fusion to a dimerizing protein, as disclosed in U.s.
Patents Nos. 5,155,027 and 5,567,584. Preferred dimerizing proteins in this regard include other disintegrin polypeptide domains or disintegrin polypeptide domain fragments. Disintegrin polypeptide domain fusions, or disintegrin polypeptide domain fragment fusions, can be expressed in genetically engineered cells to produce 4 variety of multimeric disintegrin-like analogs. Auxiliary domain polypeptides can be fused to disintegrin domain polypeptides to target them to specific cells, tissues, or macromolecules (e.g., heart, brain, spinal cord, skeletal muscle, platelets). For example, a protease polypeptide domain, or protease polypeptide fragment or protein, could ) be targeted to a predetermined cell type by fusing it to a disintegrin polypeptide domain or fragment that specifically binds to an integrin polypeptide or integrin-~ like polypeptide on the surface of the target cell. In this way, polypeptides, polypeptide fragments and proteins can be targeted for therapeutic or diagnostic purposes.
Such disintegrins or protease polypeptide domains or fragments can be fused to two or more moieties, such as an affinity tag for purification and a targeting-disintegrin domain. Polypeptide fusions can also comprise one or more cleavage sites, particularly between domains. See Tuan et al., Connective Tissue Research 34:1-9, 1996.
Polypeptide fusions of the present invention will generally contain not more than about 1,500 amino acid residues, preferably not more than about 1,200 residues, more preferably not more than about 1,000 residues, and will in many cases be considerably smaller. )
For example, residues of zdintl polypeptide can be fused to E. coli B-galactosidase (1,021 residues; see Casadaban : et al., J. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a 4-residue factor Xa cleavage site. In a second example, residues of zdintl polypeptide can be fused to maltose binding protein (approximately 370 residues), a 4-residue cleavage site, and a 6-residue polyhistidine tag.
The proteins of the present invention can also comprise non-naturally occurring amino acid residues.
Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methanoproline,
cis-4-hydroxyproline, trans—-4-hydroxyproline, N- methylglycine, allo-threonine, methylthreonine, : hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4- methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non- naturally occurring amino acid residues into proteins.
For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art.
Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al.,
Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci.
USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chen. 271:19991-8, 1996).
Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.qg., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al., Biochem.
33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein )
Sci. 2:395-403, 1993).
A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for =zdintl amino acid residues.
Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass et al., Proc.
Natl. Acad. Sci. USA 88:4498-502, 1991). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity as disclosed below to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., J.
Biol. Chem. 271:4699-708, 1996. Sites of disintegrin- integrin, or protease interaction can also be determined . by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306- 12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992;
Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related disintegrin-like molecules.
Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and ) screening, such as those disclosed by Reidhaar-Olson and
Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. : 5 Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Patent
No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).
Variants of the disclosed zdintl DNA and polypeptide sequences can be generated through DNA shuffling, as disclosed by Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant . DNAs are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed Dy reassembly using PCR, resulting in randomly introduced . point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid “evolution” of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
Mutagenesis methods as disclosed herein can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in :
host cells. Mutagenized DNA molecules that encode active polypeptides (e.g., disintegrin-cell surface binding or protease activity) can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of ) individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
Using the methods discussed herein, one of ordinary skill in the art can identify and/or prepare a variety of polypeptide fragments or variants of SEQ ID
NO:2 or that retain the disintegrin and or protease activity of the wild-type zdintl protein. Such polypeptides may include additional amino acids from, for example, a secretory domain, a propeptide domain, a protease domain, part or all of a transmembrane and intracellular domains, including amino acids responsible for intracellular signaling; a fusion domains; affinity tags; and the like.
For any zdintl polypeptide, including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above. .
PROTEIN PRODUCTION
The zdintl polypeptides of the present invention, including full-length polypeptides, biologically active fragments, and fusion polypeptides, can be produced in genetically engineered host cells according to conventional techniques. Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. ‘ Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are - 5 disclosed by Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, NY, 1989, and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987.
In general, a DNA sequence encoding a zdintl polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector. The vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
To direct a zdintl polypeptide into the secretory pathway of a host cell, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
The secretory signal sequence may be derived from another secreted protein (e.g., t-PA) or synthesized de novo. The secretory signal sequence is operably linked to the zdintl
DNA sequence, 1i.e., the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell. Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al., U.S. Patent No. 5,037,743; Holland et al., U.S. Patent No. 5,143,830). i
Polypeptides and peptide fragments of the present invention are considered biologically active in the absence of the native signal sequence.
The protease domain of zdintl can be substituted by a heterologous sequence providing a different protease domain. In this case, the fusion product can be secreted, and the disintegrin domain of zdintl can direct the protease domain to a specific tissue described above.
This substituted protease domain can be chosen from the protease domains represented by the DP protein families, or domains from other known proteases.
Cultured mammalian cells are suitable hosts within the present invention. Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al., Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation (Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al., ibid.), and liposome-mediated transfection (Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and Finer, Nature Med. 2:714-6, 1996) . The production of recombinant polypeptides in cultured mammalian cells is disclosed, for example, by
Levinson et al., U.S. Patent No. 4,713,339; Hagen et al.,
U.S. Patent No. 4,784,950; Palmiter et al., U.S. Patent
No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134.
Suitable cultured mammalian cells include the COS-1 (ATCC
No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No.
CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and
Chinese hamster ovary (e.g. CHO-Kl; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the ’ 5 art and available from public depositories such as the
American Type Culture Collection, Rockville, Maryland. In general, strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288. Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.
Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as "transfectants". Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as "stable transfectants." A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. ~ Selection is carried out in the presence of a neomycin- : type drug, such as G-418 or the like. Selection systems can also be used to increase the expression level of the ) gene of interest, a process referred to as "amplification." Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes. A preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
Other drug resistance genes (e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase) can also be used. Alternative markers that introduce ap altered phenotype, such as green fluorescent protein, or cell surface proteins, such as CD4, CD8, Class I MHC, or placental alkaline phosphatase, may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
Other higher eukaryotic cells can also be used ’ as hosts, including plant cells, insect cells and avian cells. The use of Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by
Sinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987.
Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al., U.S.
Patent No. 5,162,222 and WIPO publication WO 94/06463.
Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV). See, King, L.A. and Possee,
R.D., The Baculovirus Expression System: A Laboratory
Guide, London, Chapman & Hall; O'Reilly, D.R. et al.,
Baculovirus Expression Vectors: A Laboratory Manual, New
York, Oxford University Press., 1994; and, Richardson, C.
D., Ed., Baculovirus Expression Protocols. Methods in
Molecular Biology, Totowa, NJ, Humana Press, 1995. A second method of making recombinant zdintl baculovirus utilizes a transposon-based system described by Luckow (Luckow, V.A, et al., J Virol 67:4566-79, 1993). This - system, which utilizes transfer vectors, is sold in the
Bac-to-Bac™ kit (Life Technologies, Rockville, MD). This system utilizes a transfer vector, pFastBacl™ (Life
Technologies) containing a Tn7 transposon to move the DNA encoding the zdintl polypeptide into a baculovirus genome maintained in E. «coli as a large plasmid called a “bacmid.” See, Hill-Perkins, M.S. and Possee, R.D., J Gen
Virol 71:971-6, 1990; Bonning, B.C. et al., J Gen Virol 75:1551-6, 1994; and, Chazenbalk, G.D., and Rapoport, B.,
J Biol Chem 270:1543-9, 1995. In addition, transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed ) zdintl polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci. 82:7952- - 5 4, 1985). Using a technique known in the art, a transfer vector containing zdintl is transformed into E. Coli, and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus. The bacmid
DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect
Spodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus that expresses zdintl is subsequently produced.
Recombinant viral stocks are made by methods commonly used the art.
The recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda. See, 1in general, Glick and Pasternak, Molecular Biotechnology: Principles and
Applications of Recombinant DNA, ASM Press, Washington,
D.C., 1994. Another suitable cell line is the High FiveQO™ cell line (Invitrogen) derived from Trichoplusia ni (U.S.
Patent #5,300,435). Commercially available serum-free media are used to grow and maintain the cells. Suitable media are S£f900 II™ (Life Technologies) or ESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cell040Q5™ (JRH Biosciences, Lenexa, KS) or Express FiveO™ (Life
Technologies) for the T. ni cells. The cells are grown up from an inoculation density of approximately 2-5 x 10° cells to a density of 1-2 x 10° cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
Procedures used are generally described in available laboratory manuals (King, L. A. and Possee, R.D., ibid. ;
O'Reilly, D.R. et al., ibid.; Richardson, C. D., ibid.) .
Subsequent purification of the zdintl polypeptide from the supernatant can be achieved using methods described herein.
Fungal cells, including yeast cells, can also be
S used within the present invention. Yeast species of ) particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S.
Patent No. 4,599,311; Kawasaki et al., U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al.,
U.S. Patent No. 5,037,743; and Murray et al., U.S. Patent
No. 4,845,075. Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine). A preferred vector system for use in Saccharomyces cerevisiae is the POTI vector system disclosed by Kawasaki et al. (U.S. Patent
No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S.
Patent No. 4,599,311; Kingsman et al., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including
Hansenula polymorpha, Schizosaccharomyces pombe,
Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and Candida maltosa are known in the art.
See, for example, Gleeson et al., J. Gen. Microbiol. 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279.
Aspergillus cells may be utilized according to the methods of McKnight et al., U.S. Patent No. 4,935,349. Methods ] for transforming Acremonium chrysogenum are disclosed by
Sumino et al., U.S. Patent No. 5,162,228. Methods for : 5 transforming Neurospora are disclosed by Lambowitz, U.S.
Patent No. 4,486,533.
The use of Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO
Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior te transformation. For polypeptide production in P. methanolica, it is preferred that the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene (AUG1 or AUGZ2). Other useful promoters include those of the dihydroxyacetone synthase {(DHAS), formate dehydrogenase (FMD) , and catalase (CAT) genes. To facilitate integration of the DNA into the host chromosome, it is preferred to have the entire expression : segment of the plasmid flanked at both ends by host DNA sequences. A preferred selectable marker for use in : Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC;
EC 4.1.1.21), which allows ade? host cells to grow in the absence of adenine. For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUGl1 and AUG?) are deleted. For production of secreted proteins, host cells deficient in vacuolar protease genes (PEP4 and PRBI) are preferred.
Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 ’ milliseconds, most preferably about 20 milliseconds.
Prokaryotic host cells, including strains of the bacteria Escherichia coli, Bacillus and other genera are also useful host cells within the present invention.
Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al., ibid.) . When expressing a zdintl polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis ) against a buffered saline solution. In the latter case, the polypeptide can be recovered from the periplasmic : space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells. A variety of suitable media, including defined media and complex media,
are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins ) and minerals. Media may also contain such components as growth factors or serum, as required. The growth medium : 5 will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell. P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C. Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors. A preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% Bacto™ peptone (Difco Laboratories, Detroit, MI), 1%
Bacto™ yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine).
Protein Isolation
It is preferred to purify the polypeptides of } the present invention to 280% purity, more preferably to 290% purity, even more preferably 295% purity, and particularly preferred is a pharmaceutically pure state, that 1s greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
Expressed recombinant zdintl polypeptides (or chimeric zdintl polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples. Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, ’ agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso
Haas, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like. Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties. Examples of coupling chemistries include cyanogen bromide activation, N- : hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl : and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art.
Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity
Chromatography: Principles & Methods, Pharmacia LKB
Biotechnology, Uppsala, Sweden, 1988.
