Creation of an additional glycosylation site as a mechanism for type I antithrombin deficiency
| Autor: | A C, Fitches, K, Lewandowski, R J, Olds |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Adult
Male Protein Folding Glycosylation Adolescent Recombinant Fusion Proteins Antithrombin III Mutation Missense Transfection Exocytosis Substrate Specificity Chlorocebus aethiops Animals Humans Point Mutation Thrombophilia Venous Thrombosis Antithrombin III Deficiency Cell-Free System Pedigree Molecular Weight Amino Acid Substitution COS Cells Mutagenesis Site-Directed Female Protein Processing Post-Translational |
| Zdroj: | Thrombosis and haemostasis. 86(4) |
| ISSN: | 0340-6245 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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