PURIFICATION AND CHARACTERIZATION OF A SIALIC ACID-SPECIFIC LECTIN FROM TRITRICHOMONAS-MOIBILENSIS

New sialic acid-specific lectin has been isolated from culture superna tant of the protozoan Tritrichomonas mobilensis. It was purified by ad sorption by erythrocytes or bovine submaxillary gland mucin (BSM)-Seph arose affinity chromatography. The T. mobilenis lectin (TML) does not require bivalent cations for activity and agglutinates all human eryth rocytes. The lectin forms multimeric complexes with molecular mass 556 and 491 kDa as determined by size-exclusion chromatography. SDS/PAGE under reducing conditions disclosed a large band of 343 kDa and three bands of 246, 265 and 286 kDa which, after denaturation with urea, wer e split into three subunits of 56, 61 and 66 kDa; under non-reducing c onditions there were two bands, of 360 and 260 kDa. Western blots perf ormed with anti-TML monoclonal antibodies revealed bands identical wit h those in the silver-stained gels, suggesting homogeneity of the BSM -Sepharose-purified lectin. TML is a highly glycosylated protein with approx. 8% of N-linked glycosides found by protein-N-glycanase F treat ment; the total amount of saccharides revealed by chemical deglycosyla tion was 20%. Haemagglutination-inhibition studies documented exclusiv e specificity for sialic acid (NeuAc). Both (alpha 2-->6)- and (alpha 2-->3)-linked and free NeuAc were eight times more potent inhibitors t han N-glycolylneuraminic acid. The lectin does not require O-acetyl gr oups on NeuAc for recognition. A spectrum of mono- and oligo-saccharid es other than sialic acid had no inhibitory effect at 200 mM. Anti-TML monoclonal antibodies strongly inhibited the lectin activity. TML was stable at temperatures below 4 degrees C and lyophilized with 3% (w/w ) glycerol.