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)

A new ligand for syndecan (a cell surface heparan sulfate-rich proteoglycan) has been discovered. In the solid-phase binding assay utilizing small nitrocellulose discs to immobilize matrix molecules, binding of syndecan to neurite growth-promoting protein, p30/amphoterin, was observed. This binding was strongly dependent on the concentration of amphoterin used to coat the discs, but was saturable with an excess amount of syndecan. The interaction was inhibitable with heparan sulfate and heparin but less effectively with chondroitin sulfate, indicating that heparan sulfate chains of syndecan were involved in the binding. Anti-amphoterin antibodies inhibited the binding partially. Mouse mammary epithelial cells were shown to bind amphoterin directly but not after trypsin treatment or in the presence of heparin and to produce amphoterin in the extracellular space. Both syndecan and amphoterin were found to localize on lateral surfaces of newly adhered mammary epithelial cells. Toward confluency amphoterin amounts decreased. Because amphoterin can be localized to the same sites with syndecan and because of their interaction, amphoterin is a new putative pericellular ligand for syndecan. These interactions may be involved in the regulation of cell behavior.
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PMID:Neurite growth-promoting protein (amphoterin, p30) binds syndecan. 136 84

A simple panning procedure that allows for the evaluation of interactions between various heparin-like molecules and basic FGF has been developed. This assay measures the ability of compounds to inhibit the interaction of transfected human lymphoblastoid cells, UC 729-6 (UC cells), expressing hamster syndecan and basic FGF-coated plastic plates. The transfected cells bind rapidly to basic FGF-coated plates while the control cells do not bind well. Binding of the transfected cells to basic FGF was inhibited by heparin and heparin sulfate (HS), but not by chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid. There was little inhibition of binding by chemically modified heparin such as completely desulfated, N-acetylated heparin, completely desulfated, N-sulfated heparin, and N-desulfated, N-acetylated heparin. These results suggested that both the N-sulfate and O-sulfate groups of heparin are required for binding to basic FGF. In addition, inhibition by oligosaccharides derived from depolymerized heparin increased with fragment size; partial inhibition was observed with oligosaccharides as small as hexamers. The biochemical basis for the binding of transfected cells to basic FGF was established by showing a significant increase of 35SO4 incorporation into HS. In particular, the level of 35SO4-HS in the trypsin-releasable (cell surface) pool increased fivefold. This increase was accounted for by demonstration of the presence of HS on immunoprecipitated syndecan from the transfected cells.
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PMID:A cell-based assay for evaluating the interaction of heparin-like molecules and basic fibroblast growth factor. 151 58

Proteoglycan accumulation by thioglycollate-elicited mouse peritoneal macrophages and a panel of murine monocyte-macrophage cell lines has been examined to determine whether these cells express plasma membrane-anchored heparan sulfate proteoglycans. Initially, cells were screened for heparan sulfate and chondroitin sulfate glycosaminoglycans after metabolic labeling with radiosulfate. Chondroitin sulfate is secreted to a variable extent by every cell type examined. In contrast, heparan sulfate is all but absent from immature pre-monocytes and is associated predominantly with the cell layer of mature macrophage-like cells. In the P388D1 cell line, the cell-associated chondroitin sulfate is largely present as a plasma membrane-anchored proteoglycan containing a 55 kD core protein moiety, which appears to be unique. In contrast, the cell-associated heparan sulfate is composed of a proteoglycan fraction and protein-free glycosaminoglycan chains, which accumulate intracellularly. A fraction of the heparan sulfate proteoglycan contains a lipophilic domain and can be released from cells following mild treatment with trypsin, suggesting that it is anchored in the plasma membrane. Isolation of this proteoglycan indicates that it is likely syndecan-4: it is expressed as a heparan sulfate proteoglycan at the cell surface, it is cleaved from the plasma membrane by low concentrations of trypsin, and it consists of a single 37 kD core protein moiety that co-migrates with syndecan-4 isolated from NMuMG mouse mammary epithelial cells. Northern analysis reveals that a panel of macrophage-like cell lines accumulate similar amounts of syndecan-4 mRNA, demonstrating that this proteoglycan is expressed by a variety of mature macrophage-like cells. Syndecan-1 mRNA is present only in a subset of these cells, suggesting that the expression of this heparan sulfate proteoglycan may be more highly regulated by these cells.
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PMID:Membrane-anchored proteoglycans of mouse macrophages: P388D1 cells express a syndecan-4-like heparan sulfate proteoglycan and a distinct chondroitin sulfate form. 822 71

