Biochemical and functional characterization of the Tn-specific lectin from Salvia sclarea seeds.
SSL, the lectin isolated from Salvia sclarea seeds, recognizes the Tn antigen (GalNAcalpha-O-Ser/Thr), a specific marker of many human carcinomas. Two-dimensional electrophoresis, amino-acid and amino-sugar analysis, and MALDI-TOF MS showed that SSL is an acidic (pI 5.5), 60-61-kDa dimeric glycoprotein composed of apparently identical subunits linked by a single disulfide bond. The apparent molecular mass of SSL in solution determined by equilibrium sedimentation analytical ultracentrifugation was 59 +/- 9 kDa. This value did not change in the pH range 2.5-8.5, indicating that SSL does not associate into higher order structures. Tandem mass spectrometry and methylation analysis of N-glycans released from SSL by hydrazinolysis indicated that SSL possesses 2-3 glycosylation sites occupied with the typical plant glycans Manalpha1-6[(Manalpha1-3)(Xylbeta1-2)]Manbeta1-4 -GlcNAcbeta1-4(Fucalp ha1-3)GlcNAc and [(Manalpha1-3/6)(Xylbeta1-2)]Manbeta1-4-GlcNAcbeta1 -4(Fucalpha1-3)Glc NAc. The influence of adjacent Tn structures on the binding of two Tn-specific lectins (SSL and the isolectin B4 from Vicia villosa) and an anti-Tn monoclonal antibody (mAb 83D4) was evaluated using synthetic Tn glycopeptides. The binding of both lectins to the synthetic Tn glycopeptides was independent of the density of Tn structures. On the other hand, mAb 83D4 only reacted with glycopeptides displaying two or three consecutive Tn structures.
Medeiros
A
A
Depto. de Bioquímica, Facultad de Medicina, Montevideo, CP, Uruguay; Instituto de Biomedicina, CSIC, Valencia, Spain.
Bianchi
S
S
Calvete
J J
JJ
Balter
H
H
Bay
S
S
Robles
A
A
Cantacuzène
D
D
Nimtz
M
M
Alzari
P M
PM
Osinaga
E
E
D000911:Antibodies, Monoclonal / Q000276:immunology; D015295:Antigens, Tumor-Associated, Carbohydrate / Q000378:metabolism*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D000939:Epitopes / Q000276:immunology; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D037102:Lectins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D008969:Molecular Sequence Data; D037121:Plant Lectins; D010944:Plants / Q000196:embryology / Q000378:metabolism*; D012639:Seeds / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D015295:Antigens, Tumor-Associated, Carbohydrate; D004220:Disulfides; D000939:Epitopes; D006020:Glycopeptides; D037102:Lectins; D037121:Plant Lectins; C040286:Tn antigen