P. Peake et al., MECHANISM OF INTERACTION OF THE 85B SECRETED PROTEIN OF MYCOBACTERIUM-BOVIS WITH FIBRONECTIN, Infection and immunity, 61(11), 1993, pp. 4828-4834
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.