The localization of heparin-binding fragments on human C4b-binding protein.
C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP interacts with C C4b on a domain located in a 48-kDa chymotryptic fragment. We now demonstrate that C4BP contains heparin-binding fragments, which are located within the C4b binding domain. We have used an assay using heparin coupled to Sepharose CL-6B to show that 125I-C4BP binds to heparin in a time-dependent, saturable, and reversible manner. Binding could be inhibited by purified 48-kDa fragments and direct binding on the 48-kDa fragments to heparin-Sepharose was demonstrated by SDS-PAGE. mAb against native C4BP and the isolated 160-kDa central core fragment were evaluated for their ability to block the binding of 125I-C4BP to heparin and C4b. The relative efficacy of mAb against intact C4BP in blocking C4BP binding to heparin-Sepharose was similar to that for blocking 125I-C4BP binding to C4b. In addition, heparin blocked the binding of 125I-C4BP to C4b and vice versa. It is therefore likely that the heparin-binding fragments are localized on or close to the C4b-binding site of C4BP.
Hessing
M
M
Department of Haematology, University Hospital Utrecht, The Netherlands.
Vlooswijk
R A
RA
Hackeng
T M
TM
Kanters
D
D
Bouma
B N
BN
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D006023:Glycoproteins / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D007158:Immunologic Techniques; D066298:In Vitro Techniques; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D017293:Protein S; D013696:Temperature
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D010446:Peptide Fragments; D017293:Protein S; D015935:Complement C4b; D006493:Heparin