Immunochemical studies on the combining site of the A + N blood type specific Moluccella laevis lectin.
The specificity of the anti A+N lectin of Moluccella laevis (MLL) was examined by hemagglutination experiments with enzyme-modified human erythrocytes and by inhibition of hemagglutination. In addition, binding to various glycoproteins and inhibition by different sugars and glycoproteins were examined by enzyme immunoassay with antibodies to the lectin. Treatment of AMM erythrocytes with proteolytic enzymes increased their agglutinability by MLL 4-16-fold; similar treatment of ONN cells decreased their agglutinability 8-16-fold. This is in line with the known location and enzyme sensitivity of A and N specificity determinants. Treatment of the erythrocytes with sialidase increased their agglutinability and abolished the distinction between N and M cells. Hapten inhibition of hemagglutination of AMM and ONN erythrocytes by the lectin, and its binding to glycoproteins measured by enzyme immunoassay, confirmed the high specificity of MLL for N-acetyl-D-galactosamine (200-500 times more than for D-galactose) and suggested the presence of hydrophobic interactions around HO-2 of the D-galactose unit. The methyl alpha-glycosides of D-galactose and of N-acetyl-D-galactosamine were better inhibitors than the corresponding beta-glycosides; this preference was abolished, and sometimes reversed, when the p-nitrophenyl glycosides of the same monosaccharides were tested, stressing again the importance of hydrophobic interactions in the binding of carbohydrates to MLL. The lectin reacted well with ONN substance and with glycophorin A of the N phenotype (GPAN), but did not react with OMM substance or GPAM. The strongest inhibitor was asialo ovine submaxillary mucin, which contains many unsubstituted alpha-D-GalpNAc-(1-->3)-Ser/Thr residues; calculated per N-acetyl-D-galactosamine residue, it was 1500 stronger than free N-acetyl-D-galactosamine. In accordance with this result, it was found that the lectin strongly agglutinates Tn cells. The specificity of MLL can, thus, be defined as anti-Tn, crossreactive with blood types A and N, and with sialosyl-Tn. The N-specificity can best be explained by assuming that GPAN contains a small number of unsubstituted or partially sialylated alpha-D-GalpNAc-(1-->3)-Ser/Thr residues, which are present in smaller proportions, if at all, in GPAM.
Duk
M
M
Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw.
Mitra
D
D
Lisowska
E
E
Kabat
E A
EA
Sharon
N
N
Lis
H
H
D000918:Antibody Specificity / Q000276:immunology; D001665:Binding Sites / Q000502:physiology; D001789:Blood Group Antigens / Q000276:immunology*; D002240:Carbohydrate Sequence; D004797:Enzyme-Linked Immunosorbent Assay; D006021:Glycophorins / Q000302:isolation & purification; D006023:Glycoproteins / Q000302:isolation & purification; D006801:Humans; D007120:Immunochemistry; D037102:Lectins / Q000032:analysis*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification
D001789:Blood Group Antigens; D006021:Glycophorins; D006023:Glycoproteins; D037102:Lectins; D010446:Peptide Fragments