title: Creation of an additional glycosylation site as a mechanism for type I antithrombin deficiency.
abstract: We report the identification of a new mutation resulting in type I antithrombin (AT) deficiency and the mechanism by which the deficiency arose. The single base substitution of G to A at nucleotide 2709 was identified in a proband with a family history of venous thrombosis. The mutation results in a substitution of 82 Ser by Asn, creating a new glycosylation site. Expression studies were then carried out, to confirm Asn-linked glycosylation occurred at this consensus site and that this resulted in the AT deficient phenotype. Cell-free translations using rabbit reticulocyte lysate in the presence of microsomes demonstrated that the 82 Asn variant was post-translationally processed efficiently. The 82 Asn variant protein was of a higher molecular weight than normal AT. consistent with the addition of a fifth glycan chain. Incubation of translation product with endoglycosidase H, confirmed that the higher molecular weight product had resulted from additional carbohydrate. Expression of the 82 Asn variant in COS-7 cells resulted in intracellular accumulation, with a low level of secretion of the protein into culture supernatant, consistent with type I AT deficiency. The addition of an extra carbohydrate side chain to residue 82 of antithrombin may block post-translational folding. trapping the variant intracellulary.
authors.affiliation: Department of Pathology, Dunedin School of Medicine, University of Otago, New Zealand. alison.fitches@stonebow.otago.ac.nz
authors.forename: A C
authors.initials: AC
authors.lastname: Fitches
authors.forename: K
authors.initials: K
authors.lastname: Lewandowski
authors.forename: R J
authors.initials: RJ
authors.lastname: Olds
mesh_terms: D000293:Adolescent; D000328:Adult; D019943:Amino Acid Substitution; D000818:Animals; D000990:Antithrombin III / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D020152:Antithrombin III Deficiency / Q000145:classification / Q000235:genetics*; D019556:COS Cells; D002474:Cell-Free System; D002522:Chlorocebus aethiops; D005089:Exocytosis; D005260:Female; D006031:Glycosylation; D006801:Humans; D008297:Male; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D010375:Pedigree; D017354:Point Mutation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D013379:Substrate Specificity; D019851:Thrombophilia / Q000235:genetics*; D014162:Transfection; D020246:Venous Thrombosis / Q000209:etiology* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D000990:Antithrombin III