Characterization of a β-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to β-glucan-binding proteins.
Spodoptera frugiperda β-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against β-1,3-glucan (laminarin), but cannot hydrolyze yeast β-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive β-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of β-1,3-glucanases and β-glucan-binding protein supports the assumption that the β-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived β-1,3-glucanases by the loss of an extended N-terminal region and the β-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells.
Bragatto
Ivan
I
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, CP 26077, 05513-970 São Paulo, Brazil.
Genta
Fernando A
FA
Ribeiro
Alberto F
AF
Terra
Walter R
WR
Ferreira
Clélia
C
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000737:chemistry / Q000378:metabolism; D043326:Glucan 1,3-beta-Glucosidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005936:Glucans; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D007814:Larva / Q000201:enzymology; D037102:Lectins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism; D016415:Sequence Alignment; D018411:Spodoptera / Q000201:enzymology* / Q000254:growth & development
D002352:Carrier Proteins; D005936:Glucans; D019476:Insect Proteins; D037102:Lectins; D011134:Polysaccharides; C037937:glucan-binding proteins; C008247:laminaran; D043326:Glucan 1,3-beta-Glucosidase