Abstract
Factor H is a regulator of complement activation and, in this capacity, it prevents activation of the alternative pathway on host cells and tissues when it recognizes markers on these surfaces. This report describes the binding characteristics and location of the site on factor H that is responsible for host recognition. Factor H was found to bind a variety of polyanions, including heparin, heparan sulfate, dextran sulfate, and clusters of sialic acid. In heparin-agarose binding assays it exhibited an affinity for heparin only 2-fold weaker than that of antithrombin III. Factor H exhibited little or no affinity for polyaspartic acid or bacterial colominic acid (polysialic acid). Factor H (Mr 150,000 with approximate dimensions of 30 x 600 A) is composed of 20 highly homologous domains (SCRs) that are arranged as beads on a string. Polyanions were found to block a tryptic cleavage site in domain 15, and a photoaffinity-tagged heparin probe labeled the region between domains 12 and 15. Affinity chromatography of tryptic fragments on heparin-Sepharose confirmed that this region contained the heparin-binding site. CNBr cleavage at Met787 located between SCRs 13 and 14 split the photoaffinity-tagged region. Sequence analysis strongly suggests that domain 13 contains the primary site of polyanion binding. Factor H expresses its complement regulatory function through a site located in domains 4-6 where C3b binds. Thus, the polyanion-binding site that regulates the affinity of factor H for C3b appears to reside more than 200 A away from the C3b-binding site.
Highlights
Activation of the alternative pathway of complement repaffinity o[ factor H for C3b appears to reside more resents a natural defense system for recognition and destructhan 200A away from the C3b-binding site
Factor H is a regulatory protein of the complement system and a member of a superfamily of proteins composed of repeating 60 amino acid domains (short consensusrepeats (SCR)’ or complement control protein (CCP) domains) (reglycoproteins (C3and factors B, D, H, I, and P),but recognizes a wide variety of foreign organisms without prior contact or participation of memory cells
We recently showed that the site responsible thfhaeitpoeap;rriSonAp-liS2o’Dn1a,tSesAu; lSfAoDsS,uAmc,ciniNni-mimhyaidldlryyolxd2ye-s-(uNpc--csaiuznliifdmaoitsedadyllhi-ec4py-alaarziminiddowos)aietlhtihctyyhlle-ilcpa,ch3io’d--di-wfoer reversing this show that inhibition this sities is located on factor H, and located far from the C3b-binding toactivatable, disulfide-containing, radioiodinated reagent SASD cou- site
Summary
The ability to bind C3b and exhibited cofactor activity for Binding to Heparin-Agarose-Factor H has been demonstrated tobind to heparin-agarose (lo), and thpisroperty was used to examine the relative affinity and thespecificity of the binding site for other polyanions. Tag migrated with either the 117-82 kDa group or appeared in the region of the gel with fragments less than 25 kDa (see Fig. 5,30-min and 60-minpanelsC).onsistent with the altered cleavage pattern in the presence of heparin shown, the 30-min panel of Fig. 5 shows that factor H bearing covalently bound heparin fragments A radiolabeled, heparin-directed, photoaffinity probe tagged the region between SCRs 12-15 as demonstrated by tryptic digestion and SDS gel electrophoresis of the fragments the label subsequently appeared near the bottom of the gel, (Fig. 5 and Table I). CNBr digestion of photonot in the 66-kDa fragment This suggests that the heparin- affinity-tagged factor H demonstrated that cleavage of the directed labeling occurred in an approximately 16,000-dalton protein at Met787split thephotoaffinity-tagged region. AndDorothy Peterson for their excellent technical work and Melanie McDade for secretarial assistance
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