Biosynthesis and processing of phytohemagglutinin in developing bean cotyledons.

Area of Science:

• Plant molecular biology
• Protein biosynthesis
• Glycobiology

Background:

• Phytohemagglutinin (PHA) is a tetrameric isolectin found in Phaseolus vulgaris cotyledons.
• PHA consists of erythroagglutinating (E) and lymphocyte-mitogenic (L) subunits with distinct molecular weights.
• PHA is a glycoprotein with complex N-acetylglucosamine, mannose, fucose, and xylose-containing oligosaccharide side chains.

Purpose of the Study:

• To elucidate the biosynthesis and processing of PHA glycopolypeptides.
• To identify the stages of cotranslational and post-translational modification of PHA.
• To investigate the role of glycosylation in PHA subunit size determination.

Main Methods:

• Analysis of PHA synthesis in polysomes attached to the endoplasmic reticulum.
• Investigation of glycosylation using tunicamycin.
• Enzymatic digestion of oligosaccharides with alpha-mannosidase and endo-beta-N-acetylglucosaminidase H.
• Tracking PHA transport through the Golgi apparatus to protein bodies.

Main Results:

• PHA polypeptides are synthesized on ER-bound polysomes and glycosylated cotranslationally, typically acquiring two high-mannose type oligosaccharides.
• Differences in E and L subunit molecular weights are not solely due to glycosylation.
• Oligosaccharide chains are modified in the Golgi and further trimmed in protein bodies, increasing subunit mobility in gels.

Conclusions:

• PHA biosynthesis involves cotranslational glycosylation and extensive post-translational modification of oligosaccharide chains.
• Glycosylation is a key but not the sole determinant of PHA subunit size heterogeneity.
• Protein body storage is associated with final oligosaccharide trimming and maturation of PHA.