CRYPTIC SELF-ASSOCIATION SITES IN TYPE-III MODULES OF FIBRONECTIN

The first type III module of fibronectin (Fn) contains a cryptic site that binds Fn and its N-terminal 29 kDa fragment and is thought to be important for fibril formation (Morla, A., Zhang, Z., and Ruoslahti, E . (1994) Nature 367, 193-196; Hocking, D. C., Sottile, J., and McKeown -Longo, P. J. (1994) J. Biol. Chem. 269, 19183-19191). A synthetic 31- mer peptide (NAPQ...TIPG) derived from the middle of domain III1 was a lso shown to bind Fn, but the site of its interaction was not determin ed (Morla, A., and Ruoslahti, E. (1992) J. Cell Biol. 118, 421-429). B y affinity chromatography on peptide-agarose, we tested a set of fragm ents representing the entire light chain of plasma Fn. Only 40-kDa Hep -2 (III12-15) failed to bind. The concentration of urea required for p eak elution of Fn and the other fragments decreased in the order Fn > 42-kDa GBF (I6II1-2I7-9) > 19-kDa Fib-2 (I-10-12) > 110-kDa CBF(III2-1 0) > 29-kDa Fib-1 (I-1-I-5). Neither Fn nor any of the fragments bound immobilized intact III1, confirming the cryptic nature of this activi ty. In an effort to detect interactions between other Fn. domains, all fragments were coupled to Sepharose, and each fragment was tested on each affinity matrix before and after denaturation. The only interacti on detected was that of fluid phase III1 with immobilized denatured 11 0-kDa CBF and 40 kDa Hep-2, both of which contain type III domains. An alysis of subfragments revealed this activity to be dominated by domai ns III7 and III15. Fn itself did not bind to the denatured fragments. Thus, domain III, contains two cryptic ''self-association sites,'' one that is buried in the core of the fold but recognizes many Fn fragmen ts when presented as a peptide and another that is concealed in Fn but exposed in the native isolated domain and recognizes cryptic sites in two other type III domains. These interactions between type III domai ns could play an important role in assembly of Fn multimers in the ext racellular matrix.