A novel glycosylphosphatidylinositol-anchored glycoside hydrolase from Ustilago esculenta functions in β-1,3-glucan degradation.
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 enzymatically 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 U. 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 glycosylphosphatidylinositol (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 catabolism of β-1,3-glucan after its release in the extracellular medium.
Iwate Biotechnology Research Center, Iwate, Japan.
Masahiro
M
Nakajima
Tetsuro
T
Yamashita
Machiko
M
Takahashi
Yuki
Y
Nakano
Takumi
T
Takeda
D001236:Aspergillus oryzae / Q000235:genetics; D002845:Chromatography; D017931:DNA Primers / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006868:Hydrolysis; D013058:Mass Spectrometry; D016133:Polymerase Chain Reaction; D011509:Proteoglycans; D014588:Ustilago / Q000201:enzymology* / Q000378:metabolism*; D047071:beta-Glucans / Q000378:metabolism*
D017931:DNA Primers; D017261:Glycosylphosphatidylinositols; D011509:Proteoglycans; D047071:beta-Glucans; C010770:polysaccharide-K; D006026:Glycoside Hydrolases