title: The EC domains of human fibrinogen420 contain calcium binding sites but lack polymerization pockets.
abstract: The extended (E) isoform unique to Fibrinogen420 (Fib420) is distinguished from the conventional chain of Fibrinogen340 by the presence of an additional 236-residue carboxyl terminus globular domain (EC). A recombinant form of EC (rEC), having a predicted mass of 27,653 Daltons, was expressed in yeast (Pichia pastoris) and purified by anion exchange column chromatography. Purified rEC appears to be predominantly intact, as judged by N-terminal sequence analysis, mass spectral analysis of the C-terminal cyanogen bromide (CNBr) fragment, and comparison of recognition by epitope-specific monoclonal antibodies. Carbohydrate determination, coupled with analysis of CNBr digestion fragments, confirms N-linked glycosylation at Asn667, the site at which sugar is attached in E. Analysis of CNBr digestion fragments confirms that two disulfide bridges exist at cysteine pairs E613/644 and E780/793. In the presence of 5 mmol/L EDTA, rEC is highly susceptible to plasmic degradation, but Ca2+ (5 mmol/L) renders rEC resistant. No protective effect from plasmic degradation was conferred to rEC by the peptides GPRPamide or GHRP, nor did rEC bind to a GPR peptide column. These results suggest that the EC domain contains a calcium-binding site, but lacks a polymerization pocket. By analogy with the site elucidated in the gammaC domain, we predict that the EC calcium binding site involves residues E772-778: DADQWEE.
authors.affiliation: Lindsley F. Kimball Research Institute of the New York Blood Center, New York, NY, USA.
authors.forename: D
authors.initials: D
authors.lastname: Applegate
authors.forename: L
authors.initials: L
authors.lastname: Haraga
authors.forename: K M
authors.initials: KM
authors.lastname: Hertzberg
authors.forename: L S
authors.initials: LS
authors.lastname: Steben
authors.forename: J Z
authors.initials: JZ
authors.lastname: Zhang
authors.forename: C M
authors.initials: CM
authors.lastname: Redman
authors.forename: G
authors.initials: G
authors.lastname: Grieninger
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001704:Biopolymers; D002118:Calcium / Q000378:metabolism*; D003553:Cystine / Q000032:analysis; D004492:Edetic Acid / Q000494:pharmacology; D005340:Fibrinogen / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D020033:Protein Isoforms / Q000737:chemistry* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D014170:Transformation, Genetic
chemical_list: D001704:Biopolymers; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D003553:Cystine; D005340:Fibrinogen; D004492:Edetic Acid; D002118:Calcium