Similar to P176, no significant binding by any rScl protein was detected for fibrinogen, decorin, heparin, collagens type I, and IV (data not shown). In general, the recombinant rScl1 constructs, derived from Scl1 proteins, bound cFn and Lm (Fig. 4a), while the Scl2-protein-based constructs

P163, P177, and P178 were ECM-binding negative. Furthermore, none of the rScl1 proteins tested bound pFn, which is in agreement with our previous reports showing that those rScl1 proteins did not bind pFn from human plasma by affinity chromatography (Han et al., 2006a; Caswell et al., 2008b). All LDL-binding constructs derived from Scl1 proteins Crenolanib supplier of the M1-, M28-, M41-, M12-, M2-, and M52-type GAS (Han et al., 2006a) showed ECM binding, although to varying degrees. However, the CFH/CFHR-1-binding rScl1s originating from the M6- and M55-type GAS (Caswell et al., 2008b) did not show any significant binding to ECM ligands. In order to determine the region of Scl1 responsible for binding

to ECM proteins, an ELISA was performed using chimeric rScl constructs generated by domain swapping (Fig. 4b). We used two types of chimeric molecules: (1) derived from the ECM-binding positive (+) construct P144 (Scl1.1 of M1-type GAS) and the ECM-binding negative (−) construct P177 (Scl2 of M4-type GAS) and (2) constructs derived from the ECM-binding positive P144 and the ECM-binding negative P179 (Scl1 of M6-type GAS). The rScl1 (+)–rScl2 (−) chimeric construct P183 (P144V/P177CL), but not P184 (P177V/P144CL), bound cFn and Lm. Likewise, the rScl1 (+)–rScl1 (−) chimeric construct P213 (P144V/P179CL), but not P212 (P179V/P144CL), selleck products bound cFn and Lm. These data strongly indicate that, indeed, the Scl1-V region is responsible for mediating interactions with ECM proteins. The present and previous results underscore the functional diversity of the Scl1-V region. Of particular interest to us is the emergence of two main binding patterns Chloroambucil among Scl1 variants. The more common pattern includes binding of plasma LDL and ECM components cFn and Lm, which may represent an intriguing adaptation of Scl1 to either the blood or the tissue environment. Our previous molecular evolutionary genetic analysis

identified an elevated constraint of the Scl1-V region sequence, suggesting that this region responds to selective pressure (Lukomski et al., 2000). Inasmuch as the amino acid sequence in the V-region differs between Scl1 proteins of different M-types, the prediction of two α-helices (Rasmussen et al., 2000; Han et al., 2006a) and the globular structure of the Scl1-V domain (Xu et al., 2002; Han et al., 2006b) seem to be conserved among all Scl1 proteins. The present work provides a platform for future investigations that will determine the Scl1-ECM-binding affinities and identify the specific amino acid sequences or structural motifs of Scl1 variants that constitute the molecular basis for the Scl1-ligand (ECM, LDL, and CFH) recognition. We thank S. Beres for providing plasmid pSB027.