Isolation of a novel N-acetylglucosamine-specific lectin from fresh sclerotia of the edible mushroom Pleurotus tuber-regium

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Abstract

An N-acetylglucosamine-binding lectin with a molecular mass of 32 kDa was isolated from fresh sclerotia of the edible mushroom Pleurotus tuber-regium. Its N-terminal sequence exhibited some similarity to that of Agaricus bisporus lectin. The isolation procedure was simple, involving (NH4)2SO4 precipitation, ion exchange chromatography on DEAE–cellulose, affinity chromatography on N-acetyl-d-glucosamine–agarose, and gel filtration by fast protein liquid chromatography on Superdex 75. The lectin exhibited hemagglutinating activity toward trypsinized rabbit erythrocytes but not toward untrypsinized rabbit erythrocytes.

Section snippets

Isolation procedure

Fresh sclerotia of P. tuber-regium collected in Mainland China (1.5 kg) were extracted with saline. After addition of (NH4)2SO4 to 30% saturation and removal of the precipitate, (NH4)2SO4 was added to 80% saturation. The precipitate was collected by centrifugation, dissolved, and then dialyzed before ion exchange chromatography on a DEAE–cellulose

Purification and N-terminal sequence of lectin

Hemagglutinating activity which could be inhibited by N-acetyl-d-glucosamine, hereinafter referred to as lectin activity, was eluted in peak D1 which was unadsorbed on DEAE–cellulose. Subsequent chromatography of D1 on immobilized N-acetyl-d-glucosamine fractionated it into a large unadsorbed peak N1 devoid of lectin activity and a small unadsorbed peak N2 with lectin activity (Fig. 1). FPLC-gel filtration of N2 on Superdex 75 yielded a large peak SU1 and a small peak SU2 (Fig. 2). Lectin

Discussion

Pleurotus tuber-regium lectin can be isolated with a relatively simple procedure involving (NH4)2SO4 precipitation, ion exchange chromatography on DEAE–cellulose, affinity chromatography on N-acetylglucosamine–agarose, and FPLC-gel filtration on Superdex 75.

Not many N-acetyl-d-glucosamine-specific lectins have been reported in the literature. What germ agglutinin is one of them. However, it also demonstrates specificity for sialic acid. It is composed of subunits with a molecular mass of 36 kDa

Acknowledgements

The award of a direct grant by the Medicine Panel, Research Committee, The Chinese University of Hong Kong and the expert secretarial assistance of Miss Fion Yung are gratefully acknowledged.

References (39)

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