Isolation and partial characterization of N-acetyl-D-galactosamine-binding lectins from Epiphragmophora trenquelleonis snail.
A human blood type A hemagglutinating activity was detected in albumin gland extracts of Epiphragmophora trenquelleonis snail separated by GalNAc-agarose affinity chromatography, of which two N-acetyl-D-galactosamine-binding lectins in the extracts were ETL1 was displaced from the affinity column with 1 mM GalNAc, and ETL2 with 20 mM GalNAc. Both lectins agglutinated specifically human blood type A and AB erythrocytes, but not type B and O erythrocytes. Gel filtration chromatography gave a native molecular weight of about 59 kDa for ETL1 and about 54 kDa for ETL2. On SDS-PAGE under nonreducing conditions, ETL1 showed two protein subunits of about 29 and 27 kDa, while ETL2 showed three protein subunits of about 27, 24, and 22 kDa. On SDS-PAGE under reducing conditions, both lectins showed four protein subunits of 17, 16, 12, and 11 kDa. By Western blot analyses developed with biotin-labeled lectins, N-linked oligosaccharides were detected in the 17- and 16-kDa protein subunits of ETL1 and ETL2, and in the 12-kDa protein subunit of ETL2. O-linked oligosaccharides were detected only in the 11-kDa protein subunit of ETL1 and ETL2. On isoelectric focusing both lectins exhibited microheterogeneity: ETL1 focused as three protein bands with pIs in the range of 5.6-6.0, while ETL2 focused as four protein bands with pIs in the range of 6.8-7.4. We suggest that native ETL1 and ETL2 are glycoprotein complexes with molecular weights of 59-54 kDa, composed of two 29-22-kDa nonreduced protein subunits held together by noncovalent hydrophobic interactions. Each of the nonreduced protein subunits seems to be composed of two 17-11-kDa reduced protein subunits held together by interchain disulfide linkages. The main differences between ETL1 and ETL2 could be due to different posttranslational modifications or to the relative contribution of one or more of their protein subunits.
Castagna
L
L
Departamento de Química Biológica, Universidad Nacional de Córdoba, Argentina.
Zarzur
J
J
Filipetti
M
M
Landa
C
C
D000116:Acetylgalactosamine / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006386:Hemagglutination Tests; D006801:Humans; D007525:Isoelectric Focusing; D037102:Lectins / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D012908:Snails / Q000378:metabolism*
D037102:Lectins; D000116:Acetylgalactosamine