Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori , That Binds to Cry1A Toxins

Abstract

We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and K d constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc.

Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

“…Differential lectin binding of B. mori polycalin suggests that it may have both N-and O-linked GalNAc, including terminal GalNAc, yet binding by Cry1Ac was not inhibited by GalNAc (Hossain et al, 2004), a result that contrasts with purification method reported here for the protein from H. armigera. The H. armigera polycalin has two Asn-Xaa-Ser/Thr sequons that may carry N-linked glycan, five predicted O-linked sites and a predicted GPI anchor site but a mucin-like region is not predicted.…”

“…This polycalin was characterised as a binding protein for chlorophyllid-A, the prosthetic group from chlorophyll (Mauchamp et al, 2006). The polycalin sequence of B. mori was subsequently matched with two peptides from a previously unidentified, very high-molecular-weight protein that bound Cry1Aa, Cry1Ab and Cry1Ac in heterologous competition binding studies (Hossain et al, 2004;Hossain et al, 2005;Pigott and Ellar, 2007).…”

Investigation of physicochemical condition to stabilize phosphatidylcholine-liposome enclosing fluorescent calcein and its exploitation for analysis of pore formation with Cry1A toxins of Bacillus thuringiensis

“…B. thuringiensis sotto T84A1, a gift from Dr. M. Ohba, and B. thuringiensis kurstaki HD-73 were used to prepare Cry1Aa9 and Cry1Ac1, respectively, as previously described (Hossain et al, 2004). The bacteria were cultured in 500 ml bot-tom-baffled flasks containing 100 ml NYS medium, as in Suzuki et al (1992).…”

Cry10Aa Protein is Highly Toxic to <i>Anthonomus grandis</i> Boheman (Coleoptera: Curculionidae), an Important Insect Pest in Brazilian Cotton Crop Fields

“…The best characterized among them are the APN receptor (Pigott and Ellar, 2007) and the cadherin-like receptor (Gahan et al, 2001;Likitvivatanavong et al, 2011;Pacheco et al, 2009;Valaitis et al, 2001) identified in lepidopterans. Other putative receptors include ALP (Fernández et al, 2006;Jurat-Fuentes and Adang, 2004;Jurat-Fuentes and Adang, 2006), a 270 kD glycoconjugate (Valaitis et al, 2001), and a 252 kD protein (Hossain et al, 2004).…”