MECHANISM OF INTERACTION OF THE 85B SECRETED PROTEIN OF MYCOBACTERIUM-BOVIS WITH FIBRONECTIN

The 85B protein of Mycobacterium bovis is a member of the secreted ant igen 85 complex, which has been identified in a number of pathogenic m ycobacteria. The 85 complex contains three components with molecular m asses of 30 to 32 kDa which share the property of binding to fibronect in, a large glycoprotein present in plasma. To investigate this activi ty we have expressed the M. bovis 85B antigen as a recombinant protein and studied its interaction with human fibronectin. Fibronectin bound to the immobilized 85B protein in a solid-phase enzyme-linked immunos orbent assay (ELISA) and in the fluid phase in a radioimmunoassay usin g I-125-labelled 85B protein. In addition, fibronectin reacted with im mobilized 85B in immunoblots and vice versa. Fibronectin also bound to three fragments of a cyanogen bromide digest of 85B which were subseq uently identified by N-terminal sequencing. These fragments contained fibronectin-reactive peptides identified in ELISAs utilizing a set of 28 overlapping 20-mer peptides encompassing the 85B sequence. Further studies showed that the 85B protein reacted with a 32-kDa polypeptide from a limited tryptic digest of fibronectin which was identified as t he collagen-binding domain. This region was confirmed as the 85B bindi ng site by the fact that gelatin but not heparin inhibited the binding of fibronectin to 85B. These data indicate that the 85B-fibronectin i nteraction involves the binding of multiple regions of the 85B protein to the collagen-binding domain of fibronectin.