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)

About 30% of human plasma protein C is smaller than the predominant form as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has been suggested that this species, referred to as beta protein C, is a degraded molecule. However, beta protein C is secreted in culture by the HepG2 cell line and is present in plasma collected directly into numerous proteinase inhibitors; the percentage of beta protein C does not change with time during culture or after blood collection. Neither thrombin, activated protein C, nor activated factor X converts the alpha form to beta in the presence or absence of calcium and phospholipids. The NH2-terminal sequences of the heavy chains of both forms are identical, and both release the same dodecapeptide and develop a functional active site when cleaved by thrombin. Both also react with antibodies to a synthetic COOH-terminal peptide. Timed digests with N-glycosidase are consistent with the interpretation that beta protein C has three N-linked oligosaccharide chains whereas alpha protein C has four. It is asparagine 329 that is not glycosylated in beta protein C since antibodies to a synthetic peptide based on the sequence around this amino acid react only with beta protein C. This site is unique in having cysteine instead of serine or threonine 2 residues distal. It is likely that the sulfhydryl group can substitute for the usual hydroxyl group as a hydrogen bond acceptor for the glycosylation reaction only until it forms a disulfide bond. The percentage of protein C that is glycosylated at this site may therefore depend at least in part on the rate of disulfide bond formation which may in turn be related to the rate of protein synthesis.
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PMID:Beta protein C is not glycosylated at asparagine 329. The rate of translation may influence the frequency of usage at asparagine-X-cysteine sites. 169 79

The protein C anticoagulant system provides important control of the blood coagulation cascade. The key protein is protein C, a vitamin K-dependent zymogen which is activated to a serine protease by the thrombin-thrombomodulin complex on endothelial cells. Activated protein C functions by degrading the phospholipid-bound coagulation factors Va and VIIIa. Protein S is a cofactor in these reactions. It is a vitamin K-dependent protein with multiple domains. From the N-terminal it contains a vitamin K-dependent domain, a thrombin-sensitive region, four EGF) epidermal growth factor (EGF)-like domains and a C-terminal region homologous to the androgen binding proteins. Three different types of post-translationally modified amino acid residues are found in protein S, 11 gamma-carboxy glutamic acid residues in the vitamin K-dependent domain, a beta-hydroxylated aspartic acid in the first EGF-like domain and a beta-hydroxylated asparagine in each of the other three EGF-like domains. The EGF-like domains contain very high affinity calcium binding sites, and calcium plays a structural and stabilising role. The importance of the anticoagulant properties of protein S is illustrated by the high incidence of thrombo-embolic events in individuals with heterozygous deficiency. Anticoagulation may not be the sole function of protein S, since both in vivo and in vitro, it forms a high affinity non-covalent complex with one of the regulatory proteins in the complement system, the C4b-binding protein (C4BP). The complexed form of protein S has no APC cofactor function. C4BP is a high molecular weight multimeric protein with a unique octopus-like structure. It is composed of seven identical alpha-chains and one beta-chain. The alpha- and beta-chains are linked by disulphide bridges. The cDNA cloning of the beta-chain showed the alpha- and beta-chains to be homologous and of common evolutionary origin. Both subunits are composed of multiple 60 amino acid long repeats (short complement or consensus repeats, SCR) and their genes are located in close proximity on chromosome 1, band 1q32. Available experimental data suggest the beta-chain to contain the single protein S binding site on C4BP, whereas each of the alpha-chains contains a binding site for the complement protein, C4b. As C4BP lacking the beta-chain is unable to bind protein S, the beta-chain is required for protein S binding, but not for the assembly of the alpha-chains during biosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Protein S and C4b-binding protein: components involved in the regulation of the protein C anticoagulant system. 183 51

Protein S is a vitamin K-dependent plasma protein that functions as a cofactor to activated protein C in the inactivation of coagulation factors Va and VIIIa. The nucleotide sequence of a full-length cDNA clone, obtained from a bovine liver library, was determined and the amino acid sequence was deduced. In addition, 95% of the structure was determined by protein sequencing. Protein S consists of 634 amino acids in a single polypeptide chain and has one asparagine-linked carbohydrate side chain. The cDNA sequence showed that the protein has a leader sequence, 41 amino acid residues long. The amino-terminal part of the molecule containing gamma-carboxyglutamic acid is followed by a region, residues 42-75, with two peptide bonds that are very sensitive to cleavage by thrombin. Residues 76-244 have four cysteinerich repeat sequences, each about 40 residues long, that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the carboxyl-terminal part of protein S is not homologous to the serine proteases.
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PMID:Primary structure of bovine vitamin K-dependent protein S. 294 May 98

