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)

Thrombomodulin is an endothelial cell thrombin receptor that serves as a cofactor for thrombin-catalyzed activation of protein C. Structural requirements for thrombin binding and cofactor activity were studied by mutagenesis of recombinant human thrombomodulin expressed on COS-7 and CV-1 cells. Deletion of the fourth epidermal growth factor (EGF)-like domain abolished cofactor activity but did not affect thrombin binding. Deletion of either the fifth or the sixth EGF-like domain markedly reduced both thrombin binding affinity and cofactor activity. Thrombin binding sequences were also localized by assaying the ability of synthetic peptides derived from thrombomodulin to compete with diisopropyl fluorophosphate-inactivated 125I-thrombin binding to thrombomodulin. The two most active peptides corresponded to (a) the entire third loop of the fifth EGF-like domain (Kp = 85 +/- 6 microM) and (b) parts of the second and third loops of the sixth EGF-like domain (Kp = 117 +/- 9 microM). These data suggest that thrombin interacts with two discrete elements in thrombomodulin. Deletion of the Ser/Thr-rich domain dramatically decreased both thrombin binding affinity and cofactor activity and also prevented the formation of a high molecular weight thrombomodulin species containing chondroitin sulfate. Substitutions of this domain with polypeptide segments of decreasing length and devoid of glycosylation sites progressively decreased both cofactor activity and thrombin binding affinity. This correlation suggests that increased proximity of the membrane surface to the thrombin binding site may hinder efficient thrombin binding and the subsequent activation of protein C. Membrane-bound thrombomodulin therefore requires the Ser/Thr-rich domain as an important spacer, in addition to EGF-like domains 4-6, for efficient protein C activation.
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PMID:Functional domains of membrane-bound human thrombomodulin. EGF-like domains four to six and the serine/threonine-rich domain are required for cofactor activity. 131 30

Modification of protein Ag by proteolysis is one of the principal steps in the presentation of Ag to Th cells. However, little is known about the enzymes participating in these events, their specificity or the characteristics of the natural fragments that they produce. Cathepsin D (CD) is an aspartyl protease identified in endosomes of APC. In this report, the role of CD in the processing of OVA has been investigated. OVA digested in vitro with purified CD was able to stimulate IL-2 secretion by three different OVA-specific I-Ad restricted Th cell hybridomas when it was presented by fixed APC. The digest of OVA was recognized in the context of I-Ad, but not by I-Ak-restricted OVA-specific Th cells. No difference was observed in the ability of OVA digested with CD to stimulate Th cells in the absence of FCS or in the presence of protease inhibitors indicating that extracellular proteases were not likely to contribute to processing of OVA. Taken together, these results suggest that CD is necessary and sufficient for the generation of an antigenic epitope from OVA. A fragment containing the epitope was isolated from the OVA digest by reverse phase HPLC. This fragment, which migrates in SDS-PAGE as a 10-kDa polypeptide, is a potent epitope. Its capacity to activate Th cells is compared to that of the tryptic peptide OVA323-339.
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PMID:Role of cathepsin D in antigen presentation of ovalbumin. 132 88

We have generated for the first time monoclonal antibodies (mAbs) specific for topoisomerase I (topo I) from scleroderma patients, and tight skin mice which develop a scleroderma-like syndrome. The epitope specificity of these antibodies has been determined using a series of fusion proteins containing contiguous portions of topo I polypeptide. Western blot analysis demonstrated that both human and mouse mAbs bound strongly to fusion protein C encompassing the NH2-terminal portion of the enzyme, and weakly to fusion proteins F and G containing regions close to the COOH-terminal end of the molecule. This crossreactivity is related to a tripeptide sequence homology in F, G, and C fusion proteins. It is interesting that a pentapeptide sequence homologous to that in fusion protein C was identified in the UL70 protein of cytomegalovirus, suggesting that activation of autoreactive B cell clones by molecular mimicry is possible. Both human and mouse mAbs exhibiting the same antigen specificity, also share an interspecies cross-reactive idiotope. These data suggest that B cell clones producing antitopo autoantibodies present in human and mouse repertoire are conserved during phylogeny, and are activated during the development of scleroderma disease.
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PMID:Antitopoisomerase I monoclonal autoantibodies from scleroderma patients and tight skin mouse interact with similar epitopes. 137 44

