Serum amyloid P component binding to C4b-binding protein.
Human C4b-binding protein (C4BP), which is a regulator of the classical complement pathway C3 convertase, forms high affinity complexes with anticoagulant protein S and with the pentraxin serum amyloid P component (SAP). SAP is a plasma protein present in all amyloid deposits. Recently, SAP was shown to inhibit the complement regulatory functions of C4BP. In this investigation, we have studied the structural requirements for the C4BP-SAP interaction. C4BP was subjected to chymotrypsin digestion, which yielded two major fragments corresponding to the central core (160 kDa) and to the cleaved-off tentacles (48 kDa). SAP-Sepharose specifically bound the 160-kDa fragment, suggesting that the central core of C4BP contains the binding site for SAP. In a quantitative affinity chromatography assay, the dissociation constants for binding of intact C4BP and of the 160-kDa central core fragment to SAP were found to be 30 and 70 nM, respectively. Recombinant C4BP composed of only alpha-chains bound SAP with similar affinity (Kd = 22 nM), whereas nonglycosylated recombinant alpha-chain C4BP (synthesized in the presence of tunicamycin) bound SAP with lower affinity (Kd = 126 nM). This suggests that the carbohydrate moiety of the central core of C4BP is important for binding of C4BP to SAP in contrast to the C4BP beta-chain, which is not required. EDTA, heparin, and phosphorylethanolamine as well as a peptide comprising amino acids 27-39 of SAP were found to completely displace C4BP from the SAP matrix. Moreover, the immobilized SAP peptide bound C4BP in a reaction that, in contrast to the C4BP-SAP interaction, was not dependent on calcium.
García de Frutos
P
P
Department of Clinical Chemistry, Lund University, University Hospital Malmö, Sweden.
Härdig
Y
Y
Dahlbäck
B
B
D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D002918:Chymotrypsin; D015935:Complement C4b / Q000378:metabolism*; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000683:Serum Amyloid P-Component / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D003169:Complement Inactivator Proteins; D006023:Glycoproteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000683:Serum Amyloid P-Component; D015935:Complement C4b; D002918:Chymotrypsin