CHEMICAL SYNTHESIS AND CHARACTERIZATION OF THE EPIDERMAL GROWTH FACTOR-LIKE MODULE OF HUMAN-COMPLEMENT PROTEASE CLR

C1r is one of the two serine proteases of C1, the first component of c omplement, in which it is associated in a calcium-dependent manner to the homologous serine protease C1s. This interaction is mediated by th e N-terminal region of C1r, which comprises a single epidermal growth factor (EGF)-like module containing the consensus sequence required fo r calcium binding, surrounded by two CUB modules. With a view to deter mine the structure of the EGF-like module of C1r and evaluate its cont ribution to calcium binding, this module [C1r(123-175)] was synthesize d by automated solid-phase methodology using the Boc strategy. A first synthesis using the Boc-His(Z) derivative gave very low yield, due to partial deprotection of His residues leading to chain termination by acetylation, and to insertion of glycine residues. This could be circu mvented by using the Boc-His(DNP) derivative and by condensation of ap propriate glycine-containing segments. The synthetic peptide was effic iently folded under redox conditions to the species with three correct disulfide bridges, as determined by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. The homogeneity of the syn thetic peptide was assessed by reversed-phase HPLC and electrospray ma ss spectrometry. One-dimensional H-1 NMR spectroscopic analysis provid ed evidence that the EGF-like module had a well defined structure, and was able to bind calcium with an apparent K-d of 10 mM. This value, c omparable to that found for the isolated EGF-like modules of coagulati on factors IX and X, is much higher than that measured for native C1r. As already proposed for factors IX and X, it is suggested that neighb ouring module(s), most probably the N-terminal CUB module, contribute( s) to the calcium binding site. (C) Munksgaard 1997.