Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recombinant human Protein C (rHPC), expressed in human kidney 293 cells, has a higher anticoagulant activity than plasma HPC, while its in vivo circulatory half-life is essentially unaltered compared to that of the natural protein. In seeking to elucidate the molecular basis for the improved efficacy of the recombinant antithrombotic drug, we focused on the carbohydrate moiety of rHPC. Protein C is a heavily post-translationally modified serine protease with four N-glycosylation sites. Glycosyl composition analysis of rHPC revealed a 5-fold higher fucose content and a 2-fold lower sialic acid content compared to plasma HPC. In addition, we found that rHPC contains N-acetylgalactosamine (2.6 mol GalNAc/mol rHPC) in its Asn-linked oligosaccharides, while plasma HPC is devoid of GalNAc. The Asn-linked oligosaccharides of rHPC were released by N-glycanase and separated into 25 fractions by high-pH anion-exchange chromatography. The most abundant oligosaccharides were structurally characterized by glycosyl composition and linkage analysis, in conjunction with 1H-NMR spectroscopy at 600 MHz. The structure of the major neutral oligosaccharide in rHPC was determined to be: [formula: see text] Two representatives of the sialylated oligosaccharides in rHPC are: [formula: see text] and [formula: see text] Thus, many of the Asn-linked oligosaccharides in rHPC were found to terminate in GalNAc beta (1-->4)GlcNAc beta (1-->.), in NeuAc alpha (2-->6)GalNAc beta (1-->4)GlcNAc beta (1-->.), and/or in GalNAc beta (1-->4)[Fuc alpha (1-->3)]GlcNAc beta (1-->.). Since the latter trisaccharide was first [Yan, S.B., Chao, B.Y. and Van Halbeek,H. (1992) J. Cell. Biochem., 16D, 151] observed in the Asn-linked oligosaccharides of rHPC derived from human kidney 293 cells, we propose to label the GalNAc beta-(1-->4)[Fuc alpha (1-->3)]GlcNAc beta (1-->.) terminal trisaccharide the PC-293 determinant. The PC-293-containing oligosaccharides may contribute to the higher anticoagulant activity of rHPC as compared to plasma HPC.
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PMID:Novel Asn-linked oligosaccharides terminating in GalNAc beta (1-->4)[Fuc alpha (1-->3)]GlcNAc beta (1-->.) are present in recombinant human protein C expressed in human kidney 293 cells. 813 Mar 92

Ecamulin, a novel prothrombin activating enzyme, has been isolated and purified 63-fold with a 57% yield from the venom of the Middle-Asian sand viper Echis multisquamatus using three-step ion-exchange chromatography. The enzyme was shown to activate prothrombin similarly to Ecarin, a prothrombin-converting enzyme from Echis carinatus venom, however, differing from the latter by structural and physico-chemical properties. The enzyme is a Zn-proteinase: it contains 1 mol Zn per 1 mol of protein. The molecular mass of the enzyme as determined by Sephacryl S-200 chromatography is 93 +/- 2 kDa. Upon SDS-PAAG electrophoresis ecamulin produces two bands with Mr of 67 and 27 kDa under non-reducing conditions, and three bands with Mr of 67, 14 and 13 kDa in the presence of DTT. During native PAGE without SDS, the activator yields one slow mobility band: two bands are observed after addition of DTT or EDTA. Carbohydrates containing N-acetyl-alpha-D-glucosamine residues are localized in the 67 kDa chain. Ecamulin has two isoforms, S2 and S3, that are distinguished by the charge and partial coagulation activities: form S2 has 250 NIH units/mg, while the S3 form has 524 NIH units/mg. The amino acid sequences of the both isoforms are similar but the more active S3 form has 4 times higher content of Gln and 4 times less of Gly than the S2 form. The isoelectric point is 4.3-4.5; E280 of 1% solution is 10.2. Forms S2 and S3 of ecamulin hydrolyze chromogenic substrates of plasma kallikrein S2302 and glandular kallikrein 2266. Ecamulin does not hydrolyze BAEE, TAME, LEE, thrombin substrates Chromozym TH and S2160, factor Xa-S2222, protein Ca-Chromozym PCa and Plasmin S2251. The amidase activity is nonreversibly inhibited by EDTA, o-phenanthroline (the activity is recovered by addition of Zn2+), Cys or DTT, EGTA, DFP, PMSF or pCMB do not inhibit the enzyme activity. Ecamulin converts prothrombin to alpha-thrombin passing by a shunt via the meizothrombin stage. The reaction of prothrombin activation does not require Ca2+, phospholipids of factor Va. Part of this work was presented at the International Conference "Fibrinogen and fibrinolysis", Yalta, September 23-28, 1995.
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PMID:[Isolation and characteristics of ekamulin--a prothrombin activator from multiscaled viper (Echis multisquamatus) venom]. 901 Dec 45

