title: Insect midgut α-mannosidases from family 38 and 47 with emphasis on those of Tenebrio molitor.
abstract: α-Mannosidases are enzymes which remove non-reducing terminal residues from glycoconjugates. Data on both GH47 and GH38 (Golgi and lysosomal) enzymes are available. Data on insect midgut α-mannosidases acting in digestion are preliminary and do not include enzyme sequences. Tenebrio molitor midgut α-mannosidases were separated by chromatography into two activity peaks: a major (Man1) and a minor (Man2). An antibody generated against a synthetic peptide corresponding to a sequence of α-mannosidase fragment recognizes Man2 but not Man1. That fragment was later found to correspond to TmMan2 (GenBank access KP892646), showing that the cDNA coding for Man2 is actually TmMan2. TmMan2 codes for a mature α-mannosidase with 107.5 kDa. Purified Man2 originates after SDS-PAGE one band of about 72 kDa and another of 51 kDa, which sums 123 kDa, in agreement with gel filtration (123 kDa) data. These results suggest that Man2 is processed into peptides that remain noncovalently linked within the functional enzyme. The physical and kinetical properties of purified Man1 and Man2 are similar. They have a molecular mass of 123 kDa (gel filtration), pH optimum (5.6) and response to inhibitors like swainsonine (Man1 Ki, 68 nM; Man2 Ki, 63 nM) and deoxymannojirimycin (Man1 Ki, 0.12 mM; Man2 Ki, 0.15 mM). Their substrate specificities are a little different as Man2 hydrolyzes α-1,3 and α-1,6 bonds better than α-1,2, whereas the contrary is true for Man1. Thus, they pertain to Class II (GH38 α-mannosidases), that are catabolic α-mannosidases similar to lysosomal α-mannosidase. However, Man2, in contrast to true lysosomal α-mannosidase, is secreted (immunocytolocalization data) into the midgut contents. There, Man2 may participate in digestion of fungal cell walls, known to have α-mannosides in their outermost layer. The amount of family 38 α-mannosidase sequences found in the transcriptome (454 pyrosequencing) of the midgut of 9 insects pertaining to 5 orders is perhaps related to the diet of these organisms, as suggested by a large number of lysosomal α-mannosidase in the T. molitor midgut.
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil.
authors.forename: Nathalia R
authors.initials: NR
authors.lastname: Moreira
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil.
authors.forename: Christiane
authors.initials: C
authors.lastname: Cardoso
authors.affiliation: Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo, C.P. 11461, 05513-970 São Paulo, Brazil.
authors.forename: Alberto F
authors.initials: AF
authors.lastname: Ribeiro
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil.
authors.forename: Clelia
authors.initials: C
authors.lastname: Ferreira
authors.affiliation: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, C.P. 26077, 05513-970 São Paulo, Brazil. Electronic address: warterra@iq.usp.br.
authors.forename: Walter R
authors.initials: WR
authors.lastname: Terra
mesh_terms: D000818:Animals; D005260:Female; D041981:Gastrointestinal Tract / Q000201:enzymology; D019476:Insect Proteins / Q000737:chemistry* / Q000302:isolation & purification; D007700:Kinetics; D007814:Larva / Q000201:enzymology; D008297:Male; D008351:Mannans / Q000378:metabolism; D013379:Substrate Specificity; D013712:Tenebrio / Q000201:enzymology* / Q000235:genetics; D043323:alpha-Mannosidase / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D019476:Insect Proteins; D008351:Mannans; C014647:mannobiose; D043323:alpha-Mannosidase
keywords: Alpha-mannosidase; Immunocytolocalization; Post-translational processing; Substrate specificity