Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.52 (PNGase F)
 1,527 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We recently developed an enzyme-linked immunosorbent assay (ELISA) for total protein S (PS) antigen using the monoclonal antibody S-12. During the screening of thrombophilic patients we identified a patient, who was using marcoumar, with 0% PS by monoclonal ELISA and 23% PS by polyclonal ELISA. Further analysis of this patient and his family showed that the patient was a compound heterozygote for type 1 PS deficiency and for an abnormal PS molecule (PS-Heerlen) that was not recognized by the S-12 antibody. Similar observations were made in two sisters from an unrelated Dutch family. Subsequent studies showed that PS Heerlen has a slightly lower molecular weight (71,000) than normal PS (73,000), binds normally to C4b-binding protein, and retains full activated protein C cofactor activity. The alteration in the PS Heerlen molecule was identified as a substitution of Ser460 by Pro, which is due to a unique T---C transition in exon 13 of the active PS-alpha gene. The substitution occurs in the consensus sequence for the potential N-linked glycosylation of Asn458. Digestion with N-glycanase showed that normal PS probably contains three N-linked oligosaccharide side chains, while PS Heerlen contains only two (Asn458 not glycosylated?). Segregation analysis in the two original families showed that the presence of the genetic abnormality was always associated with the PS-Heerlen phenotype. The frequency of the PS-Heerlen allele was found to be 0.52% in the general population and 0.67% in a population of patients with unexplained thrombophilia. There is no evidence that the PS Heerlen allele is associated with an increased risk for thrombosis.
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PMID:Heerlen polymorphism of protein S, an immunologic polymorphism due to dimorphism of residue 460. 214 91

Human protein S (HPS) has three potential N-linked glycosylation sites at Asn458, 468, 489. To study the role of glycosylation at these sites, PCR mutagenesis was used to abolish the consensus sequence of each N-linked glycosylation site (Asn458-->Gln, Ser460-->Gly; Asn468-->Gln, Thr470-->Gly; Asn489-->Gln, Thr491-->Gly) in full-length HPS cDNA. Each resulting construct was expressed in human kidney 293 cells by stable transfection of cDNA/SV40/adeno/pBR322-derived expression vectors, and conditioned medium was collected for recombinant protein purification. SDS-PAGE gels revealed that glycosylation mutants migrate identically and faster than the wild-type rHPS, showing that each of the three potential N-glycosylation sites contain a similar amount of carbohydrate. Mass spectral analysis yielded similar results and a molecular mass of approximately 78,000 for wild-type HPS. To demonstrate that the difference in mobility between wild-type and mutant protein S is due to their carbohydrate content, plasma-derived HPS and recombinant HPS were subjected to N-glycanase digestion and subsequently shown to migrate identically on SDS-PAGE gels. All forms of HPS have similar time courses for cleavage by alpha-thrombin. Functional studies indicate that wild-type rHPS possesses the same cofactor specific activity as plasma-derived HPS, as tested by a standard clotting assay. Asn458 and Ser460 mutant rHPS have only a slightly higher cofactor activity, whereas the other four mutants have similar clotting activities, compared to wild-type rHPS. In a purified component system, glycosylation mutants of protein S showed a slightly enhanced ability to stimulate APC-mediated factor Va inactivation after an initial lag phase. The interaction of rHPS glycosylation mutants with human C4b-binding protein (C4bp) was also studied by solution phase equilibrium binding assay. Two mutants (Asn458, Ser480) have marginally lower dissociated constants (Kd) with C4bp, whereas the others have the same apparent Kd as wild-type rHPS.
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PMID:The effect of N-linked glycosylation on molecular weight, thrombin cleavage, and functional activity of human protein S. 924 50