Characterisation of a secreted N-acetyl-beta-hexosaminidase from Trichinella spiralis.
A thorough investigation was conducted for glycoside hydrolase activities in the secreted proteins of Trichinella spiralis. The data demonstrated that the only secreted glycosidase with significant activity was an exo-beta-hexosaminidase with catalysis of the substrates N-acetyl-beta-D-glucosamine, N-acetyl-beta-D-galactosamine and N-acetyl-beta-D-glucosamine-6-sulphate proceeding with an efficiency similar to the human isozyme beta-hexosaminidase A (Hex A). The hydrolysis of N-acetyl-beta-D-glucosamine followed Michaelis-Menten kinetics with a K(m) of 0.187+/-0.025 mM, and catalysis was inhibited competitively by both N-acetyl-beta-d-glucosamine and N-acetyl-beta-D-galactosamine, with K(i) values of 15.75+/-0.99 and 1.17+/-0.24 mM, respectively. The enzyme was maximally active at pH 4.4, had a temperature optimum at 54 degrees C and was thermolabile. We observed no cleavage of N-acetylglucosamine beta1-4 linkages in N-acetylchitooligosaccharides, but significant hydrolysis of N-acetylglucosamine beta1-2 linked to mannose in glycans was detected indicating that the secreted enzyme is linkage specific. The enzyme was partially purified and identified by SDS-PAGE and Western blotting as a protein with an apparent molecular mass of 50 kDa. We established that the protein was glycosylated and showed that the glycan was decorated with tyvelose (3,6-dideoxy-D-arabino-hexose). Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) analysis demonstrated that the carbohydrate moeity was a tyvelose capped tetra-antennary N-glycan corresponding to the structure Tyv(4)Fuc(5)HexNAc(10)Hex(3). All our studies suggest that this is a novel variant of a secreted N-acetyl-beta-hexosaminidase.
Bruce
Alexandra F
AF
Division of Cell and Molecular Biology, Biochemistry Building, Imperial College London, South Kensington Campus, London SW7 2AZ, UK.
Gounaris
Kleoniki
K
D000818:Animals; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D054818:Hexosaminidase A; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013379:Substrate Specificity; D013696:Temperature; D017160:Trichinella spiralis / Q000201:enzymology* / Q000254:growth & development; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D006026:Glycoside Hydrolases; D054818:Hexosaminidase A; D001619:beta-N-Acetylhexosaminidases