Monoclonal Antibodies to Spirosin of Yersinia enterocolitica and Analysis of the Localization of Spirosome by Use of Them

Abstract

Two hybridomas producing monoclonal antibodies (MAbs) were prepared by fusing myeloma cells (Sp2/0-Ag14) with mouse spleen cells immunized with purified spirosin from Yersinia enterocolitica SYT-11-72 (YE72). The antibodies produced by them were designated MAbs-S5 and S27. They were IgG2a and IgGi, respectively, both with k light chains. MAbs-S5 and S27 reacted specifically with spirosin from YE72. On Western blotting after limited proteolysis with Staphylococcus aureus V8 protease, YE72 spirosin revealed peptide fragments of 35 and 37 kDa reacting markedly with MAb-S5, which suggested the presence of an antigenic determinant on these fragments. By cellular fractionation of YE72 and subsequent EIA and Western blot analysis, spirosome was shown to be present in the cytoplasm of YE72.

Purification of spirosin from Lactobacillus reuteri DSM 20016 and partial characterization based on its amino acid sequences.

“…The 95 kDa protein band stained with Coomassie Brilliant Blue R250 was cut off and analyzed for the sequence on a model HPG1005A Protein Sequencing System (Hewlett-Packard, Inc., Palo Alto, CA, USA). However, the N-terminal amino acid sequence of spirosin from L. reuteri DSM 20016 could not be determined, probably because of N-acylation as in the case of L. brevis (Yamato et al, 1994). Since the amino acid sequence from L. acidophilus ATCC 9857 could not be detected either, N-acylation seems to be a chemical feature common to the Lactobacillus spirosins.…”

“…The spirosome from Lactobacillus and Enterobacteriaceae is composed of 95 kDa protein subunits called spirosin (Ueki et al, 1982;Yamato et al, 1995). There have been many reports on morphological, biochemical and immunochemical studies of spirosome as quoted in our previous papers (Yamato et al, 1994(Yamato et al, , 1995. Though some functional aspects of spirosome have been described so far Matayoshi et al, 1989), its function is not yet well understood.…”

“…Spirosin did not bind to the antibody ligand presumably because it might have a polymeric structure in a solution, and therefore its antigenic determinant might be buried as suggested previously (Yamato et al, 1997). It was finally obtained by a preparative SDS-PAGE as described in a previous paper (Yamato et al, 1994). Each step of purification was monitored on SDS-PAGE by the method of Laemmli (1970) as shown in Fig.…”

Chromatographic Study of Spirosin by Use of Monoclonal Antibodies to Spirosin of Yersinia enterocolitica

“…In a previous paper (12), we described the preparation of a monoclonal antibody (MAb-S5) specific for spirosin of Yersinia enterocolitica SYT- and the localization of spirosome in the bacterial cytoplasm as well. Recently, Kessler et al have shown that pyruvate-formate-lyase-deactivase and acetyl-CoA reductase of E. coli reside on a polymeric protein particle encoded by adhE which they speculated might be identical to spirosome (7).…”

“…Imamura et al have demonstrated that a cytoplasmic protein designated p100-A of E. coli has a spirosome-like structure and that the N-terminal amino acid sequence of p100-A is identical to that of alcohol dehydrogenase (ADH) encoded by adhE gene (2). The identity of the N-terminal amino acid sequence of spirosin to that of ADH in E. coli (1,12) prompted us to suggest that spirosin is probably ADH. Morphological analysis in electron microscopy also supported the identity of the two substances (2,7).…”