The polypeptides of the present invention can be isolated by a combination of procedures including, but not ) limited to, anion and cation exchange chromatography, size exclusion, and affinity chromography. See Example 3 for a : 5 procedure. For example, immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags.
Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7, 1985). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents. Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol., Vol. 182, "Guide to Protein Purification”, M. Deutscher, (ed.),
Acad. Press, San Diego, 1990, pp.529-39). Within additional embodiments of the invention, a fusion of the polypeptide of interest and an affinity tag (e.g., maltose-binding protein, an immunoglobulin domain) may be constructed to facilitate purification.
Fragments/ Fusion Proteins
To direct the export of a zdintl polypeptide from the host cell, the zdintl DNA is linked to a second
DNA segment encoding a secretory peptide, such as a t-PA secretory peptide. To facilitate purification of the secreted receptor polypeptide, a C-terminal extension, such as a poly-histidine tag, substance P, Flag peptide (Hopp et al., Bio/Technology 6:1204-1210, 1988; available from Eastman Kodak Co., New Haven, CT) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the zdintl polypeptide.
Moreover, using methods described in the art, polypeptide fusions, or hybrid zdintl proteins, are constructed using regions or domains of the inventive zdintl in combination with those of other disintegrin-like molecules. (e.g. ADAM, MbC, and SVMP), or heterologous ’ proteins (Sambrook et al., ibid., Altschul et al., ibid.,
Picard, Cur. Opin. Biology, 5:511-5, 1994, and references therein). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them.
Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. For example, part or all of a domains conferring a biological function may be swapped : between zdintl of the present invention with the functionally equivalent domains from another family : member, such as ADAM, MDC, and SVMP. Such domains include, but are not limited to, conserved motifs such as the secretory signal sequence, protease, RGD, cysteine, and disintegrin domains. Such fusion proteins would be expected to have a biological functional profile that is the same or similar to polypeptides of the present invention or other known disintegrin-like family proteins (e.g. ADAMs, MDCs, and SVMPs), depending on the fusion constructed. Moreover, such fusion proteins may exhibit other properties as disclosed herein.
zdintl polypeptides or fragments thereof may also be prepared through chemical synthesis. zdintl : polypeptides may be monomers or multimers: glycosylated or non-glycosylated; pegylated or non-pegylated; and may or . 5 may not include an initial methionine amino acid residue.
Chemical Synthesis of Polypeptides
Zzdintl polypeptides, peptides, variants and or fragments thereof may also be prepared through chemical synthesis. TML polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non- pegylated; amidated or non-amidated; sulfated or non- sulfated; and may or may not include an initial methionine amino acid residue. For example, TML polypeptides can also be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. The polypeptides are preferably prepared by solid phase peptide synthesis, for example as described by Merrifield, J. Am. Chem. Soc. 85:2149, 1963. The synthesis is carried out with amino acids that are protected at the alpha-amino terminus. ) Trifunctional amino acids with labile side-chains are also protected with suitable groups to prevent undesired chemical reactions from occurring during the assembly of the polypeptides. The alpha-amino protecting group is selectively removed to allow subsequent reaction to take place at the amino-terminus. The conditions for the removal of the alpha-amino protecting group do not remove the side-chain protecting groups.
The alpha-amino protecting groups are those known to be useful in the art of stepwise polypeptide synthesis. Included are acyl type protecting groups (e.qg., formyl, trifluoroacetyl, acetyl), aryl type protecting groups (e.g., biotinyl), aromatic urethane type protecting groups fe.qg., benzyloxycarbonyl (Cbz), substituted benzyloxycarbonyl and 9-fluorenylmethyloxy- carbonyl (Fmoc)], aliphatic urethane protecting groups [e.g., t-butyloxycarbonyl {tBocj, isopropyloxycarbonyl, cyclohexloxycarbonyl] and alkyl type protecting groups ) (e.g., benzyl, triphenylmethyl). The preferred protecting groups are tBoc and Fmoc.
The side-chain protecting groups selected must remain intact during coupling and not be removed during the deprotection of the amino-terminus protecting group or during coupling conditions. The side-chain protecting groups must also be removable upon the completion of synthesis using reaction conditions that will not alter the finished polypeptide. In tBoc chemistry, the side- chain protecting groups for trifunctional amino acids are mostly benzyl based. In Fmoc chemistry, they are mostly tert-butyl or trityl based.
In tBoc chemistry, the preferred side-chain protecting groups are tosyl for arginine, cyclohexyl for aspartic acid, 4-methylbenzyl (and acetamidomethyl) for cysteine, benzyl for glutamic acid, serine and threonine, benzyloxymethyl (and dinitrophenyl) for histidine, 2-Cl- benzyloxycarbonyl for lysine, formyl for tryptophan and 2- bromobenzyl for tyrosine. In Fmoc chemistry, the . preferred side-chain protecting groups are 2,2,5,7,8- pentamethylchroman-6-sulfonyl (Pmc) or 2,2,4,6,7- pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for arginine, trityl for asparagine, cysteine, glutamine and histidine, tert-butyl for aspartic acid, glutamic acid, serine, threonine and tyrosine, tBoc for lysine and tryptophan.
For the synthesis of phosphopeptides, either direct or post-assembly incorporation of the phosphate group is used. In the direct incorporation strategy, the phosphate group on serine, threonine or tyrosine may be protected by methyl, benzyl, or tert-butyl in Fmoc
Chemistry or by methyl, benzyl or phenyl in tBoc ’ chemistry. Direct incorporation of phosphotyrosine without phosphate protection can also be used in Fmoc chemistry. - 5 In the post-assembly incorporation strategy, the unprotected hydroxyl groups of serine, threonine or tyrosine are derivatized on solid phase with di-tert- butyl-, dibenzyl- or dimethyl-N,N'- diisopropylphosphoramidite and then oxidized by tert- butylhydroperoxide.
Solid phase synthesis is usually carried out from the carboxyl-terminus by coupling the alpha-amino protected (side-chain protected) amino acid to a suitable solid support. An ester linkage is formed when the attachment is made to a chloromethyl, <chlortrityl or hydroxymethyl resin, and the resulting polypeptide will have a free carboxyl group at the C-terminus.
Alternatively, when an amide resin such as benzhydrylamine or p-methylbenzhydrylamine resin (for tBoc chemistry) and
Rink amide or PAL resin (for Fmoc chemistry) are used, an amide bond is formed and the resulting polypeptide will . have a carboxamide group at the C-terminus. These resins, whether polystyrene- or polyamide-based or } polyethyleneglycol-grafted, with or without a handle or linker, with or without the first amino acid attached, are commercially available, and their preparations have been described by Stewart et al., "Solid Phase Peptide
Synthesis" (2nd Edition), (Pierce Chemical Co., Rockford,
IL, 1984) and Bayer & Rapp Chem. Pept. Prot. 3:3 (1986); and Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach, IRL Press, Oxford, 1989.
The C-terminal amino acid, protected at the side chain 1f necessary, and at the alpha-amino group, is attached to a hydroxylmethyl resin using various activating agents including dicyclohexylcarbodiimide
(DCC), N,N'-diisopropylcarbodiimide (DIPCDI) and carbonyldiimidazole (CDI). It can be attached to chloromethyl or chlorotrityl resin directly in its cesium tetramethylammonium salt form or in the presence of triethylamine (TEA) or diisopropylethylamine (DIEA). First ) amino acid attachment to an amide resin is the same as amide bond formation during coupling reactions.
Following the attachment to the resin support, the alpha-amino protecting group is removed using various reagents depending on the protecting chemistry (e.qg., tBoc, Fmoc). The extent of Fmoc removal can be monitored at 300-320 nm or by a conductivity cell. After removal of the alpha-amino protecting group, the remaining protected amino acids are coupled stepwise in the required order to obtain the desired sequence.
Various activating agents can be used for the coupling reactions including DCC, DIPCDI, 2-chloro-1,3- dimethylimidium hexafluorophosphate (CIP), benzotriazol-1- yl-oxy-tris- (dimethylamino)-phosphonium hexafluoro- phosphate (BOP) and its pyrrolidine analog (PyBOP), bromo- tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),
O- (benzotriazol-1-yl)-1,1,3,3-tetramethyl~uronium : hexafluorophosphate (HBTU) and its tetrafluoroborate analog (TBTU) or its pyrrolidine analog (HBPyU), O-(7- - azabenzotriazol-1-yl)-1,1,3,3-tetramethyl~-uronium hexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU) or its pyrrolidine analog (HAPyU). The most common catalytic additives used in coupling reactions include 4-dimethylaminopyridine (DMAP), 3-hydroxy-3,4- dihydro-4-oxo-1,2,3-benzotriazine (HODhbt), N- hydroxybenzotriazole (HOBt) and l-hydroxy-7- azabenzotriazole (HOAt). Each protected amino acid is used in excess (>2.0 equivalents), and the couplings are usually carried out in N-methylpyrrolidone (NMP) or in
DMF, CH2Cl2 or mixtures thereof. The extent of completion
LL of the coupling reaction can be monitored at each stage, €.g., by the ninhydrin reaction as described by Kaiser et : al., Anal. Biochem. 34:595, 1970.
After the entire assembly of the desired . 5 peptide, the peptide-resin is cleaved with a reagent with proper scavengers. The Fmoc peptides are usually cleaved and deprotected by TFA with scavengers (e.qg., H20, ethanedithiol, phenol and thioanisole). The tBoc peptides are usually cleaved and deprotected with liquid HF for 1-2 hours at -5 to 0° C, which cleaves the polypeptide from the resin and removes most of the side-chain protecting groups. Scavengers such as anisole, dimethylsulfide and p- thiocresol are usually used with the liquid HF to prevent cations formed during the cleavage from alkylating and acylating the amino acid residues present in the polypeptide. The formyl group of tryptophan and the dinitrophenyl group of histidine need to be removed, respectively by piperidine and thiophenyl in DMF prior to the HF cleavage. The acetamidomethyl group of cysteine can be removed by mercury (II)acetate and alternatively by iodine, thallium (III)trifluoroacetate or silver } tetrafluoroborate which simultaneously oxidize cysteine to cystine. Other strong acids used for tBoc peptide cleavage and deprotection include trifluoromethanesulfonic acid (TFMSA) and trimethylsilyltrifluoroacetate (TMSOTF).
The disintegrin loop (residue 438 to residue 449 of SEQ ID NO:2) is of particular interest for use in assays and treatment of disorders of the heart, brain, spinal cord, and skeletal muscle. For these purposes the disintegrin loop peptide synthesized includes the terminal cysteine residues and thus, would be from residue 437 to residue 450 of SEQ ID NO:2. This peptide can be synthesized as a linear peptide or a disulfide linked peptide. Peptides having disulfide bonds between residues can be 438, 444, and 450 are of particular interest. See
Jia, L.G., ibid for additional description of peptide synthesis and disulfide linkage. : One skilled in the art will recognize that it is useful to design and synthesize new binding peptides using the integrin binding peptides of zdintl as a model. ’
Methods for synthesizing such peptides are described by P.
L. Barker et al., J. Med. Chem. 35: 2040-2048, 1992, and
L. Jia et al., J. Biol. Chem. 272: 13094-13102, 1997. As the structural conformation of the integrin binding peptide is critical, it is recognized that although some amino acid substitutions will not change the conformation of the peptides, the cyclization of the peptide is advantageously conserved. Synthetic peptides are useful as agonists or antagonsits for =zdintl and could be assayed.
ASSAYS
The activity of zdintl polypeptides «can be measured using a variety of assays that measure, for example, cell-cell interactions, proteolysis, extracellular matrix formation or remodeling.
Additionally, other biological functions associated with : disintegrin family members or with integrin/disintegrin interactions, apoptosis, proliferation or differentiation . can also be measured. Of particular interest is a change in platelet aggregation. Assays measuring platelet aggregation are well known in the art. For a general reference, see Dennis, Proc. Natl. Acad. Sci. 87: 2471- 2475, 1989.
Another assay of interest measures or detects changes in differentiation, development and/or and electrical coupling of muscle «cells or myocytes.
Additionally, the effects of a =zdintl polypeptides on cell-cell interactions of fibroblasts, myoblasts, nerve cells, white blood cells, endothelial cells and tumor cells would be of interest to measure. Yet another assays ‘ examines changes in protease activity and apoptosis.
The activity of molecules of the present . 5 invention can be measured using a variety of assays that, for example, measure neogenesis or hyperplasia (i.e., proliferation) of cardiac cells based on the tissue specificity in adult heart. Additional activities likely associated with the polypeptides of the present invention include proliferation of endothelial cells, cardiomyocytes, fibroblasts, skeletal myocytes directly or indirectly through other growth factors; action as a chemotaxic factor for endothelial cells, fibroblasts and/or phagocytic cells; osteogenic factor; and factor for expanding mesenchymal stem cell and precursor populations.
Proliferation can be measured using cultured cardiac cells or in vivo by administering molecules of the claimed invention to an appropriate animal model.
Generally, proliferative effects are observed as an increase in cell number and therefore, may include inhibition of apoptosis, as well as mitogenesis. Cultured cells include cardiac fibroblasts, cardiac myocytes, skeletal myocytes, human umbilical vein endothelial cells from primary cultures. Established cell lines include:
NIH 3T3 fibroblast (ATCC No. CRL-1658), CHH-1 chum heart cells (ATCC No. CRL-1680), H9c2 rat heart myoblasts (ATCC
No. CRL-1446), Shionogi mammary carcinoma cells (Tanaka et al., Proc. Natl. Acad. Sci. 89:8928-8932, 1992) and
LNCap.FGC adenocarcinoma cells (ATCC No. CRL-1740) .
Assays measuring cell proliferation are well known in the art. For example, assays measuring proliferation include such assays as chemosensitivity to neutral red dye (Cavanaugh et al., Investigational New Drugs 8:347-354, 1990), incorporation of radiolabelled nucleotides (Cook et al., Analytical Biochem. 179:1-7, 1989), incorporation of
S5-bromo-2'~deoxyuridine (BrdU) in the DNA of proliferating cells (Porstmann et al., J. Immunol. Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann, J. Immunol.
Methods 65:55-63, 1983; Alley et al., Cancer Res. 48:589~ 601, 1988; Marshall et al., Growth Reg. 5:69~84, 1995; and )
Scudiero et al., Cancer Res. 48:4827-4833, 1988) .
Differentiation is a progressive and dynamic process, beginning with pluripotent stem cells and ending with terminally differentiated cells. Pluripotent stem cells that can regenerate without commitment to a lineage express a set of differentiation markers that are lost when commitment to a cell lineage is made. Progenitor cells express a set of differentiation markers that may or may not continue to be expressed as the cells progress down the cell lineage pathway toward maturation.
Differentiation markers that are expressed exclusively by mature cells are usually functional properties such as cell products, enzymes to produce cell products and receptors. The stage of a cell population's differentiation is monitored by identification of markers present in the cell population. Myocytes, osteoblasts, adipocytes, chrondrocytes, fibroblasts and reticular cells are believed to originate from a common mesenchymal stem cell (Owen et al., Ciba Fdn. Symp. 136:42-46, 1988).
Markers for mesenchymal stem cells have not been well identified (Owen et al., J. of Cell Sci. 87:731-738, 1987), so identification is usually made at the progenitor and mature cell stages. The existence of early stage cardiac myocyte progenitor cells (often referred to as cardiac myocyte stem cells) has been speculated, but not demonstrated, in adult cardiac tissue. The novel polypeptides of the present invention are useful for studies to isolate mesenchymal stem cells and cardiac myocyte progenitor cells, both in vivo and ex vivo.
There is evidence to suggest that factors that stimulate specific cell types down a pathway towards . terminal differentiation or dedifferentiation affect the entire cell population originating from a common precursor ; 5 or stem cell. Thus, zdintl polypeptides may stimulate inhibition or proliferation of myocytes, smooth muscle cells, osteoblasts, adipocytes, chrondrocytes and endothelial cells. Molecules of the present invention may, while stimulating proliferation or differentiation of cardiac myocytes, inhibit proliferation or differentiation of adipocytes, by virtue of their effect on common precursor/stem cells. Thus, molecules of the present invention have use in inhibiting chondrosarcomas, atherosclerosis, restenosis and obesity.
Assays measuring differentiation include, for example, measuring cell-surface markers associated with stage-specific expression of a tissue, enzymatic activity, functional activity or morphological changes (Watt, FASEB, 5:281-284, 1991; Francis, Differentiation 57:63-75, 1994;
Raes, Adv. Anim. Cell Biol. Technol. Bioprocesses, 161- 171, 1989; all incorporated herein by reference).
In vivo assays for evaluating cardiac neogenesis or hyperplasia include treating neonatal and mature rats with the molecules of the present invention. The animals’ cardiac function is measured as heart rate, blood pressure, and cardiac output to determine left ventricular function. Post-mortem methods for assessing cardiac improvement include: increased cardiac weight, nuclei/cytoplasmic volume, staining of cardiac histology sections to determine proliferating cell nuclear antigen (PCNA) vs. cytoplasmic actin levels (Quaini et al.,
Circulation Res. 75:1050-1063, 1994 and Reiss et al.,
Proc. Natl. Acad. Sci. 93:8630-8635, 1996.)
Assays measuring in vivo effects of synthetic zdintl agonists include a Left Ventricular Hypertrophy model (A. M. Feldman et al., Circ. Res. 73: 184-192, 1993), which measures remodeling and repair after 5S congestive heart failure and chronic pressure overload. :
Proteins, including alternatively spliced peptides, of the present invention are useful for tumor suppression, and growth and differentiation either working in isolation, or in conjunction with other molecules (growth factors, cytokines, etc.) in brain, heart, spinal column, and skeletal muscle cells. Alternative splicing of zdintl may be cell-type specific and confer activity to specific tissues.
Proteins of the present invention are useful for delivery of therapeutic agents such as, but not limited to, proteases, radionuclides, chemotherapy agents, and small molecules. Effects of these therapeutic agents can . be measured in vitro using cultured cells or in vivo by administering molecules of the claimed invention to the appropriate animal model. For instance, zdintl transfected expression host cells may be embedded in an alginate environment and injected (implanted) into - recipient animals. Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation } and diffusion chambers have been described as a means to entrap transfected mammalian cells or primary mammalian cells. These types of non-immunogenic “encapsulations” or microenvironments permit the transfer of nutrients into the microenvironment, and also permit the diffusion of proteins and other macromolecules secreted or released by the captured cells across the environmental barrier to the : recipient animal. Most importantly, the capsules or microenvironments mask and shield the foreign, embedded cells from the recipient animal’s immune response. Such microenvironments can extend the life of the injected cells from a few hours or days (naked cells) to several : weeks (embedded cells).
Alginate threads provide a simple and quick . 5 means for generating embedded cells. The materials needed to generate the alginate threads are readily available and relatively inexpensive. Once made, the alginate threads are relatively strong and durable, both in vitro and, based on data obtained using the threads, in vivo. The alginate threads are easily manipulable and the methodology is scalable for preparation of numerous threads. In an exemplary procedure, 3% alginate is prepared in sterile HO, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension (containing about 5 x 10% to about 5 x 107 cells/ml) is mixed with the 3% alginate solution. One ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered CaClp solution over a time period of ~15 min, forming a "thread". The extruded thread is then transferred into a solution of 50 mM CaCly, and then into ’ a solution of 25 mM CaCly. The thread is then rinsed with deionized water before coating the thread by incubating in a 0.01% solution of poly-L-lysine. Finally, the thread is rinsed with Lactated Ringer's Solution and drawn from solution into a syringe barrel (without needle attached).
A large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer's Solution.
An alternative in vivo approach for assaying proteins of the present invention involves viral delivery systems. Exemplary viruses for this purpose include adenovirus, herpesvirus, lentivirus, vaccinia virus and adeno-associated virus (AAV) Adenovirus, a double-
stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see T.C. Becker et al., Meth. Cell Biol. ) 43:161-8%, 1994; and J.T. Douglas and D.T. Curiel, Science & Medicine 4:44-53, 1997). The adenovirus system offers : several advantages: adenovirus can (1) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection.
By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts can be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid. In an exemplary system, the essential E1 gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary) . When intravenously administered to intact animals, adenovirus primarily . targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host’s tissue (e.g., liver) will express and process (and, if a secretory signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
The adenovirus system can also be used for protein production in vitro. By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of : interest. The cells are then grown under serum-free conditions, which allows infected cells to survive for . 5 several weeks without significant cell division.
Alternatively, adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see
Garnier et al., Cytotechnol. 15:145-55, 1994). With either protocol, an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non-secreted proteins may also be effectively obtained.
Within yet another embodiment is provided an oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO:1 or a sequence complementary to SEQ ID NO:1.
Agonists and Antagonists
In view of the tissue distribution (heart, } brain, spinal cord and skeletal muscle) observed for zdintl expression, agonists (including the native disintegrin and protease domains) and antagonists have enormous potential in both in vitro and in vivo applications. Compounds identified as =zdintl agonists and antagonists are useful for studying cell-cell interactions, myogenesis, apoptosis, neurogenesis, connective tissue disorders, chondrogenesis, arthritis, tumor proliferation and suppression, extracellular matrix proteins, repair and remodeling of ischemia reperfusion and inflammation in vitro and in vivo. For example, zdint] and agonist compounds are useful as components of defined cell culture media, and may be used alone or in combination with other cytokines and hormones to replace serum that is commonly used in cell culture. Agonists are thus useful in specifically promoting the growth and/or development of cells of the myeloid lineages in culture.
Additionally, zdintl polypeptides and zdintl agonists, ) including small molecules are useful as a research reagent, such as for the expansion, differentiation, and/or cell-cell interactions of heart, brain, spinal cord, or skeletal muscle cells. =zdintl polypeptides are added to tissue culture media for these cell types.
Antagonists
Antagonists are also useful as research reagents for characterizing sites of complementary /anti- complementary interaction. Inhibitors of zdintl activity (zdintl antagonists) include anti-zdintl antibodies and soluble zdintl receptors, as well as other peptidic and non-peptidic agents (including ribozymes) . zdintl can also be used to identify inhibitors (antagonists) of its activity. Test compounds are added to the assays disclosed herein to identify compounds that inhibit the activity of zdintl. In addition to those . assays disclosed herein, samples can be tested for inhibition of zdintl activity within a variety of assays . designed to measure disintegrin/integrin binding or the stimulation/inhibition of zdintl-dependent cellular responses. For example, zdintl-responsive cell lines can be transfected with a reporter gene construct that is responsive to a zdintl-stimulated cellular pathway.
Reporter gene constructs of this type are known in the art, and will generally comprise a DNA response element operably linked to a gene encoding an assayable protein, such as luciferase, or a metabolite. DNA response elements can include, but are not limited to, cyclic AMP response elements (CRE), hormone response elements (HRE),
insulin response element (IRE) (Nasrin et al., Proc. Natl.
Acad. Sci. USA 87:5273-7, 1990) and serum response - elements (SRE) (Shaw et al. Cell 56: 563-72, 1989).
