Cloning and sequence analysis of the aminopeptidase N isozyme (APN2) from Bombyx mori midgut.
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.
Hua
G
G
Department of Microbial Chemistry, Faculty of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan.
Tsukamoto
K
K
Ikezawa
H
H
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000201:enzymology* / Q000235:genetics*; D018826:CD13 Antigens / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004064:Digestive System / Q000201:enzymology; D006801:Humans; D019476:Insect Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid