N-GLYCOSYLATION OF PHYTOHEMAGGLUTININ EXPRESSED IN BEAN COTYLEDONS ORIN TRANSGENIC TOBACCO CELLS

Phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris, is a tet rameric glycoprotein containing two closely related subunits PHA-E and PHA-L. Both subunits are glycosylated with one high-mannose and one c omplex type N-glycan per polypeptide. Complete structural analysis of these N-glycans was performed for bean PHA-E and L subunits. N-glycans released by peptide N-glycosidase A (PNGase A), either from bean PHA- E + L or PHA-L, were analysed by High-pH Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD). Agrobacterium-mediate d introduction of PHA-L cDNA into tobacco cells was previously shown t o result in the expression of two polypeptide forms. N-glycosylation o f these two recombinant PHA-L forms has been investigated using immuno - and affinodetection on blots as well as by HPAEC-PAD analysis of PNG ase A-released N-glycans. These two PHA-L forms, which probably result from a partial proteolytic maturation of a single polypeptide in the vacuole, were found to be normally N-glycosylated with both one high-m annose and one complex type N-glycan with structures similar to those found in bean PHA-L. In this paper, HPAE-PAD chromatography is a major tool in demonstrating that glycans N-linked to the recombinant PHA-L are maturated correctly in transgenic tobacco cells.