The amino acid sequence of human C1r A chain was determined, from sequence analysis performed on fragments obtained from C1r autolytic cleavage, cleavage of methionyl bonds, tryptic cleavages at arginine and lysine residues, and cleavages by staphylococcal proteinase. The polypeptide chain has an N-terminal serine residue and contains 446 amino acid residues (Mr 51,200). The sequence data allow chemical characterization of fragments alpha (positions 1-211), beta (positions 212-279) and gamma (positions 280-446) yielded from C1r autolytic cleavage, and identification of the two major cleavage sites generating these fragments. Position 150 of C1r A chain is occupied by a modified amino acid residue that, upon acid hydrolysis, yields erythro-beta-hydroxyaspartic acid, and that is located in a sequence homologous to the beta-hydroxyaspartic acid-containing regions of Factor IX, Factor X, protein C and protein Z. Sequence comparison reveals internal homology between two segments (positions 10-78 and 186-257). Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 108 and 204. Combined with the previously determined sequence of C1r B chain [Arlaud & Gagnon (1983) Biochemistry 22, 1758-1764], these data give the complete sequence of human C1r.
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PMID:Complete amino acid sequence of the A chain of human complement-classical-pathway enzyme C1r. 303 70

Cruzipain is a lysosomal enzyme of the flagellate Trypanosoma cruzi. It has three potential asparagine-glycosylation sites, two in the catalytic domain and one in the C-terminal domain. The latter appeared to have both high mannose- and complex-type oligosaccharides, whereas the catalytic domain only had compounds of the former type. The partial susceptibility of the complex-type compounds to endo-beta-N-acetylglucosaminidase H and their relative mannose and galactose content indicate that they had hybrid/monoantennary and biantennary structures. The same pattern of high mannose-type compounds was found at both domains, thus indicating that in cruzipain molecules having only high mannose-type compounds, all oligosaccharides were equally exposed to processing glycosidases and glycosyltransferases. As heterogenity of the protein C-terminal domain has already been detected, it is suggested that this feature might elicit an increased accessibility to processing enzymes responsible for complex-type oligosaccharide formation in certain cruzipain molecules or, alternatively, that a second glycosylation site with increased accessibility might be present in certain cruzipain molecules. Furthermore, the presence of complex-type oligosaccharides strongly suggests that, as in mammalian cells, T. cruzi lysosomal enzymes traverse the entire Golgi apparatus up to the trans-Golgi cisternae and the trans-Golgi network before reaching lysosomes.
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PMID:The presence of complex-type oligosaccharides at the C-terminal domain glycosylation site of some molecules of cruzipain. 777 88

Vitamin-K-dependent protein S is an anticoagulant plasma protein which functions as cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa. In addition, it interacts with C4b-binding protein (C4BP), a regulator of the complement system. Using a human protein S cDNA clone as probe, cDNA clones for rabbit protein S were isolated from a rabbit liver cDNA library. The cDNA sequence encoded the mature protein S and 12 residues of the leader sequence. The amino acid sequence of the single-chain 634-amino-acid-residue-long rabbit protein S molecule was 82% and 81% identical to those of human and bovine protein S, respectively. Northern blotting demonstrated protein S mRNA not only in liver but also in reproductive organs (testis, ovary and uterus), in lung and brain. Recombinant rabbit protein S was expressed in eucaryotic cells and found to be post-translationally modified, i.e. it had the correct amino terminus, contained N-linked carbohydrate side chains, gamma-carboxyglutamic acid residues and beta-hydroxylated aspartic acid and asparagine residues. Recombinant rabbit protein S bound calcium like its human counterpart, as judged by its migration in the presence of calcium on agarose-gel electrophoresis. Rabbit protein S has been reported to be species specific with respect to its interaction with APC and not to function with bovine APC. However, we found it to act as cofactor to both human and bovine APC, albeit it was somewhat more efficient with human than with bovine APC. Rabbit protein S, like its human and bovine counterparts, bound human C4BP in a reaction which was associated with the loss of its APC-cofactor activity. However, unlike human plasma, rabbit plasma appeared to contain only the free form of protein S as a radiolabeled rabbit protein S tracer added to rabbit plasma migrated as free protein S on agarose-gel electrophoresis. Addition of human C4BP to rabbit plasma resulted in the formation of a C4BP-protein-S complex, suggesting an explanation for the absence of complexed protein S in rabbit plasma to be sought for in the structure of rabbit C4BP.
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PMID:Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin-K-dependent protein S. 822 42

