Characterisation of a secreted N-acetyl-β-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-β-hexosaminidase with catalysis of the substrates N-acetyl-β-d-glucosamine, N-acetyl-β-d-galactosamine and N-acetyl-β-d-glucosamine-6-sulphate proceeding with an efficiency similar to the human isozyme β-hexosaminidase A (Hex A). The hydrolysis of N-acetyl-β-d-glucosamine followed Michaelis–Menten kinetics with a Km of 0.187 ± 0.025 mM, and catalysis was inhibited competitively by both N-acetyl-β-d-glucosamine and N-acetyl-β-d-galactosamine, with Ki 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 °C and was thermolabile. We observed no cleavage of N-acetylglucosamine β1–4 linkages in N-acetylchitooligosaccharides, but significant hydrolysis of N-acetylglucosamine β1–2 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 Tyv4Fuc5HexNAc10Hex3. All our studies suggest that this is a novel variant of a secreted N-acetyl-β-hexosaminidase.