Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The glycoprotein Ib (GPIb), a two-chain integral platelet membrane protein, acts as a receptor for von Willebrand factor. In order to obtain information on the domain involved in this function, as well as on the structural organization of GPIb, the protein has been purified and submitted to limited proteolysis using three different enzymes. The resulting fragments were topographically oriented by means of partial NH2-terminal sequence analysis and immunological identification using monoclonal antibodies. One of these antibodies (LJ-Ib1) inhibited the von Willebrand factor-GPIb interaction completely, one (LJ-P3) partially, and one (LJ-Ib10) had no inhibitory effect. Three distinct fragments, the 38-kDa fragment produced by Serratia marcescens protease as well as the 45- and 35-kDa fragments produced by trypsin, had the same NH2 terminus as the intact GPIb alpha-chain (apparent molecular mass = 140 kDa). These fragments and the alpha-chain reacted with the inhibitory antibodies. On the other hand, three fragments produced by Staphylococcus aureus V8 protease, one of 92 kDa similar to the previously described "macroglycopeptide" and two others of 52 and 45 kDa, had NH2-terminal sequences different from that of the GPIb alpha-chain and reacted only with the noninhibitor monoclonal antibody LJIb10. Thus, the binding domain for von Willebrand factor resides near the NH2 terminus of the GPIb alpha-chain, whereas the carbohydrate-rich region is part of the innermost portion of GPIb and does not appear to be involved in the von Willebrand factor binding function.
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PMID:The von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. Characterization by monoclonal antibodies and partial amino acid sequence analysis of proteolytic fragments. 294 38

A large-scale method for the isolation of von Willebrand factor (vWF) from human factor VIII concentrates was developed in order to study the structure of this protein and its platelet binding activity. vWF is composed of a number of glycoprotein subunits that are linked together by disulfide bonds to form a series of multimers. These multimers appear to contain an even number of subunits of 270K. Two minor components of Mr 140K and 120K were also identified, but these chains appear to result from minor proteolysis. The smallest multimer of vWF contained nearly equimolar amounts of the 270K, 140K, and 120K subunits, while the largest multimers contained less than 20% of the two minor components. Amino acid sequence analysis, amino acid composition, and cleavage by cyanogen bromide indicate that the 270K subunits are identical and each is a single polypeptide chain with an amino-terminal sequence of Ser-Leu-Ser-Cys-Arg-Pro-Pro-Met-Val-Lys and a carboxyl-terminal sequence of Glu-Cys-Lys-Cys-Ser-Pro-Arg-Lys-Cys-Ser-Lys. Platelet binding in the presence of ristocetin was 8-fold greater with multimers larger than five (i.e., containing more than 10 subunits of 270K) as compared to multimers less than three (containing less than six subunits of 270K). However, partially reduced vWF (Mr 500K), regardless of whether it was prepared from large or small molecular weight multimers, gave platelet binding similar to that of the smallest multimers. Likewise, partial proteolysis by elastase, thermolysin, trypsin, or chymotrypsin produced small "multimer-like" proteins with platelet binding properties similar to either partially reduced vWF or to the smallest multimers. We conclude that human vWF contains identical 270K subunits assembled into a multivalent structure. Disassembly by either partial reduction or partial proteolysis produces essentially monovalent protein with platelet binding properties similar to that of the smallest multimers. Multivalency is likely the primary factor responsible for the increase in biological activity with multimer size.
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PMID:Human von Willebrand factor: a multivalent protein composed of identical subunits. 301 99

