The Complement Cofactor Protein (SBP1) from the Barred Sand Bass (Paralabrax nebulifer) Mediates Overlapping Regulatory Activities of Both Human C4b Binding Protein and Factor H

Irma Gigli,Peter F. Zipfel,Claudia Kemper

doi:10.1074/jbc.273.31.19398

Irma Gigli, Peter F. Zipfel + Show 1 more

Open Access

https://doi.org/10.1074/jbc.273.31.19398

Copy DOI

Abstract

We have previously shown that serum of the teleost fish barred sand bass (Paralabrax nebulifer) cleaves the alpha'-chain of human C4b and C3b. The proteins that participate in these reactions were purified, and a specific protease and a single cofactor protein were identified. Functional characterization of the recombinantly expressed sand bass cofactor protein (SBP1) and truncated forms containing short consensus repeats (SCRs) 1-2, 1-3, 1-4, 1-5, and 12-17 revealed that SBP1 and SCRs 1-4 mediate the functional activities of the human plasma regulatory protein C4bp and factor H. They form a complex with C4b, inhibit the formation, and accelerate the decay of the classical pathway C3 convertase and display cofactor activity for the cleavage of C4b. In contrast, the interaction of SBP1 and SCRs 1-4 with human C3b in all these activities was limited. This difference is due to species-specific incompatibilities between the cofactor protein and human C3b. SBP1 and SCRs 1-5 displayed full binding and cofactor activity for methylamine-treated C3 from trout, a species closely related to the sand bass. The presence of only one cofactor in the fish plasma that combines the functional activities of C4bp and factor H demonstrates that the sand bass cofactor protein is the ancestral precursor to the two complement regulatory proteins in human plasma.

Highlights

We have previously shown that serum of the teleost fish barred sand bass (Paralabrax nebulifer) cleaves the ␣؅-chain of human C4b and C3b
1 The abbreviations used are: C, complement; C4-binding protein (C4bp), C4b binding protein; short consensus repeats (SCRs), short consensus repeat; SBP1, sand bass cofactor protein; SBP1—A PCR-generated NotI/SmaI-restricted cDNA (SB1), sand bass cofactor protein cDNA clone; Normal human serum (NHS), normal human serum; VBS, veronal-buffered saline; trout C3b-like, trout C3 converted into a C3b-like molecule by treatment with methylamine; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis
Membranes were blocked with 5% (w/v) dried milk in phosphate-buffered saline (PBS) for 30 min and incubated overnight with either a specific polyclonal rabbit antiserum raised against SBP1 [23] or a recombinantly expressed peptide representing SCRs 1–5 of SBP1

Summary

The abbreviations used are

C, complement; C4bp, C4b binding protein; SCR, short consensus repeat; SBP1, sand bass cofactor protein; SB1, sand bass cofactor protein cDNA clone; NHS, normal human serum; VBS, veronal-buffered saline; trout C3b-like, trout C3 converted into a C3b-like molecule by treatment with methylamine; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis. C4bp and factor H function as cofactors for the plasma protease factor I that mediates the degradation of C4b and C3b [5, 6, 9] During this reaction the ␣Ј-chain of C4b is cleaved into three fragments of 48 (␣2), 33 (␣3), and 27 (␣3) kDa [10]. Expressed SBP1 and truncated forms obtained by the consecutive deletion of SCRs from the C terminus of the molecule were analyzed for binding to human C4b and C3b, inhibition of the formation, and decay accelerating activity for C4b2a and C3bBb and cofactor activity in the cleavage of C4b and C3b. The functional domains of SBP1 were localized to SCRs 1– 4

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION