Factor XIIIa-catalyzed cross-linking of recombinant alpha C fragments of human fibrinogen.
Direct measurements of the structure and function of the COOH-terminal portion of the A alpha chain (residues 220-610) of human fibrinogen have been hampered by the difficulty of isolating intact fragments derived from this protease-sensitive region. Here, we overcame this problem by expressing two fragments, alpha C45K (A alpha 221-610) and a truncated version of it, alpha C30K (A alpha 368-610), in Escherichia coli. Both proteins were purified to homogeneous state, and their integrity was confirmed at protein level by sequencing. Upon treatment with factor XIIIa, the alpha C45K fragment but not the alpha C30K fragment formed polymers similar to those derived from fibrin clots. Sequence analysis of cross-linked alpha C45K polymers revealed involvement in the cross-linking reaction of at least three Gln residues (221, 237, 328) in the NH2-terminal region of the fragment and four Lys residues (539, 556, 580, 601) located in the COOH-terminal part of the molecule. In addition, a fraction of alpha C45K fragment was found in an intramolecular cross-linked form, suggesting a high level of flexibility of its polypeptide chain and consistent with the location of its donor and acceptor residues in clusters near the ends of the molecule. The alpha C30K fragment, lacking the NH2-terminal Gln residues, was not able to form polymers or internally cross-linked monomers. Thus, the C-terminal part of the A alpha chain comprises an autonomous, functionally active, and flexible region that plays a key role in alpha polymer formation and stabilization of fibrin clots by factor XIIIa.
Matsuka
Y V
YV
J. Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.
Medved
L V
LV
Migliorini
M M
MM
Ingham
K C
KC
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005340:Fibrinogen / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000737:chemistry; D006801:Humans; D066298:In Vitro Techniques; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011503:Transglutaminases / Q000378:metabolism*
D003432:Cross-Linking Reagents; D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D005973:Glutamine; D005340:Fibrinogen; D011503:Transglutaminases; D008239:Lysine