Structural requirements for the complement regulatory activities of C4BP.
C4b-binding protein (C4BP) is a regulator of the classical complement pathway C3 convertase (C4bC2a complex). It is a disulfide-linked polymer of seven alpha-chains and a unique beta-chain; the alpha- and beta-chains are composed of eight and three complement control protein (CCP) domains, respectively. To elucidate the importance of the polymeric nature of C4BP and the structural requirements for the interaction between C4b and the alpha-chain, 19 recombinant C4BP variants were created. Six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally, four variants in which double alanine residues were introduced between CCPs were functionally characterized. The smallest truncated C4BP variant still active in regulating fluid phase C4b comprised CCP1-3. The monomeric variants were less efficient than polymeric C4BP in degrading C4b on cell surfaces. All three N-terminal CCP domains contributed to the binding of C4b and were important for full functional activity; CCP2 and CCP3 were the most important. The spatial arrangements of the first CCPs were found to be important, as introduction of alanine residues between CCPs 1 and 2, CCPs 2 and 3, and CCPs 3 and 4 resulted in functional impairment. The results presented here elucidate the structural requirements of individual CCPs of C4BP, as well as their spatial arrangements within and between subunits for expression of full functional activity.
Blom
A M
AM
Lund University Wallenberg Laboratory, Department of Clinical Chemistry, University Hospital Malmö, S-205 02 Malmö, Sweden. Anna.Blom@klkemi.mas.lu.se
Kask
L
L
Dahlbäck
B
B
D001665:Binding Sites; D015935:Complement C4b / Q000378:metabolism; D003169:Complement Inactivator Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D006801:Humans; D011951:Receptors, Complement / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D003169:Complement Inactivator Proteins; D018076:DNA, Complementary; D006023:Glycoproteins; D011951:Receptors, Complement; D011994:Recombinant Proteins; D015935:Complement C4b