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Front Immunol. 2014 Aug 25;5:404. doi: 10.3389/fimmu.2014.00404. eCollection 2014.

Differential site accessibility mechanistically explains subcellular-specific N-glycosylation determinants.

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1
Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Macquarie University , Sydney, NSW , Australia.

Abstract

Glycoproteins perform extra- and intracellular functions in innate and adaptive immunity by lectin-based interactions to exposed glyco-determinants. Herein, we document and mechanistically explain the formation of subcellular-specific N-glycosylation determinants on glycoproteins trafficking through the shared biosynthetic machinery of human cells. LC-MS/MS-based quantitative glycomics showed that the secreted glycoproteins of eight human breast epithelial cells displaying diverse geno- and phenotypes consistently displayed more processed, primarily complex type, N-glycans than the high-mannose-rich microsomal glycoproteins. Detailed subcellular glycome profiling of proteins derived from three breast cell lines (MCF7/MDA468/MCF10A) demonstrated that secreted glycoproteins displayed significantly more α-sialylation and α1,6-fucosylation, but less α-mannosylation, than both the intermediately glycan-processed cell-surface glycoproteomes and the under-processed microsomal glycoproteomes. Subcellular proteomics and gene ontology revealed substantial presence of endoplasmic reticulum resident glycoproteins in the microsomes and confirmed significant enrichment of secreted and cell-surface glycoproteins in the respective subcellular fractions. The solvent accessibility of the glycosylation sites on maturely folded proteins of the 100 most abundant putative N-glycoproteins observed uniquely in the three subcellular glycoproteomes correlated with the glycan type processing thereby mechanistically explaining the formation of subcellular-specific N-glycosylation. In conclusion, human cells have developed mechanisms to simultaneously and reproducibly generate subcellular-specific N-glycosylation using a shared biosynthetic machinery. This aspect of protein-specific glycosylation is important for structural and functional glycobiology and discussed here in the context of the spatio-temporal interaction of glyco-determinants with lectins central to infection and immunity.

KEYWORDS:

N-glycan; N-glycome; N-glycosylation; glycoprotein; glycoproteome; glycosylation site; solvent accessibility; subcellular location

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