Structural studies of phosphorylated high mannose-type oligosaccharides

Area of Science:

• Biochemistry
• Cell Biology
• Glycobiology

Background:

• Phosphomannosyl residues on acid hydrolases are crucial recognition markers for lysosomal targeting.
• Newly synthesized beta-glucuronidase contains oligosaccharide units with phosphate residues in diester linkage.

Purpose of the Study:

• To obtain larger quantities of phosphorylated oligosaccharides for detailed structural analysis.
• To elucidate the structure, phosphorylation sites, and linkage types of oligosaccharides on newly synthesized beta-glucuronidase.

Main Methods:

• Isolation of total cellular glycopeptides from [2-3H]mannose-labeled mouse lymphoma cells.
• Release of phosphorylated oligosaccharides using endo-beta-N-acetylglucosaminidase CII and H.
• Purification by gel filtration and ion exchange chromatography, followed by characterization using alpha-mannosidase digestion and acetolysis.

Main Results:

• Characterized a family of phosphorylated oligosaccharides with high mannose-type cores.
• Identified major classes with single or double phosphodiester linkages, some convertible to phosphomonoesters by alpha-N-acetylglucosaminidase.
• Observed classes with phosphomonoester groups and smaller oligosaccharide cores, indicating partial processing.

Conclusions:

• Phosphorylation occurs at up to five distinct mannose residues on high mannose-type oligosaccharides.
• Individual molecules can bear one, two, or possibly three phosphate residues.
• The majority of newly synthesized phosphorylated oligosaccharides feature phosphate groups in diester linkage.