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, wa s released by phosphatidylinositol-specific phospholipase C (PI-PLC) and pu rified homogeneously, from the brush border membrane of Bombyx mori. From t he result of cDNA cloning, the primary structure of 90 kDa APN proved to co nsist of 948 amino acid residues, containing a typical metalloprotease-spec ific zinc-binding motif in the deduced sequence. Moreover, the primary sequ ence contained two hydrophobic segments on N- and C-termini. The N-terminal one showed characteristics of leader peptide for secretion and the C-termi nal one contained a possible glycosylphosphatidylinositol (GPI) anchoring s ite, 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 significa ntly homologous with those of insect and mammalian APNs, and contains four conserved segments around the zinc-binding motif, two potential N-glycosyla tion sites and four conserved Cys residues. The deduced primary sequence ha d 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, reveal ing B. mori 90 kDa APN to be the third isozyme on the midgut brush border m embrane. 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 cla ssified 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. (C) 1998 Elsevier Science Inc. All rights reserved.