Cyclic AMP response elements are reviewed in Roestler et . 5 al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener,
Molec. Endocrinol. 4 (8):1087-94; 1990. Hormone response elements are reviewed in Beato, Cell 56:335-44; 1989. The most likely reporter gene construct would contain a disintegrin that, upon binding an integrin, would signal intracellularly through, for example, a SRE reporter.
Candidate compounds, solutions, mixtures or extracts are tested for the ability to inhibit the activity of zdintl on the target cells, as evidenced by a decrease in zdintl stimulation of reporter gene expression. Assays of this type will detect compounds that directly block zdintl binding to cell-surface receptors, i.e., integrin or the anti-complementary member of a complementary/anti- complementary pair, as well as compounds that block processes in the cellular pathway subsequent to complement/anti-complement binding. In the alternative, compounds or other samples can be tested for direct } blocking of =zdintl binding to an integrin using zdintl tagged with a detectable label (e.qg., 1251, biotin, horseradish peroxidase, FITC, and the like). Within assays of this type, the ability of a test sample to inhibit the binding of labeled zdintl to the integrin is indicative of inhibitory activity, which can be confirmed through secondary assays. Integrins used within binding assays may be cellular integrins or isolated, immobilized integrins.
An amino acid sequence comprising the “ECD” integrin binding component of zdintl, (residues 443 to 445 of SEQ ID NO: 2), which is analogous to the “RGD”, integrin binding loop, may also be used as an inhibitor.
Such an inhibitor would bind an integrin other than its naturally occurring integrin by nature of its folding structure. A particular interest in such an inhibitor would be to mediate platelet aggregation. Assays measuring binding and inhibition as well as platelet aggregation are known in the art. :
A zdintl polypeptide can be expressed as a fusion with an immunoglobulin heavy chain constant region, typically an F. fragment, which contains two constant region domains and lacks the variable region. Methods for preparing such fusions are disclosed in U.S. Patents Nos. 5,155,027 and 5,567,584. Such fusions are typically secreted as multimeric molecules wherein the Fc portions are disulfide bonded to each other and two non-Ig polypeptides are arrayed in closed proximity to each other. Fusions of this type can be used to evaluate effects and potential of dimerization of zdintl with itself or other disintegrin family members. Such fusions would also be useful to isolate the corresponding integrin(s) that zdintl binds. For use in assays, the chimeras are bound to a support via the Fo region and used in an ELISA format.
A zdintl integrin-binding polypeptide can also be used for purification of integrin. The polypeptide is immobilized on a solid support, such as beads of agarose, : cross-linked agarose, glass, cellulosic resins, silica- based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use. Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation. The resulting medium will generally be configured in the form of a column, and fluids containing integrins are passed through the column one or more times to allow integrins to bind to the integrin binding loop polypeptide. The integrin is then eluted ) using changes in salt concentration, chaotropic agents (guanidine HCl), or pH to disrupt integrin-receptor - 5 binding.
An assay system that uses a ligand-binding receptor (or an antibody, one member of a complementary/ anti-complementary pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcore, Pharmacia Biosensor, Piscataway, NJ) may be advantageously employed. Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip. Use of this instrument is disclosed by Karlsson, J. Immunol.
Methods 145:229-40, 1991 and Cunningham and Wells, J. Mol.
Biol. 234:554-63, 1993. A receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell. A test sample is passed through the cell. If a ligand, epitope, or opposite member of the complementary/anti-complementary pair is } present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film. This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
Another method to assay cell-cell interactions caused by =zintl polypeptides, peptides, or variants is with a silicon-based biosensor microphysiometer which measures the extracellular acidification rate or proton “excretion associated with receptor binding and subsequent physiologic cellular responses. An exemplary device is the Cytosensor™ Microphysiometer manufactured by Molecular
Devices, Sunnyvale, CA. A variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor 5S activation, and the like, can be measured by this method.
See, for example, McConnell, H.M. et al., Science 257:1906-1912, 1992; Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S. et al., J. Immunol. Meth. 212:49-59, 1998; Van Liefde, I. et al., Eur. J. Pharmacol. 346:87-95, 1998. The microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. By measuring extracellular acidification changes in cell media over time, the microphysiometer directly measures cellular responses to various stimuli, including =zdintl polypeptide, peptide, variant, agonists, or antagonists. Preferably, the microphysiometer is used to measure responses of a zdintl- responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to zdintl polypeptide, peptide, or variant. Zdintl-responsive eukaryotic cells comprise cells into which a receptor for zdintl has been transfected creating a «cell that is ’ responsive to zdintl polypeptide, peptide, or variant; or cells naturally responsive to zdintl such as, for example, : cells derived from the kidney or small intestine.
Differences, measured by a change, for example, an increase or diminution in extracellular acidification, in the response of cells exposed to zdintl polypeptide, peptide, or variant relative to a control not exposed to zdintl polypeptide, peptide, or variant, are a direct measurement of zdintl-modulated cellular responses.
Moreover, such zdintl-modulated responses can be assayed under a variety of stimuli. Using the microphysiometer, there is provided a method of identifying agonists of zdintl polypeptide, comprising providing cells responsive to a zdintl polypeptide, culturing a first portion of the - cells in the absence of a test compound, culturing a second portion of the cells in the presence of a test . 5 compound, and detecting a change, for example, an increase or diminution, in a «cellular response of the second portion of the cells as compared to the first portion of the cells. The change in cellular response is shown as a measurable change in extracellular acidification rate.
Moreover, culturing a third portion of the cells in the presence of zdintl polypeptide and the absence of a test compound can be used as a positive control for the zdintl- respensive cells, and as a control to compare the agonist activity of a test compound with that of the zdintl polypeptide. Moreover, using the microphysiometer, there is provided a method of identifying antagonists of zdintl polypeptide, comprising providing cells responsive to a zdintl polypeptide, culturing a first portion of the cells in the presence of 2zdintl and the absence of a test compound, culturing a second portion of the cells in the presence of zdintl and the presence of a test compound, and detecting a change, for example, an increase or a diminution in a cellular response of the second portion of the cells as compared to the first portion of the cells.
The change in cellular response is shown as a measurable change in extracellular acidification rate. Antagonists and agonists, for zdintl polypeptide, can be rapidly identified using this method.
Moreover, polypeptides, peptides and variants of zdintl can be used to identify cells, tissues, or cell lines which respond to a zdintl-stimulated pathway. The microphysiometer, described above, can be used to rapidly identify ligand-responsive cells, such as cells responsive to zdintl polypeptides peptides and variants of the present invention. Cells can be cultured in the presence
» or absence of zdintl polypeptides, peptides and variants.
Those cells which elicit a measurable change in extracellular acidification in the presence of zdintl ) polypeptides, peptides and variants are responsive to zdintl. Such cell lines, can be used to identify - antagonists and agonists of zdintl polypeptide as described above.
Integrin polypeptides and other receptor polypeptides which bind disintegrin polypeptides, and variants thereof, can also be used within other assay systems known in the art. Such systems include Scatchard analysis for determination of binding affinity (see
Scatchard, Ann. NY Acad. Sci. ol: 660-72, 19438) and calorimetric assays (Cunningham et al., Science 253:545~ 48, 1991; Cunningham et al., Science 245:821-25, 1991).
A "soluble receptor" is a receptor polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains. Soluble receptors can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate, or immunoglobulin constant region sequences. Many cell-surface receptors have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. Receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.
Soluble forms of zdintl polypeptides may act as antagonsits to zdintl polypeptides, and would be useful to modulate the effects of zdintl in heart, brain, skeletal muscle and spinal cord. Additionally, soluble =zdintl i peptides and fragments can disrupt the integrin-mediated attachment of a cell to the extracellular matrix. . 5 ANTIBODIES: zdintl polypeptides can also be used to prepare antibodies that specifically bind to zdintl epitopes, peptides or polypeptides. The zdintl polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal and elicit an immune response.
Suitable antigens would include fragments of the zdintl polypeptide encoded by SEQ ID NO:2 which represent six or more contiguous hydrophilic amino acids. Such antigenic regions would be, for example, from amino acid residue 159 to 164 (SEQ ID NO:7); amino acid residue 158 to 163 (SEQ
ID NO:8); amino acid residue 518 to 523 (SEQ ID NO:9) ; amino acid residue 658 to 663 (SEQ ID NO:10); and amino acid residue 190 to 195 (SEQ ID NO:11). Antibodies generated from this immune response can be isolated and purified as described herein. Methods for preparing and isolating polyclonal and monoclonal antibodies are well known in the art. See, for example, Current Protocols in
Immunology, Cooligan, et al. (eds.), National Institutes of Health, John Wiley and Sons, Inc., 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G.
R., Ed., Monoclonal Hybridoma Antibodies: Techniques and + Applications, CRC Press, Inc., Boca Raton, FL, 1982.
As would be evident to one of ordinary skill in the art, polyclonal antibodies can be generated from inoculating a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats with a zdintl polypeptide or a fragment thereof.
The immunogenicity of a zdintl polypeptide may be increased through the use of an adjuvant, such as alum
3 CE (aluminum hydroxide) or Freund's complete or incomplete adjuvant. Polypeptides useful for immunization also include fusion polypeptides, such as fusions of zdintl or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein. The polypeptide immunogen ) may be a full-length molecule or a portion thereof. If the polypeptide portion is "hapten-like", such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
As used herein, the term "antibodies" includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments, such as F(ab')y, and Fab proteolytic fragments.
Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen- binding peptides and polypeptides, are also included.
Non-human antibodies may be humanized by grafting non- human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally “cloaking” them with a human-like surface by replacement of exposed residues, wherein the result is a “veneered” antibody). In some instances, humanized : antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
Alternative techniques for generating or selecting antibodies useful herein include in vitro exposure of lymphocytes to zdintl protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled zdintl protein or peptide). Genes encoding ’ polypeptides having potential =zdintl polypeptide binding domains can be obtained by screening random peptide - 5 libraries displayed on phage (phage display) or on bacteria, such as E. coli. Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis. These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al., US
Patent NO. 5,223,409; Ladner et al., US Patent NO. 4,946,778; Ladner et al., US Patent NO. 5,403,484 and
Ladner et al., US Patent NO. 5,571,698) and random peptide display libraries and kits for screening such libraries are available commercially, for instance from Clontech (Palo Alto, CA), Invitrogen Inc. (San Diego, CA), New . England Biolabs, Inc. (Beverly, MA) and Pharmacia LKB
Biotechnology Inc. (Piscataway, NJ) . Random peptide display libraries can be screened using the zdintil sequences disclosed herein to identify proteins which bind to zdintl. These “binding proteins” which interact with zdintl polypeptides can be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity. The binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or guantitating soluble polypeptides as marker of underlying pathology or disease.
These binding proteins can also act as zdintl “antagonists” to block zdintl binding and signal transduction im vitro and in vivo. These anti-zdintl binding proteins would be useful for inhibiting, for ’ example, platelet aggregation, apoptosis, neurogenesis, myogenesis, tumor formation, and cell-cell interactions in general.
Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules. First, antibodies herein specifically bind if they bind to a zdintl polypeptide, peptide or epitope with a binding affinity (Kg) of 10° ML or greater, preferably 10’ M1 or greater, more preferably 108 ML or greater, and most preferably 10° m1 or greater. The binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G.,
Ann. NY Acad. Sci. 51: 660-672, 1949).
