Mechanism of interaction of the 85B secreted protein of Mycobacterium bovis with fibronectin

Abstract

The 85B protein of Mycobacterium bovis is a member of the secreted antigen 85 complex, which has been identified in a number of pathogenic mycobacteria. The 85 complex contains three components with molecular masses of 30 to 32 kDa which share the property of binding to fibronectin, a large glycoprotein present in plasma. To investigate this activity 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 immunosorbent assay (ELISA) and in the fluid phase in a radioimmunoassay using 125I-labelled 85B protein. In addition, fibronectin reacted with immobilized 85B in immunoblots and vice versa. Fibronectin also bound to three fragments of a cyanogen bromide digest of 85B which were subsequently 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 the collagen-binding domain. This region was confirmed as the 85B binding site by the fact that gelatin but not heparin inhibited the binding of fibronectin to 85B. These data indicate that the 85B-fibronectin interaction involves the binding of multiple regions of the 85B protein to the collagen-binding domain of fibronectin.

Identification of a 47 kDa fibronectin‐binding protein expressed by Borrelia burgdorferi isolate B31

“…These results indicated that the region of fibronectin bound by P47 overlapped with the fibronectin domain that binds gelatin and collagen. A similar site of fibronectin binding has been reported for the 85B protein of Mycobacterium bovis (Peake et al, 1993). Interestingly, the interaction of fibronectin with P47 was enhanced by prior incubation of the fibronectin probe with heparin.…”

Cross‐Reactive Immune Responses Against Mycobacterium bovis BCG in Mice Infected with Non‐Tuberculous Mycobacteria Belonging to the MAIS‐Group

“…᭧ 1997 Blackwell Science Ltd, Scandinavian Journal of Immunology, 46, [16][17][18][19][20][21][22][23][24][25][26] levels against BCG culture filtrate antigens, similar to what we previously described in the BCG model [10]. Thus, B6 mice infected with M. scrofulaceum demonstrated the highest levels of IFN-g in response to BCG culture filtrate, BCG extract and purified Ag85 and these mice were the most competent in controlling the infection.…”

“…[24]. Ag85 complex binds to fibronectin [25] and possesses enzymatic threhalose-mycolyl-transferase activity [26]. We and others have previously shown than Ag85 complex from BCG is a potent IL-2 and IFN-g inducer in healthy individuals primo-infected with M. tuberculosis/M.…”

Characterization of the Gene Encoding the MPB51, One of the Major Secreted Protein Antigens of Mycobacterium bovis BCG, and Identification of the Secreted Protein Closely Related to the Fibronectin Binding 85 Complex

“…. was part of the most hydrophobic region of MPB51, components of the antigen 85 complex, and part of the fibronectin binding site [36]. No significant homology to the other amino acid sequences present in the latest SwissProt-NBRF dala bank was detected.…”