Prothymosin alpha (ProT alpha) is an acidic polypeptide with potentiating effects on HLA-DR-restricted in vitro cellular immune response systems such as T cell proliferative responses to soluble proteins and cellular auto- or alloantigens. Experiments were performed to investigate the effect of ProT alpha on MHC class II Ag expression in human monocytes, murine splenocytes, and tumor cell lines at both protein and molecular levels. RIA and immunofluorescence analysis revealed that ProT alpha enhances HLA-DR surface Ag expression whereas Northern blot analysis demonstrated that ProT alpha causes significant accumulation of MHC class II mRNA. The enhancing effect of ProT alpha was demonstrated convincingly using precultured human peripheral monocytes, which are known to express decreased amounts of surface HLA-DR Ag, and HLA-DR-positive human cell lines. Moreover, ProT alpha was shown to induce HLA-DR Ag expression in a priori HLA-DR-negative tumor cells. Furthermore, ProT alpha was shown to be active in vivo. Splenocytes from mice pretreated with ProT alpha expressed more surface Ilpha Ag and contained more I alpha-specific mRNA. These findings suggest that ProT alpha may be important in the regulation of the immune response by enhancing MHC class II Ag expression in APC.
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PMID:Prothymosin alpha enhances human and murine MHC class II surface antigen expression and messenger RNA accumulation. 154 15

We characterized a mutant protein C gene from an individual with no detectable protein C antigen in blood plasma. Southern blot hybridization analysis with human protein C cDNA demonstrated neither gross deletion nor rearrangement of the gene. Sequencing all the exons and exon-intron boundaries of the gene except the 3' noncoding region showed two mutant alleles. The one, derived from the mother, represents a deletion of 5 nucleotides (nt) (CCCGC) in the end of exon VI (mutation I), predicted to result in the generation of a new stop codon due to a reading frameshift and the premature termination of translation. The other, derived from the father, represents a point mutation (G to A) in exon IX (mutation II), resulting in an amino acid substitution, Gly-376(GGC) to Asp(GAC), in the catalytic domain of the protein. Allele-specific oligonucleotide probe hybridization confirmed the presence of the two mutations. Mutation I would result in a truncated polypeptide of 169 amino acid residues that lacks the heavy chain. Mutation II gives rise to an alteration of a highly conserved amino acid, Gly-376. These data indicate that this patient is a compound heterozygote of the two mutant alleles, each one inherited from each parent. Transient expression assays using COS-7 cells transfected with mutated protein C expression vectors suggested that each of the two mutations leads to the protein C deficiency by causing an impairment of secretion of the respective mutant proteins.
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PMID:Protein C deficiency Hong Kong 1 and 2: hereditary protein C deficiency caused by two mutant alleles, a 5-nucleotide deletion and a missense mutation. 161 Oct 81

Protein C (PC) is a vitamin K-dependent serine protease, a deficiency of which results in thrombus. There is no spontaneously occurring mouse model of the disease. Attempts to create such a model in mice by using anti-sense gene technology requires isolation of a normal mouse PC cDNA. When a mouse liver (BALB/c) cDNA library was screened using a human PC cDNA as a probe, nine overlapping cDNA clones were isolated and sequenced. The cloned mouse PC cDNA comprised 1,512 nucleotides and the open reading frame of the cDNA encoded a polypeptide of 461 amino acids residues including a leader peptide composed of 41 amino acids. Mouse PC exhibited high homology to both human and bovine PCs. Mouse PC also had several structural features common in other PCs; locations of 23 Cys residues, location of putative beta-hydroxy Asp71, possible carbohydrate attachment sites involving Asp residues at amino acid positions 249, 314, and 330, and location of active sites such as His212, Asp258, and Ser361. Northern blot hybridization analysis identified a single species of mouse PC mRNA (2.0 kb in length) in mouse liver.
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PMID:Isolation and characterization of a mouse protein C cDNA. 161 39

