Transient N-acetylglucosamine in the biosynthesis of phytohemagglutinin: attachment in the Golgi apparatus and removal in protein bodies.

Abstract

Cotyledons of the common bean (Phaseolus vulgaris L.) synthesize large amounts of the lectin phytohemagglutinin (PHA) during seed development . The polypeptides of PHA are synthesized by endoplasmic reticulum-bound polysomes and co-translationally glycosylated, pass through the Golgi complex, and accumulate in protein bodies, which constitute the lysosomal compartment in these cells. Some of the high-mannose sidechains of PHA are modified in the Golgi complex, and in mature PHA they contain N-acetylglucosamine, mannose, fucose, and xylose in the molar ratios 2, 3 .8, 0.6, and 0.5 . The results reported here show that the Golgi complex is also the site of additional N-acetylglucosamine incorporation into the modified sidechains. When developing cotyledons are labeled with [3H]glucosamine and glycopeptides of PHA present in the Golgi complex isolated, the radioactivity can be released as [3 H]N-acetylglucosamine by digestion of the glycopeptides with ß-N-acetylglucosaminidase, indicating that the residues are in a terminal position . Arrival of PHA in the protein bodies is followed by the slow removal of these terminal N-acetylglucosamine residues, resulting in a decrease in the Mr of the modified sidechains. The biosynthetic intermediates of the glycoproteins destined for the lysosomal compartments of animal cells contain highmannose sidechains modified by phosphate groups covered by N-acetylglucosamine that is labile to mild acid treatment . When cotyledons are labeled with ["P]orthophosphate, there is no radioactivity in PHA obtained from any of the subcellular fractions . There is also no release of radioactivity when [3 H]glucosamine-labeled glycopeptides obtained from PHA in the Golgi complex are subjected to mild acid hydrolysis . These results indicate that the sortingsignals and posttranslational processing steps for proteins that are transported to the lysosomal compartment are different in plant cells and animal cells.

A Unique β1,3-Galactosyltransferase Is Indispensable for the Biosynthesis of N-Glycans Containing Lewis a Structures in Arabidopsis thaliana

“…This could be due to one of the following scenarios: (1) in situ modification of nascent Galb1-3GlcNAc structures by FUT13 is quick and quantitative; or (2) unmodified Galb1-3GlcNAc moieties are rapidly degraded due to the action of b-galactosidases (transient galactose), in analogy to the transient modification of plant N-glycans with terminal GlcNAc residues. In the latter case, GlcNAc residues added in the Golgi are quickly removed upon arrival of the respective glycoprotein in vacuolar compartments (Vitale and Chrispeels, 1984). A similar scenario could be envisaged for the removal of galactose residues from N-glycans carrying terminal Galb1-3GlcNAc moieties.…”

Mannose Analog 1-Deoxymannojirimycin Inhibits the Golgi-Mediated Processing of Bean Storage Glycoproteins

“…Mature PHA recovered with the soluble fraction (Fig. 1, lane 8) showed the two typical bands, with mobilities which do not exactly correspond with any ofthe components present in the membrane fraction: this is due to removal of the terminal N-acetylglucosamine residues, which further increases the mobility of PHA polypeptides (30,31). A completely different situation was observed in dMM-treated cotyledons: PHA from the membrane and the soluble fractions (lanes 5 and 6 in Fig.…”

“…Digestion of PHA and PHSL with endo H was done as described by Vitale et Fig. 1) which are synthesized on the ER and undergo virtually identical processing (25,30,31). Cotranslationally a signal peptide is cleaved and two oligomannose OS are added.…”

Plant glycans: friend or foe in vaccine development?

“…A second N-acetylglucosamine (GlcNAc) is enzymatically added to the mannose core by GnTII. The terminal GlcNAc residues on N-glycans of plant glycoproteins, which are stored in the vacuoles, are often removed by exoglycosidases, resulting in Man 3 XylFucGlcNAc 2 complex-type glycans [16,17]. Alternatively, although this occurs at a very low frequency, the terminal GlcNAc residues of secreted proteins may be extended by b1,3-galactose and a1,4-fucose residues by the respective glycosyltransferases.…”