The EC domains of human fibrinogen420 contain calcium binding sites but lack polymerization pockets.
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.
Applegate
D
D
Lindsley F. Kimball Research Institute of the New York Blood Center, New York, NY, USA.
Haraga
L
L
Hertzberg
K M
KM
Steben
L S
LS
Zhang
J Z
JZ
Redman
C M
CM
Grieninger
G
G
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
D001704:Biopolymers; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D003553:Cystine; D005340:Fibrinogen; D004492:Edetic Acid; D002118:Calcium