An 784 base pair (bp) copy DNA (cDNA) for the low molecular weight hydrophobic surfactant-associated protein C (SP-C) has been isolated from a lambda gt11 cDNA library constructed from fetal rabbit lung mRNA. The cDNA, which coded for a 193 amino-acid proprotein with 6 bp 5' and 193 bp 3' untranslated segments, possesses considerable nucleic acid and predicted amino-acid homology with previously reported SP-C cDNAs. The predicted amino-acid sequence of the 35 amino-acid mature polypeptide shares 94-97% identity with human, rat and mouse SP-C and is 88-91% homologous to the mature proteins from bovine, porcine and canine lung. The last 12 amino acids of mature SP-C are highly hydrophobic and invariant. Alignment of the rabbit and human nucleic acid sequences required introduction of a 27 bp gap in the rabbit sequence at a site corresponding to the exon-intron junction of the 5th exon of the human genomic sequence. Since previous studies have identified differential splicing at the 5' and 3' ends of the human 5th exon, we investigated the potential existence of alternative splicing of rabbit SP-C mRNA. Reverse transcription (RT) of total RNA followed by polymerase chain reaction (PCR) was used to establish the relative abundance of alternative splicing products from fetal and adult lung and from rabbit kidney, placenta and liver. The relative abundance of the 250, 280 and 350 bp bands observed was the same in lung and other tissues. PCR amplification of genomic rabbit DNA indicated that the 350 bp fragment corresponds to the unspliced nascent transcript. The lack of developmental or tissue-specific abundance patterns implies the absence of secondary influences on SP-C mRNA polymorphism. Indeed, free energy of formation calculations predicted the presence of hairpin structures favouring formation of the more abundant 250 bp form. These observations plus the absence of any effect of alternative splicing on SP-C protein structure led us to conclude a physiological role is unlikely.
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PMID:cDNA sequence and alternative mRNA splicing of surfactant-associated protein C (SP-C) in rabbit lung. 164 7

A recombinant Factor VIII (Factor VIII-delta II) consists of a unique polypeptide chain of 165 kDa deleted from the major part of the B-domain and from the cleavage site at Arg-1648-Glu-1649 found in plasma-derived Factor VIII. It was expressed in mammalian cells in serum-free medium containing von Willebrand factor and purified by a one-step immunopurification. The recombinant Factor VIII was characterized as a single active peak when subjected to f.p.l.c., in contrast with the plasma-derived molecule. Its coagulant activity was decreased in the presence of EDTA, suggesting that a bivalent ion is required, as for plasma-derived Factor VIII. The activation by thrombin and the inactivation by activated protein C were studied and the resulting molecular forms were analysed by f.p.l.c. and SDS/PAGE. The results clearly demonstrate that, despite the structural differences between plasma-derived and recombinant Factor VIII, activation and inactivation of Factor VIII-delta II generate proteolysed complexes similar to that described for plasma-derived Factor VIII. Thus this deleted recombinant Factor VIII, which is processed similarly to plasma-derived Factor VIII, should be normally integrated in the regulation system of Factor X activation in the blood-coagulation cascade.
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PMID:Structural and functional characterization of Factor VIII-delta II, a new recombinant Factor VIII lacking most of the B-domain. 190 11

The enzyme NAD(P)H:flavin oxidoreductase (flavin reductase) catalyzes the reduction of soluble flavins by reduced pyridine nucleotides. In Escherichia coli it is part of a multienzyme system that reduces the Fe(III) center of ribonucleotide reductase to Fe(II) and thereby sets the stage for the generation by dioxygen of a free tyrosyl radical required for enzyme activity. Similar enzymes are known in other organisms and may more generally be involved in iron metabolism. We have now isolated the gene for the E. coli flavin reductase from a lambda gt11 library. After DNA sequencing we found an open reading frame coding for a polypeptide of 233 amino acids, with a molecular weight of 26,212 and with an N-terminal segment identical to that determined by direct Edman degradation. The coding sequence is preceded by a weak ribosome binding site centered 8 nucleotides from the start codon and by a promoterlike sequence centered at a distance of 83 nucleotides. In a Kohara library the gene hybridized to position 3680 on the physical map of E. coli. A bacterial strain that overproduced the enzyme approximately 100-fold was constructed. The translated amino acid sequence contained a potential pyridine nucleotide-binding site and showed 25% identity with the C-terminal part of one subunit (protein C) of methane monooxygenase from methanotropic bacteria that reduces the iron center of a second subunit (protein A) of the oxygenase by pyridine nucleotides.
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PMID:Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme. 205 Jun 27

T cell binding to target cells involves not only the TCR and its MHC-bound ligand, but also a collection of additional proteins on both the T cell and its target. In an attempt to identify new molecules involved in this binding, mAb were raised against APC, and screened for their abilities to inhibit T cell recognition of Ag plus MHC on B cells. Six antibodies were identified that inhibited this reaction and that bound a cell-surface glycoprotein (Lgp55), with core polypeptide Mr 30,000 and a glycosylated Mr of approximately 55,000 depending upon the cell source. The properties of Lgp55 were consistent with it being the mouse homologue of a recently identified human ligand (intercellular adhesion molecule-2) for lymphocyte functional Ag-1 because the proteins are of comparable Mr, and antibody to Lgp55, like anti-lymphocyte functional antigen-1, blocks T cell recognition of Ag presented by B cells, but not of Ag presented by mouse fibroblasts.
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PMID:Identification of a new cell surface glycoprotein with accessory function in murine T cell responses. 213 99


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