Second, antibodies are determined to : specifically bind if they do not significantly cross-react with related polypeptides. Antibodies do not significantly . cross-react with related polypeptide molecules, for example, if they detect =zdintl but not known related polypeptides using a standard Western blot analysis (Ausubel et al., ibid.). Examples of known related polypeptides are orthologs, proteins from the same species that are members of a protein family, zdintl polypeptides, and non-human =zdintl. Moreover, antibodies may be “screened against” known related polypeptides to isolate a population that specifically binds to the inventive polypeptides. For example, antibodies raised to zdintl are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to zdintl will flow through : the matrix under the proper buffer conditions. Such screening allows isolation of polyclonal and monoclonal . 5 antibodies non-crossreactive to closely related polypeptides (Antibodies: A Laboratory Manual, Harlow and
Lane (eds.), Cold Spring Harbor Laboratory Press, 1988;
Current Protocols in Immunology, Cooligan, et al. (eds.),
National Institutes of Health, John Wiley and Sons, Inc., 1995). Screening and isolation of specific antibodies is well known in the art. See, Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et al., Adv. in
Immunol. 43: 1-88, 1988; Monoclonal Antibodies:
Principles and Practice, Goding, J.W. (eds.), Academic
Press Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984.
A variety of assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to zdintl proteins or peptides.
Exemplary assays are described in detail in Antibodies: A
Laboratory Manual, Harlow and Lane (Eds.), Cold Spring . Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA), dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant zdintl protein or polypeptide.
Antibodies to zdintl may be used for tagging cells that express zdintl; for isolating zdintl by affinity purification; for diagnostic assays for determining circulating levels of zdintl polypeptides; for detecting or quantitating soluble zdintl as marker of underlying pathology or disease; in analytical methods employing FACS; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as anlagonists to block zdintl in vitro and in vivo. Suitable direct tags or labels include ) radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement /anti- complement pairs as intermediates. Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
Moreover, antibodies to zdintl or fragments thereof may be used in vitro to detect denatured zdintl or fragments thereof in assays, for example, Western Blots or other assays known in the art.
The present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of a =zacrp? polypeptide described herein. Such fragments or peptides may comprise an “immunogenic epitope,” which is a part of a protein that elicits an : antibody response when the entire protein is used as an immunogen. Immunogenic epitope-bearing peptides can be . identified using standard methods (see, for example,
Geysen et al., Proc. Nat. Acad. Sci. USA 81:3998, 1983).
In contrast, polypeptide fragments or peptides may comprise an “antigenic epitope,” which is a region of a protein molecule to which an antibody can specifically bind. Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al., Science 219:660, ; 1983). Accordingly, antigenic epitope-bearing peptides and polypeptides of the present invention are useful to - 5 raise antibodies that bind with the polypeptides described herein.
Antigenic epitope-bearing peptides and polypeptides preferably contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids of SEQ ID NO:2. Such epitope-bearing peptides and polypeptides can be produced by fragmenting a zacrpZ2 polypeptide, or by chemical peptide synthesis, as described herein. Moreover, epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268, 1993, and Cortese et al., Curr. Opin. Biotechnol. 7:616, 13896). Standard methods for identifying epitopes and producing antibodies from small peptides that comprise an epitope are described, for example, by Mole, “Epitope
Mapping,” in Methods in Molecular Biology, Vol. 10, Manson (ed.), pages 105-16 (The Humana Press, Inc. 1992), Price, “Production and Characterization of Synthetic Peptide-
Derived Antibodies,” in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.), pages 60-84 (Cambridge University Press 1995), and
Coligan et al. (eds.), Current Protocols in Immunclogy, pages 9.3.1 - 9.3.5 and pages 9.4.1 - 9.4.11 (John Wiley g¢
Sons 1997). Polypeptides, or fragments thereof, of the present invention comprising sequences of amino acids from, for example, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:8,
SEQ ID NO:10, or SEQ ID NO:11 are epitope bearing.
BIOACTIVE CONJUGATES:
Antibodies or polypeptides herein can also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For ) instance, polypeptides or antibodies of the present invention can be used to identify or treat tissues or organs that express a corresponding anti-complementary molecule (integrin or antigen, respectively, for instance). More specifically, =zdintl polypeptides or anti-zdintl antibodies, or bioactive fragments or portions thereof, can be «coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti-complementary molecule.
Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like. Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas : exotoxin, ricin, abrin and the like), as well as therapeutic radionuclides, such as iodine-131, rhenium-188 : or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance). Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin. For indirect attachment of a detectable or cytotoxic molecule, the detectable or cytotoxic molecule can be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/ anticomplementary pair. ’ In another embodiment, polypeptide-toxin fusion proteins or antibody-toxin fusion proteins can be used for . 5 targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues).
Alternatively, if the polypeptide has multiple functional domains (i.e., an activation domain or a ligand binding domain, plus a targeting domain), a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a «cell or tissue type of interest. In instances where the domain only fusion protein includes a complementary molecule, the anti- complementary molecule can be conjugated to a detectable or cytotoxic molecule. Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti- complementary-detectable/ cytotoxic molecule conjugates.
In another embodiment, zdintl-cytokine fusion proteins or antibody-cytokine fusion proteins can be used for enhancing in vivo killing of target tissues (for example, brain, heart, spinal cord and skeletal muscle malignancies), if the zdintl polypeptide or anti-zdintl antibody targets hyperproliferative brain, heart, spinal cord, or skeletal muscle cells. (See, generally, Hornick et al., Blood 89:4437-47, 1997). They described fusion proteins that enable targeting of a cytokine to a desired site of action, thereby providing an elevated local concentration of cytokine. Suitable zdintl polypeptides or anti-zdintl antibodies target an undesirable cell or tissue (i.e., a tumor or a leukemia), and the fused cytokine mediated improved target cell lysis by effector cells. Suitable cytokines for this purpose include interleukin 2 and granulocyte-macrophage colony- stimulating factor (GM-CSF), for instance. : In yet another embodiment, if the zdintl polypeptide or anti-zdintl antibody targets vascular cells or tissues, such polypeptide or antibody may be conjugated ) with a radionuclide, and particularly with a beta-emitting radionuclide, to reduce restenosis. Such therapeutic approach poses less danger to clinicians who administer the radioactive therapy. For instance, iridium-192 impregnated ribbons placed into stented vessels of patients until the required radiation dose was delivered showed decreased tissue growth in the vessel and greater luminal diameter than the control group, which received placebo ribbons. Further, revascularisation and stent thrombosis were significantly lower in the treatment group. Similar results are predicted with targeting of a biocactive conjugate containing a radionuclide, as described herein.
The bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action. :
USES OF POLYNUCLEOTIDE/POLYPEPTIDE:
Molecules of the present invention can be used to identify and isolate receptors and integrins involved in cell-cell interactions. For example, proteins and peptides of the present invention can be immobilized on a column and membrane preparations run over the column (Immobilized Affinity Ligand Techniques, Hermanson et al., eds., Academic Press, San Diego, CA, 1992, pp.195-202).
Polypeptides and peptides which bind to the zdintl polypeptides, peptides, and variants fo the present invention can then be eluted and characterized using methods known in the art. Proteins and peptides can also be radiolabeled (Methods in Enzymol., vol. 182, "Guide to : Protein Purification", M. Deutscher, ed., Acad. Press, San
Diego, 1990, 721-37) or photoaffinity labeled (Brunner et ; 5 al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et al.,
Biochem. Pharmacol. 33:1167-80, 1984) and specific cell- surface proteins can be identified.
The molecules of the present invention will be useful in repair and remodeling after an ischemic event, and/or inhibiting platelet aggregation. The polypeptides, nucleic acid and/or antibodies of the present invention can be used in treatment of disorders associated with infarct in brain or heart tissue, and/or platelet aggregation. The molecules of the present invention can be used to modulate proteolysis, apoptosis, neurogenesis, ’ myogenesis, connective tissue disorders, arthritis, chondrogenesis, cell adhesion, cell fusion, and signaling or to treat or prevent development of pathological conditions in such diverse tissue as heart, brain, spinal cord and skeletal muscle. In particular, certain diseases may be amenable to such diagnosis, treatment or } prevention. The molecules of the present invention can be used to modulate inhibition and proliferation of neurons and myocytes in heart, brain, spinal cord and skeletal muscle tissues. Disorders which may be amenable to diagnosis, treatment or prevention with zdint1l polypeptides include, for example, Alzheimers’s Disease, tumor formation, Multiple Sclerosis, Congestive Heart
Failure, Ischemic Reperfusion or infarct, and degenerative diseases.
The =zdintl molecules of the present invention may be particularly useful in the treatment of intimal hyperplasia or restenosis due to acute vascular injury.
Acute vascular injuries are those which occur rapidly (i.e. over days to months), in contrast to chronic vascular injuries (e.g. atherosclerosis) which develop over a lifetime. Acute vascular injuries often result from surgical procedures such as vascular reconstruction, wherein the techniques of angioplasty, endarterectomy, atherectomy, vascular graft emplacement or the like are ) employed. Hyperplasia may also occur as a delayed response in response to, e.g., graft emplacement or organ transplantation. The dose of zdintl in the treatment for restenosis will vary with each patient but will generally be in the range of those suggested above.
Advances in the treatment of coronary vascular disease include the use of mechanical interventions to either remove or displace offending plaque material in order to re-establish adequate blood flow through the coronary arteries. Despite the use of multiple forms of mechanical interventions, including balloon angioplasty, reduction atherectomy, placement of vascular stents, laser therapy, or rotoblator, the effectiveness of these techniques remains limited by an approximately 40% restenosis rate within 6 months after treatment.
Restenosis is thought to result from a complex interaction of biological processes including platelet . deposition and thrombus formation, release of chemotactic and mitogenic factors, and the migration and proliferation } of vascular smooth muscle cells into the intima of the dilated arterial segment.
The inhibition of platelet accumulation at sites of mechanical injury can limit the rate of restenosis in human subjects. Therapeutic use of a monoclonal antibody to platelet GpIIb/IIla is able to limit the level of restenosis in human subjects (Califf et al., N. Engl. J.
Med., 330: 956-961 (1994)). The antibody is able to bind to the GpIIb/IIla receptor on the surfaces of platelets and thereby inhibit platelet accumulation. This data suggests that inhibition of platelet accumulation at the site of mechanical injury in human coronary arteries is beneficial for the ultimate healing response that occurs.
GENE THERAPY: . 5 Polynucleotides encoding zdintl polypeptides are useful within gene therapy applications where it is : desired to increase or inhibit zdintl activity. If a mammal has a mutated or absent zdintl gene, the zdintl gene can be introduced into the cells of the mammal. In one embodiment, a gene encoding a zdintl polypeptide is introduced in vivo in a viral vector. Such vectors include an attenuated or defective DNA virus, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adenovirus, adeno-associated virus (AAV), and the like. Defective viruses, which entirely or almost entirely lack viral genes, are preferred. A defective virus is not infective after introduction into a cell. Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Examples of particular vectors include, but are not limited to, a defective herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell. Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet et al., J.
Clin. Invest. 90:626-30, 1992; and a defective adeno- associated virus vector {Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et al., J. Virol. 63:3822-8, 1989).
In another embodiment, a zdintl gene can be introduced in a retroviral vector, e.g., as described in
Anderson et al., U.S. Patent No. 5,399,346; Mann et al.
Cell 33:153, 1983; Temin et al., U.s. Patent No. 4,650,764; Temin et al., U.S. Patent No. 4,980,289;
Markowitz et al., J. Virol. 62:1120, 1988; Temin et al.,
U.S. Patent No. 5,124,263; International Patent
Publication No. WO 95/07358, published March 16, 1995 by )
Dougherty et al.; and Kuo et al., Blood 82:845, 1993.
