Purification and characterization of a sialic acid-specific lectin from Tritrichomonas mobilensis.
New sialic acid-specific lectin has been isolated from culture supernatant of the protozoan Tritrichomonas mobilensis. It was purified by adsorption by erythrocytes or bovine submaxillary gland mucin (BSM)-Sepharose affinity chromatography. The T. mobilensis lectin (TML) does not require bivalent cations for activity and agglutinates all human erythrocytes. 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, were split into three subunits of 56, 61 and 66 kDa; under non-reducing conditions there were two bands, of 360 and 260 kDa. Western blots performed with anti-TML monoclonal antibodies revealed bands identical with 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 treatment; the total amount of saccharides revealed by chemical deglycosylation was 20%. Haemagglutination-inhibition studies documented exclusive specificity for sialic acid (NeuAc). Both (alpha 2-->6)- and (alpha 2-->3)-linked and free NeuAc were eight times more potent inhibitors than N-glycolylneuraminic acid. The lectin does not require O-acetyl groups on NeuAc for recognition. A spectrum of mono- and oligo-saccharides 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.
Babál
P
P
Department of Pathology, University of South Alabama, Mobile 36617.
Pindak
F F
FF
Wells
D J
DJ
Gardner
W A
WA
D000327:Adsorption; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004187:Disaccharides; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000276:immunology / Q000502:physiology; D005732:Gangliosides; D006384:Hemagglutination; D006385:Hemagglutination Inhibition Tests; D006801:Humans; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009077:Mucins; D019158:N-Acetylneuraminic Acid; D011487:Protein Conformation; D012794:Sialic Acids; D014244:Trichomonas / Q000737:chemistry*
D000596:Amino Acids; D002241:Carbohydrates; D004187:Disaccharides; D005732:Gangliosides; D037102:Lectins; D009077:Mucins; D012794:Sialic Acids; D019158:N-Acetylneuraminic Acid