Endothelium provides a specific binding site for Factor IX/IXa which can propagate activation of coagulation by promoting Factor IXa-VIII-mediated activation of Factor X. In this report the endothelial cell Factor IX/IXa binding site has been identified and the coagulant function of the receptor blocked. Studies using [3H]Factor IX derivatized with the photoaffinity labeling agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH) and cultured bovine endothelial cells demonstrated cross-linking to a trypsin-sensitive cell surface protein of Mr approximately equal to 140,000. Immunoprecipitation of metabolically labeled endothelium with Factor IX derivatized with the cleavable cross-linking agent N-succinimidyl(4-azidophenyl)-1,3'-dithiopropionate and antibody to Factor IX demonstrated the endothelial cell origin of the Mr 140,000 cell surface protein. Blockade of the Factor IX/IXa binding protein by covalently linking SANPAH-5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa or SANPAH-Factor IX prevented both specific Factor IXa binding and effective Factor IXa-VIII-mediated activation of Factor X on endothelium. Following extraction of endothelium with detergents, Factor IX/IXa binding activity was solubilized and could be assayed using a polyvinyl chloride plate binding assay. Western blots of cell extracts demonstrated binding of 125I-Factor IX at Mr approximately equal to 140,000 which was blocked by excess Factor IX, but not antisera to Factor VIII, von Willebrand factor, alpha 2-macroglobulin, or epidermal growth factor receptor. These data indicate that endothelium provides a distinct binding site for Factor IX/IXa consisting, at least in part, of a membrane protein which can modulate the coagulant activity of Factor IXa on the cell surface.
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PMID:Identification of a factor IX/IXa binding protein on the endothelial cell surface. 303 54

In the present report we describe the isolation of a functional domain of platelet membrane glycoprotein (GP) Ib which retains von Willebrand factor (vWF)-binding activity. Glycocalicin, a proteolytic fragment of the alpha-chain of GP Ib generated by an endogenous calcium-activated protease, was submitted to digestion with trypsin. The two resulting fragments, one of 45 kDa extending between residues His1 and Arg293 and representing the amino terminus of the alpha-chain, the other of 84 kDa corresponding to the previously described macroglycopeptide, were purified to homogeneity. Glycocalicin, as well as the 45- and 84-kDa fragments, inhibited the ristocetin-dependent binding of native vWF to platelet GP Ib. The concentration inhibiting 50% of binding (IC50) was between 1 and 5 microM with all these molecules. In contrast, the binding of asialo-vWF to platelet GP Ib, measured directly in the absence of ristocetin, was blocked by glycocalicin and the 45-kDa fragment with a similar IC50, but not by the 84-kDa fragment. Both glycocalicin and the 45-kDa fragment bound to purified surface-bound vWF in a ristocetin-dependent manner and with similar affinities. Monoclonal antibodies against vWF or GP Ib inhibited this interaction in a way consistent with their inhibition of vWF binding to platelet GP Ib. These studies demonstrate that the amino-terminal extracytoplasmic region of the alpha-chain, extending between residues 1 and 293, contains a functional domain that interacts with vWF in the absence of any other structure of the GP Ib complex or any other platelet membrane component. Whereas the ristocetin-dependent binding of vWF may involve also other domains in the macroglycopeptide region, the direct vWF-GP Ib interaction appears to be mediated only by a domain in the amino-terminal region of GP Ib alpha.
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PMID:Isolation and functional characterization of the von Willebrand factor-binding domain located between residues His1-Arg293 of the alpha-chain of glycoprotein Ib. 326 68

Glycoprotein Ib is a surface membrane glycoprotein of platelets that functions as a receptor for von Willebrand factor. It is a heterodimer composed of an alpha and a beta chain linked by a disulfide bond(s). A phage lambda gt11 cDNA expression library prepared from mRNA from a human erythroleukemia cell line, HEL, was screened using an affinity-purified antibody to the glycocalicin portion of the alpha chain of glycoprotein Ib. Eleven positive clones were isolated and plaque-purified. The largest cDNA insert was 2420 nucleotides in length and coded for a leader sequence of 16 amino acids, a mature protein of 610 amino acids, and a stop codon. It also contained 42 nucleotides of 5' noncoding sequence and 497 nucleotides of 3' noncoding sequence, including a poly(A) tail. The amino acid sequence of the alpha chain of GPIb predicted from the cDNA agreed completely with the sequence of 156 amino acids that was determined by Edman degradation of peptides isolated from human platelet glycocalicin after digestion with trypsin or Staphylococcus aureus V8 protease. The extracytoplasmic domain of the alpha subunit of GPIb contains several noteworthy structural features, including a region of seven tandem repeats of 24 amino acids that are homologous with those present in leucine-rich alpha 2-glycoprotein. The extracytoplasmic domain also contains two hydrophilic regions, one rich in charged amino acids and a second rich in serine and threonine residues. The region rich in serine and threonine includes five repeats of nine amino acids as well as the majority of the O-linked carbohydrate sites present in the molecule. The extracytoplasmic domain is followed by a potential transmembrane segment of approximately 29 amino acids and a potential intracellular domain of approximately 100 amino acids located at the carboxyl end of the molecule.
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PMID:Cloning of the alpha chain of human platelet glycoprotein Ib: a transmembrane protein with homology to leucine-rich alpha 2-glycoprotein. 330 30

