GENERATION OF A MONOCLONAL-ANTIBODY THAT RECOGNIZES THE AMINO-TERMINAL DECAPEPTIDE OF THE B-SUBUNIT OF ESCHERICHIA-COLI HEAT-LABILE ENTEROTOXIN - A NEW PROBE FOR STUDYING TOXIN ASSEMBLY INTERMEDIATES

Cholera toxin and the related Escherichia coil heat-labile enterotoxin are hexameric proteins comprising one B-subunit and five B-subunits. In this paper we report the generation and characterization of a monoc lonal antibody, designated LDS47, that recognizes and precipitates in vivo assembly intermediates of the B-subunit (EtxB) of E. coil heat-la bile enterotoxin. The monoclonal antibody is unable to precipitate nat ive B-subunit pentamers, thus making LDS47 a useful probe for studying the early stages of enterotoxin biogenesis. The use of LDS47 to monit or the in vivo turnover of newly synthesized B-subunits in the peripla sm off. coil demonstrated that (i) the turnover of unassembled B-subun its followed an apparent first order process and (ii) it occurred conc omitantly with the assembly of native B-pentamers (k = 0.317 +/- 0.170 min(-1); t(1/2) = 2.2 min). No other proteins were co-precipitated wi th the newly synthesized B-subunits; a finding that implies that unass embled B-subunits do not stably associate with other periplasmic prote ins prior to their assembly into a macromolecular complex. The use of overlapping synthetic peptides corresponding to the entire EtxB polype ptide demonstrated that the epitope recognized by LDS47 is located wit hin the amino-terminal decapeptide of the B-subunit. From the x-ray st ructural analysis of the toxin (Sixma, T., Kalk, K., van Zanten, B., D auter, Z., Kingma, J., Witholt, B., and Hol, W. G. J. (1993) J. Mel. B iol. 230, 890-918), this region appears to resemble a curved finger th at clasps the adjacent B-subunit. Thus, this region might be expected to be exposed in the unfolded or unassembled subunit, but to become pa rtially buried upon assembly and thus inaccessible to recognition by t he monoclonal antibody.