We previously reported that a mouse Lewis lung carcinoma-derived stroma-inducing clone, P29, highly expresses a syndecan-like proteoglycan exhibiting specific binding to fibronectin, a major constituent of the interstitial matrix formed by the induced stromal cells, via its heparan sulphate chains [Itano, Oguri, Nakanishi and Okayama (1993) J. Biochem. (Tokyo) 114, 862-873]. On metabolic labelling of the proteoglycan with [32P]Pi, followed by identification of the radiolabelled material using glycanases, almost all the isotope was found to have been incorporated into a core portion of molecular mass 48 kDa, which was generated by digestion with heparan sulphate lyase I plus chondroitin ABC lyase. Immunoblotting of the core protein with a monoclonal antibody, F58-6G12, demonstrated that the proteoglycan was mouse syndecan-2. CsCl-density-gradient centrifugation after mild treatment of liposome-intercalated 32P-labelled syndecan-2 with trypsin resulted in clear separation of the radioactivity into a bottom fraction containing all the glycosaminoglycans (accounting for 40% of the total radioactivity) and a top fraction containing liposome-associated peptides (60%). The former isotope was shown to be linked covalently to both heparan sulphate and chondroitin sulphate chains, probably at their bridge regions. The latter was mostly attributed to phosphoserine, the one and only phosphorylated amino acid released on acid hydrolysis of this proteoglycan, strongly suggesting that the phosphorylation occurs at a specific serine residue(s) in the cytoplasmic domain of the core protein.
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PMID:Phosphorylation of a membrane-intercalated proteoglycan, syndecan-2, expressed in a stroma-inducing clone from a mouse Lewis lung carcinoma. 864 78

Heparan sulfate (HS) chains accumulate in both the medium and the cell layer of mesangial cell cultures. When given in fresh medium to quiescent cultures at naturally occurring concentrations, they suppress entry into the cell cycle and progression to DNA synthesis. We have attempted to identify the proteoglycan (PG) source of the antimitogenic HS chains from mesangial cell layers (HS(c)) and medium (HS(c)). When cells were labeled for 16 hours with [35S]sulfate, 25% of the label was found in intracellular HS chains and 5% in extracellular HSPGs. Cell-surface HSPGs accounted for the remaining 70% of the label associated with cell-layer HS and were released by either trypsin or 2% Triton X-100. About 20% of this cell-surface fraction was released by treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), and probably represents glypican-like PG; glypican mRNA was present in the cells. The remainder of this fraction could be incorporated into liposomes, indicating the presence of hydrophobic transmembrane regions suggestive of syndecans. Upon purification and deglycosylation, an antiserum to rat liver HSPGs that reacts primarily with syndecan-2 showed a strong signal corresponding to this protein and three weaker bands that may represent additional syndecans. mRNAs for syndecan-1, -2, and -4 were present in the cultures. Syndecan-1 and -2 mRNAs were increased 30 minutes after stimulation of quiescent rat mesangial cells (RMCs) with serum. Heparin, HS(c), and HS(m) all prevented this increase. Syndecan-4 mRNA was not affected by serum, heparin, or HS. In pulse-chase experiments, the amount of 35S appearing in the cellular protein-free HS fraction was accounted for almost entirely by cell-surface PGs, as matrix-associated label was a minor contribution at the end of the pulse-labeling. The appearance of [35S]HS in cell extracts was unaffected by phospholipase C treatment, indicating that turnover of the newly labeled syndecan fraction is the source of the antimitogenic HS chains.
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PMID:Heparan sulfate chains with antimitogenic properties arise from mesangial cell-surface proteoglycans. 1053 82

