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

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.

Figure 1

Secreted glycoproteins display more N-glycan type processing than microsomal glycoproteins. (A) The N-glycomes of the microsomal (top) and secreted (bottom) proteins of a panel of eight geno- and phenotypically different cultured human breast epithelial cells (i.e., MCF7, SKBR3, MDA157, MDA231, MDA468, HS578T, HMEC, and MCF10A) were profiled, see Table S1 in Supplementary Material for information of investigated cells. The relative molar abundances (mean ± SD) of more processed N-glycans comprising the complex, hybrid, and paucimannose type are presented in light red and the less processed N-glycans of the immature and high-mannose type in green (inset). Subcellular-specific N-glycosylation of boxed cell lines was investigated further in greater detail. (B) Summed m/z profiles of the N-glycomes derived from microsomal (top), cell-surface (middle), and secreted (bottom) proteins of MCF7 cells. Signals corresponding to N-glycans have been assigned as less processed (green) or more processed (light red) N-glycan types following the same classification as in (A). (C) Relative molar distribution (mean ± SD) of more (right, hybrid/complex/paucimannose, light red bars) and less (left, high mannose, green bars) processed N-glycan types of the microsomal (dotted bars), cell-surface (brick), and secreted (banded) proteins of MCF7, MDA468, and MCF10A. (D) Subcellular-specific distribution of the N-glycan determinants. The proportion of terminal α-mannosylation, α-fucosylation, and α-sialylation (non-reducing end) N-glycans of the total N-glycome (mol/mol %) on the microsome, cell-surface, and secreted glycoproteomes across MCF7 (i), MDA468 (ii), and MCF10A (iii) breast cell lines were determined from the N-glycome profiles. N-glycans may terminate with multiple monosaccharide determinants making the values sum to more than 100%. For all panels: ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 2

(A–C) The subcellular proteomes of MCF7, MDA468, and MCF10A breast epithelial cell lines were mapped according to GO terms into ER, Golgi/endosome/plasma membrane, and extracellular region classifiers. This confirmed enrichment, but not isolation, of cell-surface and secreted proteins in the respective subcellular fractions. In addition, the classification confirmed that the microsomes contained a significant proportion of ER-residing proteins. (D) The subcellular distribution of the high-mannose glycan type series on proteins derived from MCF7 into Man₅, Man₆, Man₇, Man₈, Man₉ ± Glc₁, the latter representing immature N-glycans normally only associated with intracellular ER N-glycosylation. See Figure S2 in Supplementary Material for the subcellular distribution of the high-mannose glycan type series of MDA468 and MCF10A.

Figure 3

(A) Glycosylation site accessibilities (unit-less, arbitrary values, mean ± SEM) of the microsomal (green), cell-surface (white), and secreted (light red) proteins derived from MCF7, MDA468, and MCF10A breast epithelial cell lines. (B) Correlation between the site accessibilities and the N-glycosylation processing as measured by the more processed N-glycan types (hybrid, complex, and paucimannose) as a molar proportion of the total N-glycome for the three subcellular fractions. High correlation coefficients (R²) indicate strong correlation.

Figure 4

Subcellular-specific N-glycosylation is driven by differential solvent accessibility to the N-glycosylation sites on maturely folded glycoproteins. Consequently, the N-glycans of secreted, cell-surface, and microsomal proteins receive high, intermediate, and low N-glycosylation processing, respectively, and as a result, display distinct glyco-determinant signatures.