Characterization of a membrane-bound aminopeptidase purified from Acyrthosiphon pisum midgut cells

A major binding site for toxic mannose lectins

Authors


  • Database
    The sequence described here has been deposited in the GenBank database with the accession number DQ440823

Y. Rahbé, UMR INRA-INSA de Lyon BF2I, Biologie Fonctionnelle Insectes & Interactions, Bat. Louis-Pasteur, F-69621 Villeurbanne cedex, France Fax: +33 4 72 43 85 34 Tel: +33 4 72 43 83 56 E-mail: yvan.rahbe@jouy.inra.fr

Abstract

A single membrane-bound aminopeptidase N (APN) occurs in the pea aphid (Acyrthosiphon pisum Harris) midgut, with a pH optimum of 7.0, pI of 8.1 and molecular mass of 130 kDa. This enzyme accounts for more than 15.6% of the total gut proteins. After being solubilized in detergent, APN was purified to homogeneity. The enzyme is a glycoprotein rich in mannose residues, which binds the entomotoxic lectins of the concanavalin family. The internal sequence of APN is homologous with a conservative domain in APNs, and degenerated primers of highly conserved APN motifs were used to screen a gut cDNA library. The complete sequence of APN has standard residues involved in zinc co-ordination and catalysis and a glycosyl-phosphatidylinositol anchor, as in APNs from Lepidoptera. APN has a broad specificity towards N-terminal amino acids, but does not hydrolyze acidic aminoacyl-peptides, thus resembling the mammalian enzyme (EC 3.4.11.2). The kcat/Km ratios for different di-, tri-, tetra-, and penta-peptides suggest a preference for tripeptides, and that subsites S1, S2′ and S3′ are pockets able to bind bulky aminoacyl residues. Bestatin and amastatin bound APN in a rapidly reversible mode, with Ki values of 1.8 µm and 0.6 µm, respectively. EDTA inactivates this APN (kobs 0.14 m−1·s−1, reaction order of 0.44) at a rate that is reduced by competitive inhibitors. In addition to oligopeptide digestion, APN is proposed to be associated with amino-acid-absorption processes which, in contrast with aminopeptidase activity, may be hampered on lectin binding.

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