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Structural investigation of α-l-fucosidase from the pancreas of Patiria pectinifera, based on molecular cloning

Ono, Akiko, Suzuki, Tomohiro, Gotoh, Saki, Kono, Haruka, Matsui, Megumi, Aoki, Daichi, Matsuda, Masaru, Kawagishi, Hirokazu, Ogata, Makoto
Carbohydrate research 2019 v.475 pp. 27-33
Asteroidea, alpha-L-fucosidase, ammonium sulfate, bioinformatics, carbohydrates, gel chromatography, gene expression, genes, molecular cloning, molecular weight, pH, pancreas, polyacrylamide gel electrophoresis, sequence analysis
An α-l-fucosidase (Pap-Alf) was purified from the pancreas of a starfish Patiria pectinifera by ammonium sulfate precipitation followed by several column chromatographies. The molecular mass of the purified enzyme was estimated to be 52.6 kDa by SDS-PAGE, although gel filtration analysis of the native enzyme suggests it exists as a homodimer in solution. The purified enzyme showed maximal activity at pH 5.0 and 70 °C. The enzyme was highly specific toward a fucosyl-monosaccharide (Fuc-α-pNP), but it also showed activity toward 2-sulfo-Fuc-α-pNP and fucosyl-α-lactosides (Fuc-α-Galβ1→4Glc-β-pNP). We determined the primary structure of the α-l-fucosidase and validated its expression level in starfish tissue. Whole genome sequence analysis of P. pectinifera was also performed in the present study. Detailed primary structural analysis using bioinformatics tools revealed Pap-Alf lacks the C-terminal region that is otherwise conserved in all previously described α-l-fucosidases. Quantitative gene expression analysis of Pap-Alf in each tissue indicated that the expression of Pap-Alf gene in pancreas was 5-fold higher than in ovary.