We report the amino acid sequence of a 299-residue segment from the alpha chain of the human platelet membrane glycoprotein Ib. This includes the complete sequence of the amino-terminal tryptic fragment of 290 residues comprising the von Willebrand factor-binding domain. Two primary sets of overlapping fragments were obtained by cleavage of the S-carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively. Additional fragments were obtained by treatment of native glycocalicin with trypsin, Staphylococcus aureus V8 protease, and Serratia marcescens protease. Analysis of all these fragments provided data that allowed determination of the continuous sequence corresponding to approximately half of the alpha-chain polypeptide. This region of glycoprotein Ib is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains. A hydrophilic region exists between residues 215 and 299, which contains a cluster of 10 negatively charged residues at 269-287. This area is likely to attract positively charged molecules. The hydrophilic, highly glycosylated (at serine and threonine residues) region corresponding to the previously described "macroglycopeptide" and representing the carboxyl-terminal half of the alpha chain is likely to begin at residue 292. The determined sequence of the alpha chain of glycoprotein Ib contains a region (residues 29-193) with seven repeats, which is indicative of gene duplication and is highly homologous to human leucine-rich alpha 2-glycoprotein. This protein sequence agrees completely with that deduced from the cDNA sequence reported by Lopez et al. [Lopez, J.A., Chung, D.W., Fujikawa, K., Hagen, F.S., Papayannopoulou, T. & Roth, G.J. (1987) Proc. Natl. Acad. Sci. USA 84, 5615-5619].
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PMID:Amino acid sequence of the von Willebrand factor-binding domain of platelet membrane glycoprotein Ib. 349 98

We have identified four discrete proteolytic fragments of von Willebrand factor (vWF) that define two collagen-binding domains. Two of the fragments tested, T 96 kDa and T 55 kDa, were generated by digestion with trypsin, and two, Fragments I and III, with Staphylococcus aureus V8 protease. The larger Fragment III, a disulfide-linked homodimer, extends between residues 1 and 1365 of the 2050-residue vWF subunit and comprises the sequence of all the others. T 96 kDa, also a disulfide-linked homodimer, extends between residues 449 and 728. T 55 kDa and Fragment I, both single-chain polypeptides, have a partial sequence overlap corresponding to residues 911-1114, and together extend from residue 730 to 1365. The ability of the fragments to interfere with the vWF-collagen interaction was evaluated by measuring inhibition of 125I-labeled vWF binding to fibrillar bovine collagen types I and III. All the four fragments tested inhibited binding. Native conformation was essential for expression of this function; denaturation with guanidine hydrochloride and reduction of disulfide bonds resulted in marked reduction or complete loss, respectively, of the inhibitory activity at all the concentrations tested. Two monoclonal antibodies were prepared, one directed against T 96 kDa and the other against Fragment I. Both antibodies partially inhibited vWF binding to collagen, and their inhibitory effect was enhanced when they were used together. 125I-Labeled Fragment I bound to collagen in a saturable manner, and the binding was completely blocked by both T 96 kDa and T 55 kDa. Thus, we have identified at least two distinct functional domains of vWF that concurrently mediate the vWF-collagen interaction. The two domains appear to share a common recognition site on collagen.
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PMID:Isolation and characterization of two domains of human von Willebrand factor that interact with fibrillar collagen types I and III. 349 19

Previously we have studied the binding domains on von Willebrand factor (vWF) involved in ristocetin-induced binding to platelets (ristocetin binding domain, RBD) and in the binding of vWF to collagen (collagen binding domain, CBD) using tryptic fragments of 125I-labelled vWF (21, 23). We have also reported on the RBD, CBD and the domain on vWF involved in the binding to thrombin activated platelets (thrombin binding domain, TBD) using vWF-fragments prepared by digestion with staphylococcal protease V8 (25). In the present study, we have digested 125I-vWF with TPCK-trypsin and we have performed at various times of digestion immuno-precipitation with Mab 9, the antibody inhibiting binding of vWF to thrombin activated platelets. The data were compared with the immunoprecipitation patterns simultaneously obtained with CLB-RAg 35 which inhibits binding of vWF in the presence of ristocetin and with CLB-RAg 201, which inhibits binding of vWF to collagen. At 90 min, Mab 9 and CLB-RAg 201 precipitated similar high molecular weight bands, whereas CLB-RAg 35 precipitated bands at 180 and 120 kDa. After 24 h, Mab 9 precipitated bands at 200, 155, 116 and 85 kDa; CLB-RAg 201 precipitated a band at 48 kDa and CLB-RAg 35 a band at 116 kDa. Two-dimensional electrophoresis demonstrated that the high molecular weight bands, precipitated by Mab 9 and CLB-RAg 201 at 90 min, were identical. The 116 kDa fragment recognized by CLB-RAg 35 had a different subunit composition than the 116 kDa fragment precipitated by Mab 9.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of tryptic fragments of von Willebrand factor involved in binding to thrombin-activated platelets with fragments involved in ristocetin-induced binding and binding to collagen. 355 Nov 82

The basic structure of platelet membrane glycoprotein I (GPI) and its relation to glycocalicin are now well understood. Glycocalicin is a proteolytic fragment produced by the action of an endogenous Ca2+ activated protease. GPI consists of two glycopeptides, an alpha and a beta chain connected by a disulphide bridge. Glycocalicin is the major part of the GPI alpha chain and can be split by trypsin into a heavily glycosylated trypsin-resistant fragment and a peptide containing at least one intramolecular disulphide bridge and a thrombin binding site. Both the alpha and the beta chains of GPI show hydrophobic properties and are probably integral membrane proteins. The position of the von Willebrand factor binding site within the GPI molecule is still controversial but the bulk of the evidence points to it lying within the non-glycosylated part of the glycocalicin fragment. It is however evident that the GPI beta chain may influence the GPI alpha chain in maintaining the correct conformation of the binding site. The von Willebrand factor binding site and the thrombin binding site appear to be independent but may nevertheless influence one another.
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PMID:Platelet membrane glycoprotein I: structure and function. The domain of glycoprotein I involved in the von Willebrand receptor. 622

We have identified two functional domains on the von Willebrand factor (VWF) moiety of the Factor VIII-von Willebrand factor complex (FVIII-VWF), one interacting with blood platelets, and one interacting with vessel wall collagens, by means of two monoclonal antibodies directed against the VWF molecule, CLB-RAg 35 and CLB-RAg 201. The monoclonal antibody CLB-RAg 35 inhibited virtually all platelet adherence to artery subendothelium and to purified vessel wall collagen type III, at relatively high wall shear rates. CLB-RAg 35 also inhibited the ristocetin-induced platelet aggregation and the binding of FVIII-VWF to the platelet in the presence of ristocetin but did not affect the binding of FVIII-VWF to collagen. The monoclonal antibody CLB-RAg 201 inhibited the binding of FVIII-VWF to purified vessel wall collagen type I and III and all platelet adherence to collagen type III and the platelet adherence to subendothelium that was mediated by FVIII-VWF in plasma. The two functional domains on FVIII-VWF that were recognized by CLB-RAg 35 and CLB-RAg 201 were identified by means of immunoprecipitation studies of trypsin-digested FVIII-VWF. The domains resided on different polypeptide fragments, with a Mr of 48,000 for the collagen binding domain and a Mr of 116,000 for the platelet binding domain. The 116,000-mol wt fragment consisted of subunits of 52,000/56,000 mol wt and 14,000 mol wt after reduction. The 52,000/56,000-mol wt subunits possessed the epitope for CLB-RAg 35.
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PMID:Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen. 633 19


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