Identification of residues 413-433 of plasma protein S as essential for binding to C4b-binding protein.
Reversible association of protein S with C4b-binding protein (C4BP) in plasma down-regulates protein S activity, since free protein S but not the protein S.C4BP complex is an anticoagulant cofactor for activated protein C. To identify regions on the surface of protein S that mediate complex formation with C4BP, a number of nonoverlapping synthetic pentadecapeptides comprising protein S sequences were prepared and tested for their ability to inhibit complex formation. The most potent pentadecapeptide, residues 420-434 (PSP-420) (SGIKEIIQEKQNKHC), gave half-maximal effect at 20 microM. A peptide with the reverse sequence, 434-420, did not inhibit. A peptide containing the sequence of protein S residues 408-434 inhibited complex formation by > 95% with 50% inhibition at 5 microM peptide. Biotinylated C4BP bound specifically to plates coated with PSP-420 but not with the 434-420 peptide; and biotinylated PSP-420 bound to plates coated with C4BP. Rabbit antibodies were raised against several keyhole limpet hemocyanin-conjugated peptides, and each was tested for ability to inhibit complex formation. Anti-PSP-420 antibody potently inhibited complex formation with half-maximal effect at 25 nM IgG. A monoclonal antibody (LJ-56) made against PSP-420 showed high affinity for protein S and inhibited complex formation; this monoclonal antibody specifically recognized free protein S but not the protein S.C4BP complex. These results imply that the PSP-420 sequence is surface-exposed, capable of binding to C4BP, and essential for protein S binding to C4BP.
Fernández
J A
JA
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Heeb
M J
MJ
Griffin
J H
JH
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D000939:Epitopes; D006023:Glycoproteins; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D017293:Protein S / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D000939:Epitopes; D006023:Glycoproteins; D010446:Peptide Fragments; D017293:Protein S; D015935:Complement C4b