title: Deletion or replacement of the second EGF-like domain of protein S results in loss of APC cofactor activity.
abstract: Human protein S (PS), a cofactor of anticoagulant-activated protein C (APC), is a modular protein containing 4 epidermal growth factor (EGF)-like domains. EGF1 appears to mediate PS interaction with APC, but the roles of EGFs 2, 3, and 4 are less clear. We synthesized PS variants lacking single EGF domains (EGF2, 3, or 4) and assessed their APC cofactor activity in a factor Va inactivation assay. The variant lacking EGF2 (variant 134) showed the most dramatic loss of activity (approximately 10% of recombinant wild-type PS activity). Replacement of EGF2 by an additional EGF3 (variant 1334) resulted in a comparable loss of activity, suggesting that the loss of a specific rather than "spacer" function of EGF2 was responsible. We confirmed that the variant 134 had a functional gamma-carboxyglutamic acid (Gla) domain and that EGF1 was correctly folded. This is the first clear evidence that EGF2 is required for the expression of PS activity.
authors.affiliation: Department of Haematology, Faculty of Medicine, Imperial College, Hammersmith Hospital, London, United Kingdom.
authors.forename: Blandine
authors.initials: B
authors.lastname: Mille-Baker
authors.forename: Suely M
authors.initials: SM
authors.lastname: Rezende
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Simmonds
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Mason
authors.forename: David A
authors.initials: DA
authors.lastname: Lane
authors.forename: Michael A
authors.initials: MA
authors.lastname: Laffan
mesh_terms: D015055:1-Carboxyglutamic Acid / Q000737:chemistry; D001665:Binding Sites; D004815:Epidermal Growth Factor / Q000737:chemistry* / Q000502:physiology; D015943:Factor Va / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D006801:Humans; D016296:Mutagenesis; D017354:Point Mutation; D011486:Protein C / Q000502:physiology*; D017510:Protein Folding; D017293:Protein S / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: D011486:Protein C; D017293:Protein S; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D015943:Factor Va