Molecular cloning of a GPI-anchored aminopeptidase N from Bombyx mori midgut: a putative receptor for Bacillus thuringiensis CryIA toxin.
An aminopeptidase N (APN) with a molecular weight of 110kDa was released from the midgut membrane of Bombyx mori by phosphatidylinositol-specific phospholipase C (PI-PLC), and purified to a homogeneous state. This 110-kDa APN was different from the 100-kDa APN that we previously reported, in chromatographic behaviors, substrate specificity, and N-terminal and internal amino acid sequences. However, the N-terminal sequence of 110-kDa APN, DPAFRLPTTTRPRHYQVTLT, was highly homologous with those of Manduca sexta and Heliothis virescens APNs, which were identified as a receptor for an insecticidal toxin of Bacillus thuringiensis. From a B. mori midgut cDNA library, we cloned the 110-kDa APN cDNA that possessed a 2958-bp open reading frame encoding a 111573-Da polypeptide of 986 residues. The sequence of the eicosa-peptide Asp42Thr61 deduced from the cDNA was completely matched with the N-terminal sequence of the mature 110-kDa APN. One potential N-glycosylation site, HEXXHXW zinc-binding motif and characteristic proline-rich repeats were observed in the ORF. Moreover, the primary sequence contained two hydrophobic peptides on N- and C-termini. The N-terminal peptide sequence showed characteristics of leader peptide for secretion and the C-terminal peptide contained a possible glycosylphosphatidylinositol (GPI) anchoring site. Taken together, the deduced amino acid sequence suggests that the 110-kDa APN is a GPI-anchored protein and a specific receptor protein for B. thuringiensis CryIA delta-endotoxin.
Hua
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Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan.
Tsukamoto
K
K
Rasilo
M L
ML
Ikezawa
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism; D001427:Bacterial Toxins; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics*; D018826:CD13 Antigens / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004064:Digestive System / Q000201:enzymology; D004731:Endotoxins / Q000378:metabolism; D017344:Genes, Insect; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006460:Hemolysin Proteins; D006801:Humans; D019476:Insect Proteins; D018402:Manduca / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C095416:Cry toxin receptors; D017931:DNA Primers; D018076:DNA, Complementary; D004731:Endotoxins; D017261:Glycosylphosphatidylinositols; D006460:Hemolysin Proteins; D019476:Insect Proteins; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens