Oligosaccharide specificities of Phaseolus vulgaris leukoagglutinating and erythroagglutinating phytohemagglutinins. Interactions with N-glycanase-released oligosaccharides

Abstract

The structural determinants required for interaction of oligosaccharides with leukoagglutinating phytohemagglutinin (L-PHA) and erythroagglutinating phytohemagglutinin (E-PHA) from Phaseolus vulgaris have been studied by immobilized lectin affinity chromatography. Homogeneous oligosaccharides of known structure, purified following release from Asn with N-glycanase and reduction with NaBH4, were tested for their ability to interact with columns of L- and E-PHA-agarose. The characteristic elution position obtained for each oligosaccharide was reproducible and correlated with specific structural features. In virtually all cases, L- and E-PHA yielded identical results, indicating that their specificities for reduced oligosaccharides are similar. Both lectins retarded oligosaccharides bearing alpha 2,3- but not alpha 2,6-linked sialic acid. Desialylated oligosaccharides containing one, two, three, or four peripheral N-acetyllactosamine-type branches were retarded to varying extents by both lectins; however, this interaction was decreased or eliminated by removal of Gal. Desialylated oligosaccharides containing a bisecting GlcNAc residue attached to the beta-linked core Man displayed the greatest interaction with both lectins. Structures containing terminal sulfate or GalNAc did not interact with either lectin. In some instances, the specificities of L- and E-PHA lectins for free, reduced oligosaccharides differed from those established using glycopeptides. Therefore, the structural requirements for interaction with lectins such as L- and E-PHA must be fully and systematically defined using the appropriate authentic standards in order to use lectin affinity chromatography for the fractionation and characterization of free oligosaccharides.