Characterization of a membrane-bound aminopeptidase purified from Acyrthosiphon pisum midgut cells: a major binding site for toxic mannose lectins

P.T. Cristofoletti (1) , F.A. Mendonça de Sousa (2) , Yvan Rahbé (2) , W.R. Terra (1)

1 Departamento de Bioquímica
2 BF2I - Biologie Fonctionnelle, Insectes et Interactions

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.

Mots clés: aminopeptidase N; Aphididae; glycosylphosphatidylinositol (GPI) anchor; mannose lectin receptor; substrate specificity

Mots clés: 1-1-1 Article périodique à comité de lecture

Domaines: Interactions entre organismes

P.T. Cristofoletti, F.A. Mendonça de Sousa, Yvan Rahbé, W.R. Terra. Characterization of a membrane-bound aminopeptidase purified from Acyrthosiphon pisum midgut cells: a major binding site for toxic mannose lectins. FEBS Journal, 2006, 273 (24), pp.5574-5588. ⟨10.1111/j.1742-4658.2006.05547.x⟩. ⟨hal-00391293⟩