1. A Novel Glycosylphosphatidylinositol-Anchored Glycoside Hydrolase from Ustilago esculenta Functions in β-l,3-Glucan Degradation.
- Author
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Nakajima, Masahiro, Yamashita, Tetsuro, Takahashi, Machiko, Nakano, Yuki, and Takeda, Takumi
- Subjects
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GLYCOSYLPHOSPHATIDYLINOSITOL , *GLYCOSIDASES , *USTILAGO , *GLUCAN 1,3-beta-glucosidase , *CHEMICAL decomposition , *FUNGAL cultures , *GEL permeation chromatography , *ION exchange chromatography - Abstract
A glycoside hydrolase responsible for laminarin degradation was partially purified to homogeneity from a Ustilago esculenta culture filtrate by weak-cation-exchange, strong-cation-exchange, and size-exclusion chromatography. Three proteins in enzy-matically active fractions were digested with chymotrypsin followed by liquid chromatography-tandem mass spectrometry (LC/ MS/MS) analysis, resulting in the identification of three peptide sequences that shared significant similarity to a putative β-1,3-glucanase, a member of glucoside hydrolase family 16 (GH16) from Sporisorium reilianum SRZ2. A gene encoding a laminarin-degrading enzyme from 17. esculenta, lam16A, was isolated by PCR using degenerate primers designed based on the S. reilianum SRZ2 β-1,3-glucanase gene. Lam16A possesses a GH16 catalytic domain with an N-terminal signal peptide and a C-terminal gly-cosylphosphatidylinositol (GPI) anchor peptide. Recombinant Lam16A fused to an N-terminal FLAG peptide (Lam16A-FLAG) overexpressed in Aspergillus oryzae exhibited hydrolytic activity toward β-1,3-glucan specifically and was localized both in the extracellular and in the membrane fractions but not in the cell wall fraction. Lam16A without a GPI anchor signal peptide was secreted extracellularly and was not detected in the membrane fraction. Membrane-anchored Lam16A-FLAG was released completely by treatment with phosphatidylinositol-specific phospholipase C. These results suggest that Lam16A is anchored in the plasma membrane in order to modify β-1,3-glucan associated with the inner cell wall and that Lam16A is also used for the catab-olism of β-1,3-glucan after its release in the extracellular medium. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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