A T cell hybridoma raised against the synthetic glycopeptide T(72)(Tn) was used to study whether the initial TCR signaling events are markedly different when the hybridoma is stimulated with glycopeptides closely related to the cognate glycopeptide antigen. T(72)(Tn) has an alpha-D-GalNAc group O-linked to the central threonine in the decapeptide VITAFTEGLK, and the hybridoma is known to be highly specific for this carbohydrate group. T(72)(Tn)-pulsed APC induced tyrosine phosphorylation of the TCR-zeta 21- and 23-kDa proteins and the downstream p42/44 MAP kinase and strong IL-2 secretion. APC pulsed with T(72)(alpha-D-GlcNAc), which differs from T(72)(Tn) solely by the orientation of a hydroxy group in the carbohydrate structure, completely failed to induce detectable tyrosine phosphorylation and IL-2 secretion. APC pulsed with S(72)(Tn), which differs from T(72)(Tn) by not having a methyl group in the serine amino acid side chain to which the glycan is attached, induced partial tyrosine phosphorylation of the TCR-zeta 21-kDa protein, no tyrosine phosphorylation of the MAP kinases and no IL-2 production. Molecular modeling of the MHC/glycopeptide complex revealed that the dramatic difference between the stimulatory power of T(72)(Tn) and T(72)(alpha-D-GlcNAc) is mainly due to very small differences in the TCR exposed carbohydrate structure.
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PMID:Radically altered T cell receptor signaling in glycopeptide-specific T cell hybridoma induced by antigen with minimal differences in the glycan group. 1174 36

Platelet- and plasma-derived factor Va (FVa) serve essential cofactor roles in prothrombinase-catalyzed thrombin generation. Platelet-derived FV/Va, purified from Triton X-100 platelet lysates was composed of a mixture of polypeptides ranging from approximately 40 to 330 kDa, mimicking those visualized by Western blotting of platelet lysates and releasates with anti-FV antibodies. The purified, platelet-derived protein expressed significant cofactor activity such that thrombin activation led to only a 2-3-fold increase in cofactor activity yet expression of a specific activity identical to that of purified, plasma-derived FVa. Physical and functional differences between the two cofactors were identified. Purified, platelet-derived FVa was 2-3-fold more resistant to activated protein C-catalyzed inactivation than purified plasma-derived FVa on the thrombin-activated platelet surface. The heavy chain subunit of purified, platelet-derived FVa contained only a fraction ( approximately 10-15%) of the intrinsic phosphoserine present in the plasma-derived FVa heavy chain and was resistant to phosphorylation at Ser(692) catalyzed by either casein kinase II or thrombin-activated platelets. MALDI-TOF mass spectrometric analyses of tryptic digests of platelet-derived FV peptides detected an intact heavy chain uniquely modified on Thr(402) with an N-acetylglucosamine or N-acetylgalactosamine, whereas Ser(692) remained unmodified. N-terminal sequencing and MALDI-TOF analyses of platelet-derived FV/Va peptides identified the presence of a full-length heavy chain subunit, as well as a light chain subunit formed by cleavage at Tyr(1543) rather than Arg(1545) accounting for the intrinsic levels of cofactor activity exhibited by native platelet-derived FVa. These collective data are the first to demonstrate physical differences between the two FV cofactor pools and support the hypothesis that, subsequent to its endocytosis by megakaryocytes, FV is modified to yield a platelet-derived cofactor distinct from its plasma counterpart.
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PMID:Unique in vivo modifications of coagulation factor V produce a physically and functionally distinct platelet-derived cofactor: characterization of purified platelet-derived factor V/Va. 1459 14