Alternatively, the vector can be introduced by lipofection : in vivo using liposomes. Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Felgner et al., Proc. Natl. Acad.
Sci. USA 84:7413-7, 1987; Mackey et al., Proc. Natl. Acad.
Sci. USA 85:8027-31, 1988). The use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. More particularly, directing transfection to particular cells represents one area of benefit. For instance, directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting. Targeted peptides (e.g., hormones or neurotransmitters), proteins such as antibodies, or non-peptide molecules can be i coupled to liposomes chemically.
It is possible to remove the target cells from } the body; to introduce the vector as a naked DNA plasmid; and then to re-implant the transformed cells into the body. Naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.qg., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun Or use of a DNA vector transporter. See, e.g., Wu et al., J.
Biol. Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4, 1988.
Antisense methodology can be used to inhibit zdintl gene transcription, such as to inhibit cell proliferation in vivo. Polynucleotides that are complementary to a segment of a zdintl-encoding - 5 polynucleotide (e.g., a polynucleotide as set froth in SEQ
ID NO:1) are designed to bind to zdintl-encoding mRNA and to inhibit translation of such mRNA. Such antisense polynucleotides are used to inhibit expression of zdintl polypeptide-encoding genes in cell culture or in a subject.
The present invention also provides reagents which will find use in diagnostic applications. For example, the zdintl gene, a probe comprising zdintl DNA or
RNA or a subsequence thereof can be used to determine if the =zdintl gene is present on chromosome 2g33 or if a mutation has occurred. Detectable chromosomal aberrations at the zdintl gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements.
Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al., ibid.; Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
Transgenic mice, engineered to express the zdintl gene, or fragments thereof, and mice that exhibit a complete absence of zdintl gene function, referred to as "knockout mice" (Snouwaert et al., Science 257:1083, 1992), can also be generated (Lowell et al., Nature 366:740-42, 1983) by one skilled in the art. These mice can be employed to study the zdintl gene, gene fragments, and the protein encoded thereby in an in vivo system.
CHROMOSOMAL LOCALIZATION: )
Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high- resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250:245-50, 1990). Partial or full knowledge of a gene's sequence allows one to design PCR primers suitable for use with chromosomal radiation hybrid mapping panels. Radiation hybrid mapping panels are commercially available which cover the entire human genome, such as the Stanford G3 RH Panel and the
GeneBridge 4 RH Panel (Research Genetics, Inc.,
Huntsville, AL). These panels enable rapid, PCR-based chromosomal localizations and ordering of genes, sequence- tagged sites (STSs), and other nonpolymorphic and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers. The precise knowledge of a i gene’s position can be useful for a number of purposes, including: 1) determining if a sequence is part of an . existing contig and obtaining additional surrounding genetic sequences in various forms, such as YACs, BACs or cDNA clones; 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region; and 3) cross-referencing model organisms, such as mouse, which may aid in determining what function a particular gene might have.
Sequence tagged sites (STSs) can also be used independently for chromosomal localization. An STS is a
DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome. An STS is defined by a pair of - oligonucleotide primers that are used in a polymerase chain reaction to specifically detect this site in the . 5 presence of all other genomic sequences. Since STSs are based solely on DNA sequence they can be completely described within an electronic database, for example,
Database of Sequence Tagged Sites (dbSTS), GenBank, (National Center for Biological Information, National
Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), and can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences.
For pharmaceutical use, the proteins of the present invention can be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as powders, ointments, drops or transdermal patch) bucally, or as a pulmonary or nasal inhalant. Intravenous administration will be by bolus injection or infusion over a typical period of one to several hours. In general, pharmaceutical formulations will include a =zdintl protein, alone, or in conjunction with a dimeric partner, in combination with a pharmaceutically acceptable vehicle, such as saline, buffered saline, 5% dextrose in water or the like.
Formulations may further include one or more excipients, preservatives, solubilizers, buffering agents, albumin to prevent protein loss on vial surfaces, etc. Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of
Pharmacy, Gennaro, ed., Mack Publishing Co., Easton, Pa, 19th ed., 1995. Therapeutic doses will generally be in the range of 0.1 to 100 pg/kg of patient weight per day,
preferably 0.5-20 mg/kg per day, with the exact dose determined by the clinician according to accepted standards, taking into account the nature and severity of : the condition to be treated, patient traits, etc.
Determination of dose is within the level of ordinary 3 skill in the art. The proteins may be administered for acute treatment, over one week or less, often over a period of one to three days or may be used in chronic treatment, over several months or years. In general, a therapeutically effective amount of zdintl is an amount sufficient to produce a clinically significant change in extracellular matrix remodeling, scar tissue formation, tumor suppression, platelet aggregation, apoptosis, myogenesis, neurogenesis, electrical coupling, blood flow and/or cell proliferation in brain, heart, spinal cord, and skeletal muscle.
The invention is further illustrated by the following non-limiting examples.
Example 1 ]
Extension of EST Sequence
The novel zdintl polypeptide-encoding polynucleotides of the present invention were initially identified by querying an EST database. This query identified an expressed sequence tag (EST) to nucleotide 1097 to nucleotide 1415 of SEQ ID NO: 1. A cDNA clone, corresponding to this EST was obtained and the deduced amino acid sequence was determined to be incomplete.
Primers ZC17,991 (SEQ ID NO:4) and 2Cl17,992 (SEQ ID NO:5) were used to screen an arrayed fetal brain cDNA plasmid library to identify clones of zdintl. Thermocycler conditions were as follows: one cycle at 94°C for 1 minute
30 seconds; followed by thirty cycles at 94°C for 10 seconds, 64°C for 20 seconds, 72°C for 30 seconds, followed by one cycle at 72°C for 5 minutes, followed by a 4°C hold.
A sample of the reaction contents was electrophoresed on a 4% agarose gel to identify positive pools. These pools were screened by polymerase chain reaction with 2C17,992 (SEQ ID NO:5) and the vector primer 2ZC13,006 (SEQ ID
NO:6). Thermocycler conditions were as follows: one cycle at 94°C for 1 minute 30 seconds; followed by five cycles at 94°C for 10 seconds, 68°C for 2 minutes, followed by twenty-five cycles at 94°C for 10 seconds, 62°C for 20 seconds, 72°C for 2 minutes, followed by one cycle at 72°C for 10 minutes, followed by a 4°C hold. A sample of the reaction contents was electrophoresed on a 1% agarose gel and a band of ~1.5 kb was further electrophoresed on a 1% preparative gel and the resulting band gel purified using commercially available gel purification reagents and protocol (QIAEX II Gel Extraction Kit; Qiagen, Inc., Santa
Clarita, CA). This fragment was sequenced and was determined to extend the amino acid sequence of zdintl in the 5' direction. : Example 2
Tissue Distribution
Analysis of tissue distribution was performed by the Northern blotting technique using Human Multiple
Tissue and Master Dot Blots from Clontech (Palo Alto, CA), and a human vascular tissue blot prepared in-house. The human vascular blot was prepared from the following cell lines: Human Umbilical Vein Endothelial Cells (Cascade
Biologics, Inc., Portland, OR.), Human Pulmonary Artery
Endothelial Cells (Cascade Biologics, Inc., Portland,
OR.), Human Aortic Endothelail Cells, (Cascade Biologics,
Inc., Portland, OR.), Aortic Smooth Muscle Cells {Clonetics, San Diego, CA.), Human Intestinal Smooth
Muscle Cells (American Type Culture Collectio, Manasas,
Virginia), Normal Human Lung Fibroblast, Clonetics, San
Diego, CA) and Normal Human Dermal Fibroblast-Neonatal, )
Clonetics, San Diego, CA.). Messenger RNA was extracted and blots prepared by methods known in the art. The probe was obtained by restriction digest of the . original cDNA clone with a restriction endonuclease, PstI. The reaction mixture was electrophoresed on a preparative agarose gel and two bands, corresponding to a 239 base pair fragment and a 223 base pair fragment from the cDNA clone, were gel purified using commercially available gel purification reagents and protocol from Qiagen, Inc. 2A probe was made by pooling the purified DNA from both bands and was random prime labeled with 3%p using a commercially available kit (Rediprime DNA labeling system; Amersham Corp., Arlington
Heights, IL). The probe was then purified using a NUCTRAP push column (Stratagene Cloning Systems, La Jolla, CA).
EXPRESSHYB (Clontech) solution was used for pre- hybridization and hybridization. The hybridization solution consisted of 8 ml EXPRESSHYB, 80 Hl Sheared ’
Salmon Sperm DNA (10 mg/ml, 5 Prime-3 Prime, Boulder, CO), 48 pl Human Cot-1 DNA (1 mg/ml, Gibco BRL), and 57 ul labeled probe (2.3 x 107° CPM/ul). Hybridization took place overnight at 50°C, and the blots were then washed in 2X SSC and 0.1% SDS at ambient room temperature, then 2X
SSC and 0.1% SDS at 60°C, followed by 0.1X SSC and 0.1%
SBS at 60°C. The blots were exposed overnight and developed. Strong signals of three transcript sizes, approximately 3.0 kb, 4.4 kb, and 7.5 kb, were observed in heart on the multiple tissue Northern blots. Faint signals of the same transcript sizes were observed in brain and spinal cord. An fainter signal of the three transcript sizes was observed in skeletal muscle. The Master Dot Blot showed strong signals in brain, heart, fetal brain, and : fetal heart. For the human vascular blot, a strong signal at 3-3.5kb in human aortic endothelial cells and weaker . 5 signals in aortic smooth muscle cells and normal human lung fibroblast cells was observed.
Example 3
Protein Purification
Purification conditions for zdintl with N- and
C-terminal EE tags:
E. coli, Pichia, CHO and BHK cells are transfected with expression vectors containing the DNA sequence of SEQ ID NO:1, or a portion thereof, operably linked to a polynucleotide encoding a Glu-Glu tag. Zdintl protein is expressed in conditioned media of E. coli,
Pichia methanolica, and or chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells. For zdintl expressed in
E. coli and Pichia, the media is not concentrated prior to purification. Unless otherwise noted, all operations are carried out at 4°C. A total of 25 liters of conditioned medium from BHK cells is sequentially sterile filtered through a 4 inch, 0.2 mM Millipore (Bedford, MA) OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor, MI)
Supercap 50. The material is then concentrated to about 1.3 liters using a Millipore ProFlux A30 tangential flow concentrator fitted with a 3000 kDa cutoff Amicon (Bedford, MA) S10Y3 membrane. The concentrated material is again sterile-filtered with the Gelman filter, as described above. A mixture of protease inhibitors is added to the concentrated conditioned medium to final concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, IN), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim) . A 50.0 ml sample of anti-EE
Sepharose, prepared as described below, is added and the mixture gently agitated on a Wheaton (Millville, NJ) 5S roller culture apparatus for 18.0 h at 4°C. :
The mixture is then poured into a 5.0 x 20.0 cm
Econo-Column (Bio-Rad, Laboratories, Hercules, CA), and the gel is washed with 30 column volumes of phosphate buffered saline (PBS). . The unretained flow-through fraction is discarded. Once the absorbance of the effluent at 280 nM is less than 0.05, flow through the column is reduced to zero, and the anti-EE Sepharose gel is washed with 2.0 column volumes of PBS containing 0.2 mg/ml of EE peptide (AnaSpec, San Jose, CA). The peptide that is used has the sequence GluTyrMetProValAsp. After 1.0 h at 4°cC, flow is resumed and the eluted protein collected. This fraction is referred to as the peptide elution. The anti-
EE Sepharose gel is then washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and the glycine wash 1s collected separately. The pH of the glycine-eluted fraction is adjusted to 7.0 by the addition of a small volume of 10X
PBS and stored at 4°C for future analysis, if needed.
The peptide elution is concentrated to 5.0 ml using a 15,000 molecular weight cutoff membrane concentrator (Millipore, Bedford, MA), according to the manufacturer’s instructions. The concentrated peptide elution is then Separated from free peptide by chromatography on a 1.5 x 50 cm Sephadex G-50 (Pharmacia,
Piscataway, NJ) column equilibrated in PBS at a flow rate of 1.0 ml/min using a BioCad Sprint HPLC (PerSeptive
BioSystems, Framingham, MA). Two-ml fractions are collected and the absorbance at 280 nM monitored. The first peak of material absorbing at 280 nM and eluting near the void volume of the column is collected. This fraction is pure zdintl NEE or zdintl CEE. The pure material is concentrated as described above, analyzed by
SDS-PAGE and Western blotting with anti-EE antibodies, ’ aliquoted, and stored at -80°C according to standard procedures. . 5
Preparation of anti-EE Sepharose:
A 100 ml bed volume of protein G-Sepharose (Pharmacia, Piscataway, NJ) is washed 3 times with 100 ml of PBS containing 0.02% sodium azide using a 500 ml 10 Nalgene 0.45 micron filter unit. The gel is washed with 6.0 volumes of 200 mM triethanolamine, pH 8.2 (TEA, Sigma,
St. Louis, MO). and an equal volume of EE antibody solution containing 900 mg of antibody is added. After an overnight incubation at 4°C, unbound antibody is removed 15 by washing the resin with 5 volumes of 200 mM TEA as described above. The resin is resuspended in 2 volumes of
TEA, transferred to a suitable container, and dimethylpimilimidate-2HC1 (Pierce, Rockford, IL), dissolved in TEA, is added to a final concentration of 36 20 mg/ml of gel. The gel is rocked at room temperature for 45 min and the liquid is removed using the filter unit as . described above. Nonspecific sites on the gel are then blocked by incubating for 10 min at room temperature with volumes of 20 mM ethanolamine in 200 mM TEA. The gel is then washed with 5 volumes of PBS containing 0.02% sodium azide and stored in this solution at 4°C.
Purification of untagged zdintl
E. coli, Pichia, CHO and BHK cells are transfected with expression vectors containing the DNA sequence of SEQ ID NO:1, or a portion thereof. The procedure described below is used for protein expressed in conditioned medium of E. coli, Pichia methanolica, and
Chinese hamster ovary (CHO) and baby hamster kidney (BHK)
cells. For zdintl expressed in E. coli and Pichia, however, the medium is not be concentrated prior to purification. Unless otherwise noted, all operations are carried out at 4°C. A total of 25 liters of conditioned medium from BHK cells is sequentially sterile filtered ) through a 4 inch, 0.2 mM Millipore (Bedford, MA) OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor, MI)
Supercap 50. The material is then be concentrated to about 1.3 liters using a Millipore ProFlux A30 tangential flow concentrator fitted with a 3000 kDa cutoff Amicon (Bedford, MA) S10Y3 membrane. The concentrated material is again be sterile-filtered with the Gelman filter as described above. A mixture of protease inhibitors is added to the concentrated conditioned medium to final concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA, Sigma Chemical Co. St. Louis, MO), 0.001 mM leupeptin (Boehringer-Mannheim, Indianapolis, IN), 0.001 mM pepstatin (Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim) .
The procedures outlined below are adaptations of those used to purify metalloprotease/disintegrins from
Crotalus viridus and Crotalus atrox venom (Liu et al., .
Toxicol. 33: 1289-1298, 1995; Shimokawa et al., Arch
Biochem Biophys 343: 35-43, 1997). A combination of . procedures including, but not limited to, anion and cation exchange chromatography, size exclusion, and affinity chromography is used to purify untagged zdintl.
Concentrated conditioned medium is diluted 1/10 in line with 10 mM borate buffer, pH 9.0, 0.1 M NaCl, and 300 2.0 mM CaClp; using the BioCad Sprint HPLC (PerSeptive
BioSystems, Framingham, MA). The material is pumped onto a 3.5 x 20 cm Poros HQ (PerSeptive BioSystems,
Framingham, MA) column at 5 ml/min. The column is washed with loading buffer, and when the absorbance of the effluent is less than 0.05, the column is developed with a linear gradient of NaCl from 0.1 M to 1.0 M NaCl. : Fractions containing zdintl are identified by SDS-PAGE and
Western blotting with anti-zdintl peptide antibodies. . 5 zdintl-containing fractions are pooled together, and concentrated using an Amicon stirred cell concentrator fitted with a YM-10 membrane. The Poros HQ pool is then chromatographed on a Sephadex G-75 column equilibrated in mM sodium phosphate, pH 7.0. Fractions containing 10 zdintl are identified and pooled together, as described above, and applied to a 1.0 x 5 cm Poros HA hydroxyapatite column at 1.0 ml/min using the BioCad Sprint HPLC. The column is washed with loading buffer and developed with a linear gradient from 10 mM to 500 mM sodium phosphate.
Fractions contained pure zdintl are identified by SDS-PAGE and Western blotting, as described above. The purified material is aliquoted and stored as described above.
Example 4
Chromosomal Assignment and Placement of Zdintl
Zdintl was mapped to chromosome 2 using the commercially avallable version of the "Stanford G3
Radiation Hybrid Mapping Panel” (Research Genetics, Inc.,
Huntsville, AL). The "Stanford G3 RH Panel" contains
PCRable DNAs from each of 83 radiation hybrid clones of the whole human genome, plus two control DNAs (the RM donor and the A3 recipient). A publicly available WWW server (http://shgc-www.stanford.edu) allows chromosomal localization of markers.
For the mapping of zdintl with the "Stanford G3
RH Panel", 20 pl reactions were set up in a PCRable 09g- well microtiter plate (Stratagene, La Jolla, CA) and used in a "RoboCycler Gradient 96" thermal cycler (Stratagene).
Each of the 85 PCR reactions consisted of 2 pl 10X KlenTaqg
PCR reaction buffer (CLONTECH Laboratories, 1Inc., Palo
Alto, CA), 1.6 pl dNTPs mix (2.5 mM each, PERKIN-ELMER,
Foster City, CA), 1 pl sense primer, 2C20,843 (SEQ ID
NO:12), 1 pl antisense primer, 2C20,844 (SEQ ID NO:13), 2 pl "RediLoad" (Research Genetics, Inc., Huntsville, AL), 0.4 pl 50X Advantage KlenTaq Polymerase Mix (Clontech )
Laboratories, 1Inc.), 25 ng of DNA from an individual hybrid clone or control and distilled water for a total volume of 20 pl. The reactions were overlaid with an equal amount of mineral oil and sealed. The PCR cycler conditions were as follows: an initial 1 cycle 5 minute denaturation at 940¢, 35 cycles of a 45 seconds denaturation at 94°C, 45 seconds annealing at 66°C and 1 minute and 15 seconds extension at 72°C, followed by a final 1 «cycle extension of 7 minutes at 729C. The reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD).
The results showed linkage of Zdintl to the framework marker SHGC-56733 with a LOD score of >12 and at a distance of 0 cR 10000 from the marker. The use of surrounding markers positions Zdintl in the 2g33 region on the integrated LDB chromosome 2 map (The Genetic Location
Database, University of Southhampton, WWW server: http://cedar.genetics. soton.ac.uk/public html/).
Example 5
Synthesis of Peptides
Zdintl-1, a peptide corresponding to amino acid residue 437 (Cys) to amino acid residue 450 (Cys) of SEQ
ID NO: 2, is synthesized by solid phase peptide synthesis using a model 431A Peptide Synthesizer (Applied
Biosystems/Perkin Elmer, Foster City, CA). Fmoc-Glutamine resin (0.63 mmol/g; Advanced Chemtech, Louisville, KY) is used as the initial support resin. 1 mmol amino acid cartridges (Anaspec, Inc. San Jose, CA) are used for synthesis. A mixture of 2(l-Hbenzotriazol-y-yl 1,1,3,3- tetrahmethylhyluronium hexafluorophosphate (HBTU) , 1- ) hydroxybenzotriazol (HOBt), 2m N,N-Diisolpropylethylamine,
N-Methylpyrrolidone, Dichloromethane (all from Applied . 5 Biosystems/Perkin Elmer) and piperidine (Aldrich Chemical
Co., St. Louis, MO), are used for synthesis reagents.
The Peptide Companion software (Peptides
International, Louisville, KY) is used to predict the aggregation potential and difficulty level for synthesis for the =zdint-1 peptide. Synthesis is performed using single coupling programs, according to the manufacturer’s specifications.
The peptide is cleaved from the solid phase following standard TFA cleavage procedure (according to
Peptide Cleavage manual, Applied Biosystems/Perkin Elmer).
Purification of the peptide is done by RP-HPLC using a
C18, 10 um semi-peparative column (Vydac, Hesperial, CA).
Eluted fractions from the column are collected and analyzed for correct mass and purity by electrospray mass spectrometry. Pools of the eluted material are collected.
If pure, the pools are combined, frozen and lyophilized.
Example 6 } Anticoagulant Activity of zdintl
The ability of the zdintl protein to inhibit clotting is measured in a one-stage clotting assay using wild-type zdintl as a control. Recombinant proteins are prepared essentially as described above from cells cultured in media containing 5mg/ml vitamin K. Varying amounts of the zdintl or recombinant wild-type =zdintl are diluted in 50 mM Tris pH 7.5, 0.1% BSA to 100 ml. The mixtures are incubated with 100 ml of zdintl-deficient plasma and 200 ml of thromboplastin C (Dade, Miami, FL; contains rabbit brain thromboplastin and 11.8 mM Ca*™).
The clotting assay is performed in an automatic coagulation timer (MLA Electra 800, Medical Laboratory
Automation Inc., Pleasantville, NY), and clotting times are converted te units of zdintl activity using a standard curve constructed with 1:5 to 1:640 dilutions of normal pooled human plasma (assumed to contain one unit per ml zdintl activity; prepared by pooling citrated serum from healthy donors).
Zdintl activity is seen as a reduction in clotting time over control samples.
Example 7
Inhibition of Platelet Accumulation with zdintl
Zdintl is analyzed for its ability to inhibit platelet accumulation at sites of arterial thrombosis due to mechanical injury in non-human primates. A model of aortic endarterectomy is utilized in baboons, essentially as described by Lumsden et al. (Blood 81: 1762-1770 (1993)). A section cof baboon aorta 1-2 cm in length is removed, inverted and scraped to remove the intima of the artery and approximately 50% of the media. The artery is - reverted back to its correct orientation, cannulated on both ends and placed into an extracorporeal shunt in a . baboon, thereby exposing the mechanically injured artery to baboon blood via the shunt. Just prior to opening of the shunt to the circulating blood, "1In-labeled autologous platelets are injected intravenously into the animal. The level of platelet accumulation at the site of the injured artery is determined by real-time gamma camera imaging.
Evaluation of zdintl for inhibition of platelet accumulation is done using bolus injections of zdintl or saline control and are given just prior to the opening of the shunt. The injured arteries are measured continuously for 60 minutes. ’ 2dintl activity is seen as an inhibition of platelet accumulation. . 5
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
Claims (35)
1. An isolated polypeptide molecule comprising a contiguous sequence of 14 amino acids of SEQ ID NO:2.
2. An isolated polypeptide molecule according to claim 1, wherein the polypeptide molecule comprises residues 437 to 450 of SEQ ID NO:2.
