RESUMO
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.
Assuntos
Toxinas Bacterianas , Bombyx/enzimologia , Bombyx/genética , Antígenos CD13/genética , Proteínas de Insetos , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Animais , Bacillus thuringiensis , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Sequência de Bases , Antígenos CD13/isolamento & purificação , Antígenos CD13/metabolismo , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Sistema Digestório/enzimologia , Endotoxinas/metabolismo , Genes de Insetos , Glicosilfosfatidilinositóis/metabolismo , Proteínas Hemolisinas , Humanos , Manduca/enzimologia , Manduca/genética , Dados de Sequência Molecular , Peso Molecular , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
An aminopeptidase N (APN) isozyme having the molecular weight of 90 kDa, was released by phosphatidylinositol-specific phospholipase C (PI-PLC) and purified homogeneously, from the brush border membrane of Bombyx mori. From the result of cDNA cloning, the primary structure of 90 kDa APN proved to consist of 948 amino acid residues, containing a typical metalloprotease-specific zinc-binding motif in the deduced sequence. Moreover, the primary sequence contained two hydrophobic segments on N- and C-termini. The N-terminal one showed characteristics of leader peptide for secretion and the C-terminal one contained a possible glycosylphosphatidylinositol (GPI) anchoring site, suggesting that the APN encoded by the cDNA is not only a zinc-binding enzyme, but also a GPI-anchored protein. The primary sequence is significantly homologous with those of insect and mammalian APNs, and contains four conserved segments around the zinc-binding motif, two potential N-glycosylation sites and four conserved Cys residues. The deduced primary sequence had 30.7% identity with that of B. mori 110 kDa APN, and did not contain the N-terminal and internal amino acid sequences of B. mori 100 kDa APN, revealing B. mori 90 kDa APN to be the third isozyme on the midgut brush border membrane. On the other hand, the primary sequence of 90 kDa APN showed high homology with Manduca sexta APN2 (65.1% identity) and Plutella xylostella APN2 (63.8% identity). It appears that the B. mori 90 kDa APN should be classified in the insect apn2 cluster and differentiated from insect apn1 and mammalian apn clusters by phylogenetic analysis. These results suggest that 90 kDa APN isozyme encoded by the cDNA is a product of B. mori apn2 gene.