Purification and properties of an N-acetylglucosamine-specific lectin from Psathyrella velutina mushroom

N Kochibe,K L Matta

doi:10.1016/s0021-9258(17)31239-5

Abstract

A lectin in the fruiting bodies of Psathyrella velutina was purified by affinity chromatography on a chitin column and subsequent ion-exchange chromatography. P. velutina lectin (PVL) tends to aggregate irreversibly in buffered saline, but the addition of glycerol (10%, v/v) to lectin solutions was found to prevent aggregate formation. PVL is assumed to occur as a monomer of a polypeptide of Mr = 40,000 as determined by gel filtration and by gel electrophoresis in the presence of sodium dodecyl sulfate. PVL is specific for N-acetylglucosamine (GlcNAc). It was determined by equilibrium dialysis to have four binding sites/polypeptide molecule showing an average intrinsic association constant of K0 = 6.4 x 10(3) M-1 toward this sugar. The binding specificity of the lectin was studied by hemagglutination inhibition assays and by avidin-biotin-mediated enzyme immunoassays using various GlcNAc-containing saccharides. The results indicate that methyl N-acetyl beta-glucosaminide was a slightly better inhibitor than the corresponding alpha-anomer. PVL binds well to oligosaccharides bearing nonreducing terminal beta-GlcNAc linked 1----6 or 1----3 but poorly to those having a 1----4 linkage, such as N-acetylated chito-oligosaccharides. It also binds to the subterminal GlcNAc moiety when it is substituted at the C-6 position but does not interact with the moiety when substituted either at C-3 or C-4. Thus, these results show that PVL is quite different in its binding specificity from other GlcNAc-binding lectins of higher plants since they bind preferentially to beta-GlcNAc in 1----4 linkage and they have a high affinity for chitin oligosaccharides.

Highlights

Sults indicate that methyl N-acetyl /3-glucosaminide was a slightly better inhibitor than the corresponding a-anomer
Amongthese, the followingoligosaccharides were synthesized by previously reported methods: oligosaccharides V and VI [13], IX [14], X [15], XI [16], XI1 (171,XI11 [18], XIV-XVI [19], and VIL3GlcNAc and methyl N-acetyl CY- and pglucosaminides were purchased from Sigma and were recrystallized from methanol-diethyl ether mixtures
The acid-treated extract of the fruiting bodies was subjected directly to affinity chromatography using a chitin column, and the active fractions after each chromatographic step were concentrated by ultrafiltration

Summary

Introduction

Purified PVL (about 200 pg) in the same solution containing 10 mM GlcNAc wasapplied to thecolumn, and theA2mof the eluate was monitored. Inhibitor solution (25 pl) of known concentration was diluted serially and mixed with 25 pl containing 8 units of the purified PVL. Inhibition Studies in Avidin-Biotin-mediated Enzyme-linked Zmmunosorbent Assay(ELZSA)-Biotinylated PVL was prepared by reaction with biotinyl-N-hydroxysuccinimideeste(rNHS-biotin, Sigma).

Results

Conclusion