Kininogens, the high molecular weight precursor of vasoactive kinins, bind to a wide variety of cells in a specific, reversible, and saturable manner. The cell docking sites have been mapped to domains D3 and D5(H) of kininogens; however, the corresponding cellular acceptor sites are not fully established. To characterize the major cell binding sites for kininogens exposed by the endothelial cell line EA.hy926, we digested intact cells with trypsin and other proteases and found a time- and concentration-dependent loss of (125)I-labeled high molecular weight kininogen (H-kininogen) binding capacity (up to 82%), indicating that proteins are crucially involved in kininogen cell attachment. Cell surface digestion with heparinases similarly reduced kininogen binding capacity (up to 78%), and the combined action of heparinases and trypsin almost eliminated kininogen binding (up to 85%), suggesting that proteoglycans of the heparan sulfate type are intimately involved. Consistently, inhibitors such as p-nitrophenyl-beta-d-xylopyranoside and chlorate interfering with heparan sulfate proteoglycan biosynthesis reduced the total number of kininogen binding sites in a time- and concentration-dependent manner (up to 67%). In vitro binding studies demonstrated that biotinylated H-kininogen binds to heparan sulfate glycosaminoglycans via domains D3 and D5(H) and that the presence of Zn(2+) promotes this association. Cloning and over-expression of the major endothelial heparan sulfate-type proteoglycans syndecan-1, syndecan-2, syndecan-4, and glypican in HEK293t cells significantly increased total heparan sulfate at the cell surface and thus the number of kininogen binding sites (up to 3. 3-fold). This gain in kininogen binding capacity was completely abolished by treating transfected cells with heparinases. We conclude that heparan sulfate proteoglycans on the surface of endothelial cells provide a platform for the local accumulation of kininogens on the vascular lining. This accumulation may allow the circumscribed release of short-lived kinins from their precursor molecules in close proximity to their sites of action.
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PMID:High molecular weight kininogen utilizes heparan sulfate proteoglycans for accumulation on endothelial cells. 1084 88

(35)S-Radiolabeled cultured Sertoli cells from immature rat testis were extracted with detergent and the different proteoheparan sulfate (HSPG) forms of the extract were discriminated and quantified on the basis of their high anionic charge, hydrodynamic size, lipophilic properties, susceptibility to trypsin and phosphatidylinositol phospholipase C (PI-PLC). Trypsin released 50% of total cellular HSPG corresponding to 80% of total hydrophobic HSPG. Trypsin-accessible HSPG were presumed to be integral membrane species. Trypsin-resistant HSPG, probably intracellular, distributed into non-lipophilic (37.5%) and lipophilic (12.5%) populations. Biochemical analysis of PG copurified with plasma membrane confirmed the existence of hydrophobic HSPG integrated into this structure. Among hydrophobic HSPG accessible to trypsin, 35% were PI-PLC released and radiolabeled by [(3)H]inositol indicating that about one third of integral membrane HSPG were intercalated into the plasma membrane through a phosphatidylinositol anchor (glypican type). PI-PLC-resistant forms represented HSPG inserted into the membrane through a hydrophobic segment of the core protein (syndecan type). No lipophilic PG was present in other cell compartments (culture medium, cell periphery, extracellular matrix). (125)I-Iodinated hydrophobic HSPG were deglycanated and submitted to SDS-polyacrylamide gel electrophoresis. In the glypican family, a core protein (64--65 kDa) was detected, whereas in the syndecan family, bands of 60 and 68 kDa were observed which may correspond to self-association of different core proteins. In Sertoli cell, specific functional attributes of different integral membrane HSPG forms remain to be investigated.
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PMID:Biochemical characterization of integral membrane heparan sulfate proteoglycans in Sertoli cells from immature rat testis. 1134 81

