Membrane‐penetrating trehalase from silkworm Bombyx mori. Molecular cloning and localization in larval midgut

Abstract

The main blood sugar in insects, trehalose, differs from glucose in mammals. To incorporate trehalose into cells and utilize it, tissue cells possess the enzyme trehalase (EC3.2.1.28), which catalyses trehalose into glucose, in the organellar membrane or in the cytoplasm. Soluble and membrane-bound trehalase proteins have been isolated from insects. To date, however, only genes encoding the soluble trehalase have been reported in insects. Soluble trehalase is therefore believed to become localized on the cell surface via modification. In contrast, cDNAs encoding trehalase localized on the apical cell surface via the glycosylphosphatidylinositol-anchor have been isolated from mammalian small intestines. The amino acid sequence contains a specific hydrophobic region and an upstream omega site, which is cleaved for glycosylphosphatidylinositol-attachment, at the C-terminus. Here, we describe a cDNA from the silkworm Bombyx mori that encodes a novel trehalase (type-2) with one transmembrane domain and lacking the omega site. Immunoblotting and immunohistochemical analyses demonstrated that in the midgut tissue of Bombyx larvae, soluble trehalase-1 is present mainly in goblet cell cavities, but membrane-bound trehalase-2 is predominantly seen on the visceral muscle surrounding the midgut. To our knowledge, this is the first report of a cDNA encoding trehalase that penetrates the cell membrane in insects and its cellular localization.

Molecular Cloning of cDNA for Trehalase from the European Honeybee,Apis melliferaL., and Its Heterologous Expression inPichia pastoris

“…Figure 3 shows the multiple alignment of the sequences for our honeybee LG2 trehalase and the honeybee trehalase homolog in LG14, together with those for 7 representative trehalases from GH37. The honeybee trehalase showed amino acid identity to the GH37 trehalases from silkworm B. mori (39 and 51% to trehalase-1 33) and -2, 7) respectively), human 34) (40%), rabbit 22) (40%), and mealworm beetle Tenebrio molitor 35) (39%), brine shrimp A. franciscana 19) (38%), and E. coli (TreA, 36) 28%). Therefore, the honeybee trehalase as well as the homolog of LG14 was categorized as a new GH37 member based on amino acid sequence similarity.…”

“…18) The silkworm trehalase-2 (bound type) possessing a putative hydrophobic transmembrane region on the C-terminal side of the amino acid sequence, 7) as well as the trehalase of the brine shrimp Artemia franciscana, 19) is considered to be anchored to cell membrane by penetration of the specific hydrophobic region into the membrane, 20) while the trehalases of rabbit and human are bound to the brash-border membrane via glycosylphosphatidylinositol. 21,22) In our previous paper, 13) the bound trehalase was purified from the European honeybee, and the kinetic study indicated that two carboxy groups were involved directly in the catalysis as catalytic ionizable groups, but the catalytic amino acid residues are obscure in all of the GH37 enzymes.…”

“…Key words: trehalase; honeybee; cDNA; heterologous expression Trehalases (,-trehalase, ,-trehalose glucohydrolase, EC 3.2.1.28) are widely distributed in microorganisms, 1,2) plants, 3,4) invertebrates, [5][6][7] and vertebrates. [8][9][10] The primary structures of trehalase reported are classified into two groups: glycoside hydrolase family 37 (GH37) 11) for most of trehalases, and GH65 for some acid trehalases together with phosphorylases.…”

Catalytic Reaction Mechanism Based on α-Secondary Deuterium Isotope Effects in Hydrolysis of Trehalose by European Honeybee Trehalase

“…While trehalases are widely distributed in microorganisms, 6,7) plants, 8,9) invertebrates, [10][11][12] and vertebrates, [13][14][15] their physiological functions are poorly characterized, except that trehalase(s) in flying insects can be regarded as particularly significant in the rapid supply of glucose by hydrolyzing trehalose. However, several trehalases from different sources 16) have been reported to be capable of hydrolyzing specifically synthetic substrates, for instance, -D-glucopyranosyl -D-galactopyranoside, -D-xylopyranosyl -D-glucopyranoside, and -D-glucopyranosyl 6-deoxy--D-glucopyranoside.…”

Comparison of gut morphology and distribution of trehalase activity in the gut of wood-feeding and fungus-growing termites (Isoptera: Termitidae)

“…crassus and B. prabhae. The high trehalase activity in the midgut must considered in terms of the activity recorded in the lumen of the midgut and in the haemocoel around the midgut (Mitsumasu et al, 2005;Tatun et al, 2008). The latter may serve to supply glucose to the gut cells for their basic energy requirements and to other tissues to be used in reproduction and colony defence (soldier caste).…”