As part of a general project aimed at elucidating the initiation of mucin-type O-glycosylation in helminth parasites, we have characterized a novel ppGalNAc-T (UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) from the cestode Echinococcus granulosus (Eg-ppGalNAc-T1). A full-length cDNA was isolated from a library of the tissue-dwelling larval stage of the parasite, and found to code for a 654-amino-acid protein containing all the structural features of ppGalNAc-Ts. Functional characterization of a recombinant protein lacking the transmembrane domain showed maximal activity at 28 degrees C, in the range 6.5-7.5 pH units and in the presence of Cu2+. In addition, it transferred GalNAc to a broad range of substrate peptides, derived from human mucins and O-glycosylated parasite proteins, including acceptors containing only serine or only threonine residues. Interestingly, the C-terminal region of Eg-ppGalNAc-T1 bears a highly unusual lectin domain, considerably longer than the one from other members of the family, and including only one of the three ricin B repeats generally present in ppGalNAc-Ts. Furthermore, a search for conserved domains within the protein C-terminus identified a fragment showing similarity to a recently defined domain, specialized in the binding of organic phosphates (CYTH). The role of the lectin domain in the determination of the substrate specificity of these enzymes suggests that Eg-ppGalNAc-T1 would be involved in the glycosylation of a special type of substrate. Analysis of the tissue distribution by in situ hybridization and immunohistochemistry revealed that this transferase is expressed in the hydatid cyst wall and the subtegumental region of larval worms. Therefore it could participate in the biosynthesis of O-glycosylated parasite proteins exposed at the interface between E. granulosus and its hosts.
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PMID:Characterization of a UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase with an unusual lectin domain from the platyhelminth parasite Echinococcus granulosus. 1514 32

To study the relationship between the structure of dermatan sulfate (DS) derivatives and their anti-thrombotic activities, DS-derived oligosaccharides (with different structures and relative molecular weight (M(r))) were prepared, and the effects of the DS-derived oligosaccharides on the activities of heparin cofactor II (HCII), activated protein C (APC), blood platelet, and vascular endothelial cells were studied. The major disaccharides of DS and polysulfated dermatan sulfate (PSDS) were IdoA-1-->3-GalNAc-4-OSO(3) and IdoA-2OSO(3)-1-->3-GalNAc4, 6-diOSO(3), respectively. The results showed that the consequence of the thrombotic inhibitory effects of DS and its derivatives were as follows: PSDS>low molecular weight polysulfated dermatan sulfate (LPSDS)>DS. Both DS and PSDS inhibited platelet aggregation in the concentration-dependent manner, and the IC(50) value of DS and PSDS is 12.7+/-1.3 and 28.6+/-0.9 mg/mL, respectively. DS oligosaccharides (DSOSs) and PSDS oligosaccharides (PSDSOSs) both significantly inhibited P-selectin expression on platelet surface (P<0.01), while DSOSs have no different effect compared with PSDSOSs. DSOSs and PSDSOSs significantly enhanced the activity of HCII in inhibiting thrombin in the plasma. The most active PSDSOS was PSDSOS(1) with M(r) of 4959, which enhanced the HCII activity by 91% (P<0.01). The experiments on APC activity showed that DS and its derivatives enhanced APC activity. The most active PSDSOS was PSDSOS(3) with M(r) of 2749, which enhanced the APC activity to 331+/-27% (P<0.01). DSOSs and PSDSOSs enhanced tissue plasminogen activator (t-PA) activity and reduced the plasminogen activator inhibitor (PAI) activity from cultured human umbilical vein endothelial cells (HUVEC), resulting in the ratio of t-PA/PAI going up. PSDSOSs which have the same M(r) as DSOSs produced more active effects in above assays, except for platelet aggregation.
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PMID:The relationship between the structure of dermatan sulfate derivatives and their antithrombotic activities. 1709 36

Human protein C (hPC) is glycosylated at three Asn-X-Ser/Thr and one atypical Asn-X-Cys sequons. We have characterized the micro- and macro-heterogeneity of plasma-derived hPC and compared the glycosylation features with recombinant protein C (tg-PC) produced in a transgenic pig bioreactor from two animals having approximately tenfold different expression levels. The N-glycans of hPC are complex di- and tri-sialylated structures, and we measured 78% site occupancy at Asn-329 (the Asn-X-Cys sequon). The N-glycans of tg-PC are complex sialylated structures, but less branched and partially sialylated. The porcine mammary epithelial cells glycosylate the Asn-X-Cys sequon with a similar efficiency as human hepatocytes even at these high expression levels, and site occupancy at this sequon was not affected by expression level. A distinct bias for particular structures was present at each of the four glycosylation sites for both hPC and tg-PC. Interestingly, glycans with GalNAc in the antennae were predominant at the Asn-329 site. The N-glycan structures found for tg-PC are very similar to those reported for a recombinant Factor IX produced in transgenic pig milk, and similar to the endogenous milk protein lactoferrin, which may indicate that N-glycan processing in the porcine mammary epithelial cells is more uniform than in other tissues.
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PMID:N-glycosylation microheterogeneity and site occupancy of an Asn-X-Cys sequon in plasma-derived and recombinant protein C. 1934 21