3. An isolated polypeptide molecule according to claim 1 wherein the polypeptide molecule is between 82 and 232 amino acids in length.
4. An isolated polypeptide molecule according to claim 3 wherein the polypeptide molecule is residues 164 to 382 of SEQ ID NO:2.
5. An isolated polypeptide molecule according to claim 3 wherein the polypeptide molecule is residues 383 to 464 of SEQ ID NO:2.
6. An isolated polypeptide molecule according to claim 3 wherein the polypeptide molecule is residues 465 to 696 of SEQ ID NO:2.
7. A isolated polypeptide molecule selected from the group consisting of: a) a polypeptide molecule comprising residues 164 to 382 of SEQ ID NO:2; b) a polypeptide molecule comprising residues 383 to 464 of SEQ ID NO:2;
c) a polypeptide molecule comprising residues 465 to 696 of SEQ ID NO:2; d) a polypeptide molecule comprising residues 438 to 449 of SEQ ID NO:2; ’ e) a polypeptide molecule comprising residues 164 to 464 of SEQ ID NO:2; f) a polypeptide molecule comprising residues 164 to 696 of SEQ ID NO:2; g) a polypeptide molecule comprising residues 383 to 696 of SEQ ID NO:2; h) a polypeptide molecule comprising residues 164 to 449 of SEQ ID NO:2; i) a polypeptide molecule comprising residues 438 to 696 of SEQ ID NO:2; and 3) a polypeptide molecule comprising residues 1 to 696 of SEQ ID NO:2.
8. An isolated polynucleotide molecule encoding a polypeptide molecule, wherein the polypeptide molecule comprises a contiguous sequence of 14 amino acids of SEQ ID NO:2.
9. An isolated polynucleotide molecule according to claim 8, wherein the polypeptide molecule comprises - residues 437 to 450 of SEQ ID NO:2.
10. An isolated nucleotide molecule according to claim 8, wherein the polypeptide molecule is between 82 and 232 amino acids in length.
11. An isolated polynucleotide molecule according to claim 10, wherein the polypeptide molecule is residues 164 to 382 of SEQ ID NO:2.
12. An 1isclated polynucleotide molecule according to claim 10, wherein the polypeptide molecule is residues 383 } to 464 of SEQ ID NO:2.
13. An isolated polynucleotide molecule according to claim 10, wherein the polypeptide molecule is residues 465 to 696 of SEQ ID NO:2.
14. A isolated polynucleotide molecule encoding a polypeptide molecule, wherein the polypeptide molecule is selected from the group consisting of: a) a polypeptide molecule comprising residues 164 to 382 of SEQ ID NO:2; b) a polypeptide molecule comprising residues 383 to 464 of SEQ ID NO:2; c) a polypeptide molecule comprising residues 465 to 696 of SEQ ID NO:2; d) a polypeptide molecule comprising residues 438 to 449 of SEQ ID NO:2; e) a polypeptide molecule comprising residues 164 to 464 of SEQ ID NO:2; f) a polypeptide molecule comprising residues 164 to 696 of SEQ ID NO:2; g) a polypeptide molecule comprising residues 383 ’ to 696 of SEQ ID NO:2; h) a polypeptide molecule comprising residues 164 to 449 of SEQ ID NO:2: i) a polypeptide molecule comprising residues 438 to 696 of SEQ ID NO:2; and 3) a polypeptide molecule comprising residues 1 to 696 of SEQ ID NO:2.
15. An isolated polynucleotide encoding a fusion protein having a first segment and a second segment, wherein the first segment comprises a first polypeptide encoding a ] polypeptide having a protease domain and the second segment comprises a second polynucleotide encoding a polypeptide that has a contiguous sequence of 14 amino acids between residues 383 and 464 of SEQ ID NO:2, and wherein the first segment 1s positioned amino-terminally to the second segment.
16. An isolated polynucleotide according to claim 15, wherein the protease domain is selected from the group consisting of; a) a protease domain that is a member of the Disintegrin Proteases; and b) a protease domain that is at least 80% identical to amino acid residues 164 to 382 of SEQ ID NO:2.
17. An isolated polynucleotide molecule encoding a polypeptide molecule wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide molecule that encodes a polypeptide molecule that is at least 80 % identical to residues 383 to 464 of SEQ ID NO:2; and b) a polynucleotide molecule that is complementary to a).
18. An isolated polynucleotide molecule according to claim 17 wherein the polynucleotide molecule is selected from the group consisting of: a) a polynucleotide molecule that encodes ga polypeptide molecule that is at least 80 &% identical to residues 383 to 696 of SEQ ID NO:2; and b) a polynucleotide molecule that is complementary to a).
19. An isolated polynucleotide molecule according to claim 17, wherein the polynucleotide molecule is selected . from the group consisting of: a) a polynucleotide molecule that encodes a polypeptide molecule that is at least 80 % identical to residues 1 to 696 of SEQ ID NO:2; and b) a polynucleotide molecule that is complementary to a).
20. An expression vector comprising the following operably linked elements: a) a transcription promoter; b) a DNA segment encoding the polypeptide of claim 1; and c) a transcription terminator.
21. An expression vector of claim 20 wherein the DNA segment further encodes an affinity tag.
22. A cultured cell into which has been introduced an expression vector according to claim 21, wherein said cell expresses the polypeptide encoded by the DNA segment.
23. A method of producing a polypeptide comprising ) culturing a cell according to claim 22, whereby said cell expresses the polypeptide encoded by the DNA segment; and recovering the polypeptide.
24. A method for modulating cell-cell interactions by combining the polypeptide according to claim 1, with cells in vivo and in vitro.
25. A method for modulating cell-cell interactions according to claim 24, whereby the cells are derived from tissues selected from the group consisting of: a) tissues from heart; b) tissues from brain; c) tissues from spinal cord; and d) tissues from skeletal muscle.
26. An isolated polypeptide molecule comprising a contiguous sequence of amino acids, wherein the contiguous sequence of amino acids is selected from the group consisting of: a) SEQ ID NO:7; b) SEQ ID NO:8; © ¢c) SEQ ID NO:9; d) SEQ ID NO:10; and e) SEQ ID NO:11.
27. An isolated polynucleotide molecule encoding the isolated polypeptide molecule of claim 26.
28. An isolated polypeptide molecule substantially as described herein with reference to the accompanying figures.
29. An isolated polynucleotide molecule encoding a polypeptide molecule substantially as described herein with reference to the accompanying figures. AMENDED SHEET
30. An isolated polynucleotide encoding a fusion protein having a first segment and a second segment substantially as described herein with reference to the accompanying figures.
31. An expression vector substantially as described herein with reference to the accompanying drawings.
32. A cultured cell into which has been introduced an expression vector substantially as described herein with reference to the accompanying figures.
33. A method of producing a polypeptide substantially as described herein with reference to the accompanying figures.
34. A method for modulating cell-cell interactions substantially as described herein with reference to the accompanying figures.
35. An isolated polynucleotide molecule substantially as described herein with reference to the accompanying figures. AMENDED SHEET
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US11388398A | 1998-07-10 | 1998-07-10 |
Publications (1)
Publication Number | Publication Date |
---|---|
ZA200007766B true ZA200007766B (en) | 2001-12-21 |
Family
ID=22352096
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
ZA200007766A ZA200007766B (en) | 1998-07-10 | 2000-12-21 | Disintegrin homologs. |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP1144441A3 (en) |
JP (1) | JP2002526033A (en) |
AU (1) | AU4983399A (en) |
CA (1) | CA2332311A1 (en) |
IL (1) | IL140607A0 (en) |
WO (1) | WO2000002912A2 (en) |
ZA (1) | ZA200007766B (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
NZ506817A (en) | 1998-02-11 | 2003-08-29 | Immunex Corp | Metalloprotease-disintegrin SVPH3-13 and SVPH3-17 nucleic acids, and their corresponding polypeptides and methods for their production |
EP1268756A2 (en) * | 2000-04-03 | 2003-01-02 | Bristol-Myers Squibb Company | Methods and compositions for modulating integrin-mediated cell-cell interactions |
US20020081685A1 (en) * | 2000-08-03 | 2002-06-27 | Fox Brian A. | Disintegrin homologs, ZSNK10, ZSNK11, and ZSNK12 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9608130D0 (en) * | 1996-04-19 | 1996-06-26 | Univ Sheffield | Adam proteins and uses thereof |
JPH11155574A (en) * | 1997-12-01 | 1999-06-15 | Eisai Co Ltd | New protein belonging to mdc gene family and dna coding for the protein |
NZ506817A (en) * | 1998-02-11 | 2003-08-29 | Immunex Corp | Metalloprotease-disintegrin SVPH3-13 and SVPH3-17 nucleic acids, and their corresponding polypeptides and methods for their production |
-
1999
- 1999-07-09 JP JP2000559141A patent/JP2002526033A/en active Pending
- 1999-07-09 AU AU49833/99A patent/AU4983399A/en not_active Abandoned
- 1999-07-09 CA CA002332311A patent/CA2332311A1/en not_active Abandoned
- 1999-07-09 IL IL14060799A patent/IL140607A0/en unknown
- 1999-07-09 WO PCT/US1999/015638 patent/WO2000002912A2/en active Application Filing
- 1999-07-09 EP EP99933873A patent/EP1144441A3/en not_active Withdrawn
-
2000
- 2000-12-21 ZA ZA200007766A patent/ZA200007766B/en unknown
Also Published As
Publication number | Publication date |
---|---|
AU4983399A (en) | 2000-02-01 |
EP1144441A2 (en) | 2001-10-17 |
CA2332311A1 (en) | 2000-01-20 |
IL140607A0 (en) | 2002-02-10 |
JP2002526033A (en) | 2002-08-20 |
EP1144441A3 (en) | 2001-11-28 |
WO2000002912A2 (en) | 2000-01-20 |
WO2000002912A3 (en) | 2001-09-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20060024805A1 (en) | Disintegrin homologs | |
WO1999050415A2 (en) | Protease-activated receptor par4 (zchemr2) | |
US6762044B2 (en) | Mammalian adhesion protease peptides | |
EP1198560B1 (en) | Mammalian adhesion protease | |
AU7480198A (en) | Novel tumor antigens | |
ZA200007766B (en) | Disintegrin homologs. | |
US20020081685A1 (en) | Disintegrin homologs, ZSNK10, ZSNK11, and ZSNK12 | |
US20030153064A1 (en) | Disintegrin homologue, MAHBP | |
US20050239166A1 (en) | Polynucleotides encoding human secreted protein, ZZP1 | |
US20030096393A1 (en) | Disintegrin homolog, ZSNK16 | |
US20040259163A1 (en) | Antibodies to mammalian adhesion protease peptides | |
US6440697B1 (en) | Ring finger protein zapop3 | |
MXPA01000349A (en) | Disintegrin homologs | |
CA2379652A1 (en) | Tryptase-like polypeptide ztryp1 | |
US20020142439A1 (en) | Anti-angiogenic intestinal peptides, zdint5 | |
CA2368681A1 (en) | Anti-angiogenic intestinal peptides, zdint5 | |
AU1607800A (en) | Ring finger protein zapop3 | |
US20020042095A1 (en) | Secreted protein, ZSIG89 | |
JP2004500087A5 (en) | ||
AU2182900A (en) | Pancreatic polypeptide zsig66 | |
WO2000050594A2 (en) | Mammalian alpha-helical protein, zsig83 | |
WO2001042292A2 (en) | Secreted polypeptide zsig87 |