Identification of the second heparin-binding domain in human complement factor H.
Complement factor H (fH) regulates activation of the alternative pathway of C, reducing the amount of C3b deposited on sialic acid-rich surfaces. Heparin binding has been used as a model for examining the sialic acid-binding characteristics of fH. We have previously shown that of the 20 short consensus repeat (SCR) modules of fH, SCR 7 contains an important heparin binding site, but other SCRs also play a role in heparin binding. To localize the other sites, we prepared recombinant truncated and SCR deletion mutants of fH and tested them by heparin-agarose affinity chromatography. The 5 C-terminal SCRs were found to contain a heparin binding site as an SCR 7 deletion mutant of the N terminal 15 SCRs did not bind heparin, but a construct consisting of SCRs 16-20 was shown to bind heparin. Double deletion of SCRs 7 and 20 from fH abrogated binding to heparin, indicating that SCR 20 contains a heparin binding site. This finding was confirmed with the observation that attachment of SCR 20 to a group of nonbinding SCRs produced a heparin-binding protein. A protein consisting of SCRs 19 and 20 did not bind heparin, whereas SCRs 18-20 did, indicating that, although SCR 20 contains a heparin binding site, at least two nonspecific adjacent SCRs are required. fH-related protein-3 (FHR-3) possesses an SCR homologous to SCR 7 of fH and bound heparin, whereas FHR-4, which lacks such an SCR, did not. Thus, fH contains two separate heparin binding sites, which are located in SCRs 7 and 20.
Blackmore
T K
TK
Department of Microbiology and Infectious Diseases, Flinders Medical Centre, Bedford Park, South Australia. tim.blackmore@flinders.edu.au
Hellwage
J
J
Sadlon
T A
TA
Higgs
N
N
Zipfel
P F
PF
Ward
H M
HM
Gordon
D L
DL
D001053:Apolipoproteins / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics / Q000276:immunology; D001798:Blood Proteins / Q000378:metabolism; D017242:Complement Factor H / Q000235:genetics / Q000378:metabolism*; D016384:Consensus Sequence / Q000276:immunology; D006493:Heparin / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid / Q000276:immunology; D017384:Sequence Deletion / Q000276:immunology
D001053:Apolipoproteins; D001798:Blood Proteins; C493495:CFH protein, human; C105970:CFHR3 protein, human; C105092:CFHR4 protein, human; D011994:Recombinant Proteins; D017242:Complement Factor H; D006493:Heparin