We describe five families presenting with type II hereditary protein C deficiency characterized by normal antigen and amidolytic activity levels but low anticoagulant activity. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining amplification by the polymerase chain reaction (PCR), denaturing gradient gel electrophoresis of the amplified fragments, and direct sequencing of fragments displaying altered melting behavior. We detected five novel mutations. Three were located in the C-terminal part of the propeptide encoded by exon III: Arginine (Arg)-5 to tryptophan (Trp), Arg-1 to histidine (His), and Arg-1 to cysteine (Cys) mutations. The two others, located in exon IX, affected Arg 229 and serine (Ser) 252, which were respectively replaced by glutamine (Gln) and asparagine (Asn). DNA studies of the other exons from affected individuals showed no other abnormalities. These novel mutations provide further insight into the importance of the affected amino acids located close to the active site, near Asp 257, one of the three amino acids of the catalytic triad. The low anticoagulant activity of the abnormal protein C indicated that Arg 229 and Ser 252 play a key role during the interaction between protein C and its cofactor protein S, phospholipids, or factors Va and VIIIa. The Arg-1 to Cys mutation led to the dimerization of protein C with another plasmatic component, as evidenced by the presence in the plasma of a high molecular weight form of protein C that disappeared after reduction. No molecular mass abnormalities were observed in heavy and light chains of all other protein C mutants. In the five families explored, 9 (64%) of the 14 subjects bearing the mutations reported thrombotic events. This suggests that the protein C amino acids affected by the mutations are very important for the in vivo expression of the antithrombotic properties of protein C.
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PMID:Five novel mutations located in exons III and IX of the protein C gene in patients presenting with defective protein C anticoagulant activity. 832 21

Human peripheral blood monocytes activated by GM-CSF plus IL-4 have recently been found to exhibit characteristics of putative dendritic cells (DC). These cytokine-activated monocytes (CAM) may express novel activation Ag that contribute significantly to their antigen presentation potency. To examine that possibility, mAb specific for CAM were derived. Seven mAb that stained CAM but not unactivated monocytes and other peripheral blood mononuclear cell types were identified. Further screening with a panel of cell lines identified two CAM-specific mAb. The first mAb, 2.1D10, was found to be mannose-receptor specific. A second mAb, 6.3B7, immunoprecipitated a 190-kDa Ag. It stained neither activated B cells nor the putative peripheral blood precursor DC population. Furthermore, 6.3B7 did not recognize determinants in asparagine-linked carbohydrate chains or in sialic acid-containing structures. These mAb against CAM membrane proteins may provide new insights into the requirements for optimal antigen presentation by macrophages and other APC types.
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PMID:Monoclonal antibodies against human dendritic cell-like peripheral blood monocytes activated by granulocyte/macrophage-colony-stimulating factor plus interleukin 4. 951 3

