Figure 5: Glycomic analysis of total protein glycosylation in GRASP-depleted cells.

HeLa cells expressing RNAi targeting the indicated GRASP proteins were collected and processed for glycomic analysis by mass spectrometry. (a) The major high-mannose and complex glycans were quantified relative to an external standard and normalized to protein content. Double-knockdown cells exhibit significantly decreased glycan abundance. Glycan structural representations are consistent with the nomenclature proposed by the Consortium for Functional Glycomics: GlcNAc, blue square; Man, green circle; Gal, yellow circle; SA, purple diamond; Fuc, red triangle. (b) The fold change for each glycan indicated in panel a was calculated (knockdown/control). Average fold changes±s.e.m. are plotted; negative values indicate decreased abundance relative to control. Single knockdowns exhibit relatively minor alteration in the pool of major high-mannose and complex glycans, but the double-knockdown cells are significantly reduced in both high-mannose and complex glycans. In the double-knockdown cells, the fold-reduction in complex glycans is greater than the reduction in high-mannose glycans (Student’s t-test, **P<0.001). (c) LLO and FOS were quantified by mass spectrometry relative to an external standard and normalized to protein content (Dol, dolichol pyrophosphate). Total LLO levels are significantly lower than FOS levels, note change in y axis scale. All quantifiable LLO species were reduced in the GRASP double-knockdown cells, while some LLOs increased and some decreased relative to control in the single-knockdown cells. The abundance of all quantifiable FOS species was dramatically increased in double-knockdown cells, while single knockdown had relatively little effect on FOS levels. (d) Fold changes in LLO and FOS, calculated as in b, reveals the inverse relationship expected for precursor and product. Comparison with the fold change in glycoprotein glycan abundance (b) emphasizes that LLO availability limits glycosylation. When LLOs are decreased by knockdown, glycoprotein glycosylation is also decreased (55 alone and 55+65) and vice versa (65 alone), although the impact of either single knockdown is much smaller than the double knockdown.