The cFn comprises a large group of isoforms produced from splicing events that may or may not include the type III repeats called

extra domains, EDA and EDB, lacking in pFn (Pankov & Yamada, 2002). It is currently not known whether the presence of the extra domains or suprastructural organization is responsible for the selective binding of cFn to Scl1 protein. In addition to cFn, P176 also bound Lm. The cFn and Lm binding to P176 was concentration-dependent, indicating binding specificity (Fig. 1a, inset). The laminins comprise a family of A, B1, and B2 heterotrimeric glycoproteins that are constituents of basal lamina and are found in virtually all human tissues (Alberts et al., 1994). Various isoforms of laminin exist that are associated with characteristic selleck inhibitor tissue distribution. Early studies by Switalski et al. (1984) described GAS binding to Lm, although the GAS product responsible for this binding was not identified. Terao et al. (2002) identified a GAS Lm-binding protein, designated Lbp, which was recently characterized as primarily a zinc-binding protein with capacity to bind Lm (Linke et al., 2009). GAS interactions with Lm were also attributed to another streptococcal protein Shr that primarily binds human plasma hemoproteins (Fisher et al., 2008). Thus, unrelated surface proteins of GAS possess binding capacities toward ECM components Fn and Lm. Because both cFn and

Lm contain the collagen-binding Sunitinib mouse domains (Alberts et al., 1994), we could not exclude a possibility that the CL region of Scl1 was responsible for ECM binding. Therefore, we constructed a chimeric recombinant protein by domain swapping consisting of the V-region of P176 and the CL-region of the ECM-binding negative protein P163. The resulting construct P181 bound cFn and Lm, indicating that ECM binding is mediated by the P176 V-region (Fig. 1a). We next devised a competition Adenylyl cyclase assay to investigate whether cFn and Lm binding is localized to the same site within the P176 V-region (Fig. 1b). First,

immobilized P176 was incubated with one of the primary ECM ligands, cFn or Lm, and then incubated with an alternate secondary ECM ligand. Sets of triplicate wells were immunoreacted with antibodies specific for both ECM ligands to assess the presence of cFn and Lm attached to P176. Immunoreactivities of the same amounts of P176-cFn and P176-Lm were considered as 100% binding (Fig. 1b; bars 1–2). Preincubation of P176 with cFn did not prevent Lm binding (Fig. 1b; bar 4); nor did Lm displace the cFn from P176 (Fig. 1b; bar 3). Likewise, preincubation with Lm did not prevent cFn binding to P176 (Fig. 1b; bar 5); nor was cFn able to displace the Lm from P176 (Fig. 1b; bar 6). Our data suggest that under these experimental conditions, the cFn and Lm did not compete for binding to P176. Binding between the rScl1.