Owing to the high efficacy of L-asparaginase in the treatment of acute lymphatic leukaemia the enzyme was introduced into the chemotherapy schedules for remission induction of this disease shortly after results of large-scale clinical trials had become available. Since asparaginase monotherapy was associated with a high response rate but short remission duration, the enzyme is currently employed within the framework of combination chemotherapy schedules which achieve treatment response in about 90% and long-term remissions in the majority of patients. Recently initiated clinical trials have still confirmed the eminent value of asparaginase in the combination chemotherapy of acute lymphatic leukaemia and of some subtypes of non-Hodgkin lymphoma, and its important role as an essential component of multimodal treatment protocols. Despite the unique mechanism of action of this cytotoxic substance which shows relative selectivity with regard to the metabolism of malignant cells, some patients experience toxic effects during asparaginase therapy. Immunological reactions toward the foreign protein include enzyme inactivation without any clinical manifestations as well as anaphylactic shock. Severe functional disorders of organ systems result from the impaired homeostasis of the amino acids asparagine and glutamine. The changes affecting the proteins of the coagulation system have considerable clinical impact as they may induce bleeding as well as thromboembolic events and may be associated with life-threatening complications when the central nervous system is involved. Risk factors predisposing to thromboembolic complications are hereditary resistance against activated protein C and any other hereditary thrombophilia. Other organ systems potentially affected by relevant functional disorders are the central nervous system, the liver, and the pancreas, with patients who have a history of pancreatic disorders carrying an especially high risk of developing pancreatitis. Studies on the mechanisms of action and the occurrence of resistance phenomena have shown that a treatment response may only be expected if the malignant cells are unable to increase their asparagine synthetase activity to an extent providing enough asparagine to the cell; one may thus conclude that the enzyme-induced asparagine depletion of the serum constitutes the decisive cytotoxic mechanism. Independent of the asparagine depletion related cytotoxicity however, there are other mechanisms of clinical relevance like induction of apoptosis. Besides this, further influences on signal transduction cannot be excluded. Only few publications have dealt with the question of minimum trough activities to be ensured before each subsequent asparaginase dose in order to maintain uninterrupted asparagine depletion under treatment, and answers to this problem are not definitive. Clinical studies using enzymes from E. coli strains indicate that a trough activity of 100 U/l will suffice for complete asparagine depletion of the fluid body compartments with the preparations studied. These findings have been transferred to enzymes from other E. coli strains as well as those isolated from Erwinia chrysanthemi and to the PEG-conjugated E. coli asparaginases. It might be desirable to countercheck the results for confirmation or correction. The dosage and administration schedule of the various enzyme preparations required for complete asparagine depletion over a period of time have been insufficiently defined. While pharmacokinetic studies showed clinically relevant differences in biological activity and activity half-lives for enzymes from different biological sources, the findings of recently published clinical trials indicate that the therapeutic efficacy is affected when different asparaginase preparations are given by identical therapy schedules. (ABSTRACT TRUNCATED)
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PMID:Use of L-asparaginase in childhood ALL. 976 45

Recombinant factor VIII SQ (r-VIII SQ), ReFacto, is a recombinant factor VIII product similar to the smallest active factor VIII protein found in plasma-derived factor VIII (p-VIII) concentrates. The protein comprises two polypeptide chains of 80 and 90 kDa and lacks the major part of the heavily glycosylated B-domain i.e. amino acids Gln744 to Ser1637. r-VIII SQ retains six potential glycosylation sites for N-linked oligosaccharides at asparagine residues 41, 239, 582, 1685, 1810 and 2118. We describe a thorough comparison of the characteristics of r-VIII SQ with those of p-VIII. The primary and secondary structures of r-VIII SQ were in good agreement with that of B-domain-deleted p-VIII (p-VIII-LMW) as shown by SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mapping, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amount of the single chain primary translation product of 170 kDa and also the product specific sequence of 14 amino acids, the SQ-link, in the C-terminal end of the 90 kDa chain. It was shown that r-VIII SQ had a high specific activity of about 14,000 IU VIII:C/mg as determined by use of a chromogenic substrate assay. The r-VIII SQ protein was comparable to p-VIII forms with a retained B-domain, in terms of potency measured by a chromogenic substrate or a two-stage clotting assay, in interactions with thrombin, and with activated protein C (APC) in combination with Protein S. The ability of r-VIII SQ to participate as a cofactor in factor Xa generation in a mixture of factors IXa and X, phospholipid and calcium was in conformity with that of p-VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid vesicles and von Willebrand factor (vWF) as shown in gel filtration studies. The same kinetics in binding to von Willebrand factor was found for r-VIII SQ and p-VIII as determined by real-time biospecific interaction analysis (BIA) with use of the BIAcore instrument. The apparent association rate constant was 4 x 10(6) M(-1)s(-1). Two dissociation rate constants were found, 1 X 10(-2)s(-1) and 4 x 10(-4)s(-1). The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.
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PMID:Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ. 1120 95


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