Insulin-like growth factor (IGF) binding protein-related protein-1 (IGFBP-rP1) was previously identified as tumor-derived adhesion factor (TAF) secreted from human bladder carcinoma cells. It exhibits growth-stimulatory activity in synergy with insulin or IGFs. In the present study, we found that IGFBP-rP1 was proteolytically cleaved to a two-chain form. The cleavage sequence suggested that a trypsin-like serine proteinase may be responsible for the processing. The cleavage of IGFBP-rP1 led to an almost complete loss of both insulin/IGF-1-binding activity and insulin/IGF-1-dependent growth-stimulatory activity. On the other hand, the cell attachment activity of IGFBP-rP1 was markedly increased by the proteolytic processing. Syndecan-1 was thought to be a cell surface receptor for both intact and cleaved IGFBP-rP1 forms. Although the proteolytic cleavage of IGFBP-rP1 decreased its heparin-binding activity, the cleaved form could bind syndecan-1 efficiently. Thus the proteolytic processing of IGFBP-rP1 seems to modulate its insulin/IGF-dependent and -independent biological functions.
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PMID:Proteolytic processing of IGFBP-related protein-1 (TAF/angiomodulin/mac25) modulates its biological activity. 1452 55

Shedding (i.e. proteolysis of ectodomains of membrane proteins) plays an important pathophysiological role. In order to study the feasibility of identifying shed proteins, we analyzed serum-free media of human mammary epithelial cells by mass spectrometry following induction of shedding by the phorbol ester, 4 beta-phorbol 12-myristate 13-acetate (PMA). Different means of sample preparation, including biotinylation of cell surface proteins, isolation of glycosylated proteins, and preparation of crude protein fractions, were carried out to develop the optimal method of sample processing. The collected proteins were digested with trypsin and analyzed by reversed-phase capillary liquid chromatography interfaced to an ion-trap mass spectrometer. The resulting peptide spectra were interpreted using the program SEQUEST. Analyzing the sample containing the crude protein mixture without chemical modification or separation resulted in the greatest number of identifications, including putatively shed proteins. Overall, 45 membrane-associated proteins were identified including 22 that contain at least one transmembrane domain and 23 that indirectly associate with the extracellular surface of the plasma membrane. Of the 22 transmembrane proteins, 18 were identified by extracellular peptides providing strong evidence they originate from regulated proteolysis or shedding processes. We combined results from the different experiments and used a peptide count method to estimate changes in protein abundance. Using this approach, we identified two proteins, syndecan-4 and hepatoma-derived growth factor, whose abundances increased in media of cells treated with PMA. We also detected proteins whose abundances decreased after PMA treatment such as 78 kDa glucose-regulated protein and lactate dehydrogenase A. Further analysis using immunoblotting validated the abundance changes for syndecan-4 and 78 kDa glucose-regulated protein as a result of PMA treatment. These results demonstrate that tandem mass spectrometry can be used to identify shed proteins and to estimate changes in protein abundance.
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PMID:A proteomic approach to characterize protein shedding. 1567 59

Cell surface proteoglycans play an important part in the functional and metabolic behaviour of leucocytes. We studied the expression of cell surface proteoglycans in human monocytes, in monocyte-derived immature and mature dendritic cells and in macrophages by metabolic labelling with [(35)S]-sulphate, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. Immature dendritic cells had the highest metabolic activity for the synthesis of cell surface proteoglycans. The major part of these proteoglycans was in phosphatidylinositol-anchored form and was released after treatment with phospholipase C. A minor part was released by trypsin. Digestion with chondroitinase ABC and mild HNO(2) treatment showed that cell surface proteoglycans had a higher proportion of chondroitin sulphate, both in the phospholipase C and trypsin fractions, suggesting that at least some glypicans contained chondroitin sulphate chains. RT-PCR detected the transcripts of glypicans 1, 3, 4 and 5 and all syndecans. Immature dendritic cells expressed a most complex spectrum of glypicans and syndecans, glypican-1 and syndecan-1 being expressed preferentially by this type of cells. Mature dendritic cells expressed glypican-3, which was not present in other lineages. These results suggest that different mononuclear cells synthesize cell surface proteoglycans actively with characteristic expression of different syndecans and glypicans genes, depending on the degree of cell differentiation and/or maturation.
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PMID:Cell surface proteoglycan expression during maturation of human monocytes-derived dendritic cells and macrophages. 1673 18


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