Localization of a novel 252-kDa plasma membrane protein that binds Cry1A toxins in the midgut epithelia of Bombyx mori

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We previously demonstrated that a novel 252-kDa protein (P252) isolated from brush border membranes (BBM) of Bombyx mori, hybrid Shurei×Shogetsu, specifically bound Cry1Aa, Cry1Ab and Cry1Ac toxins with a K_d of 29, 179 and 20 nM, respectively. P252 was found in a Triton X-100-soluble fraction of BBM from first, third and fifth instar larvae, suggesting that it may be an important element of midgut epithelial cell membranes. P252 was not partitioned into a Triton X-100-insoluble BBM fraction and nearly identical partitioning of P252 was observed using detergents CHAPS and Igepal. These results suggested that P252 localized in non-raft regions of BBM. Immunofluorescence analysis using anti-P252 antiserum demonstrated the existence of P252 in BBMV. Cy3-labeled Cry1Aa, Cry1Ab and Cry1Ac were shown to bind to BBMV, and the addition of anti-P252 antiserum reduced the number of BBMV showing Cy3 fluorescence by 30%. This clearly suggested an important role for P252 in Cry1A binding to BBMV. CD spectra of a mixture of purified P252 with either Cry1Aa, Cry1Ab or Cry1Ac were compared with those of respective free Cry1A toxins and only one of the mixtures (Cry1Aa/P252) was shown to be significantly changed compared to that of native Cry1Aa.
2005-02-25