Molecular-mass heterogeneity of Griffonia simplicifolia lectin IV subunits. Differences in the oligosaccharide moieties in the N-terminal region.
Lectin IV of Griffonia simplicifolia (Mr approximately 56,000), which has a strong affinity for both the Lewis b and Y blood-group determinants, is a dimeric protein of two subunits, alpha (29 kDa) and beta (27 kDa), separable by SDS/PAGE and containing covalently linked oligosaccharide. After digestion with N-glycanase, the protein migrates as a single band with a mobility identical with that of the beta-subunit. After cleavage with hydroxylamine of 3H-labelled, but otherwise intact, lectin, the radioactively labelled oligosaccharide was found to be associated with two blocked N-terminal peptides separable by h.p.l.c. and having identical amino acid compositions. One of these had three or four glucosamine residues per molecule, whereas the other had only one or two. Sequence analyses of these, as well as of a 21 kDa hydroxylamine-cleaved fragment and of the intact lectin pretreated with pyroglutamate aminopeptidase, have provided a unique sequence for residues 1-62 of the two subunits. Evidence is presented for two sites of N-linked oligosaccharide attachment at Asn-5 and Asn-18. Whereas the alpha-subunit has oligosaccharide linked to both sites, the beta-subunit has carbohydrate associated with only one (Asn-18). Sugar analyses of the whole lectin reveal a monosaccharide composition of (Xyl)3(Fuc)2(Man)10(GlcNAc)6, representing 6.4% of the mass of the molecule. Taken together with the susceptibility of the Asn-5 linkage (but not of Asn-18) to N-glycanase digestion, the observations indicate that the structures of the oligosaccharides at residues 5 and 18 are different.
Nikrad
P V
PV
Department of Chemistry, University of Alberta, Edmonton, Canada.
Pearlstone
J R
JR
Carpenter
M R
MR
Lemieux
R U
RU
Smillie
L B
LB
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006031:Glycosylation; D019811:Hydroxylamine; D006898:Hydroxylamines; D037102:Lectins / Q000737:chemistry / Q000302:isolation & purification*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000302:isolation & purification*; D037121:Plant Lectins
D000596:Amino Acids; D002241:Carbohydrates; C045475:Griffonia simplicifolia lectins; D006898:Hydroxylamines; D037102:Lectins; D046911:Macromolecular Substances; D009844:Oligosaccharides; D037121:Plant Lectins; D019811:Hydroxylamine; D010450:Endopeptidases