Abnormal accumulation and recycling of glycoproteins visualized in Niemann–Pick type C cells using the chemical reporter strategy

Abstract

Niemann-Pick type C (NPC) disease is characterized by impaired cholesterol efflux from late endosomes and lysosomes and secondary accumulation of lipids. Although impaired trafficking of individual glycoproteins and glycolipids has been noted in NPC cells and other storage disorders, there is currently no effective way to monitor their localization and movement en masse. Using a chemical reporter strategy in combination with pharmacologic treatments, we demonstrate a disease-specific and previously unrecognized accumulation of a diverse set of glycoconjugates in NPC1-null and NPC2-deficient fibroblasts within endocytic compartments. These labeled vesicles do not colocalize with the cholesterol-laden compartments of NPC cells. Experiments using the endocytic uptake marker dextran show that the endosomal accumulation of sialylated molecules can be largely attributed to impaired recycling as opposed to altered fusion of vesicles. Treatment of either NPC1-null or NPC2-deficient cells with cyclodextrin was effective in reducing cholesterol storage as well as the endocytic accumulation of sialoglycoproteins, demonstrating a direct link between cholesterol storage and abnormal recycling. Our data further demonstrate that this accumulation is largely glycoproteins, given that inhibitors of O-glycan initiation or N-glycan processing led to a significant reduction in staining intensity. Taken together, our results provide a unique perspective on the trafficking defects in NPC cells, and highlight the utility of this methodology in analyzing cells with altered recycling and turnover of glycoproteins.

Selective Exo-Enzymatic Labeling Detects Increased Cell Surface Sialoglycoprotein Expression upon Megakaryocytic Differentiation

“…Next, HEL cells were either incubated with Ac 4 ManNAz for 24 h, or SEEL labeled with rat ST6Gal1 or human ST3Gal1 in the presence of CMP-Neu5Ac9N 3 followed by treatment with S-DIBO for biotinylation (Fig. 2).…”

“…CMP-Neu5Ac9N 3 , S-DIBO-Biotin, Ac 4 ManNAc, and Ac 4 ManNAz were synthesized as previously described (16,30). Vibrio cholerae neuraminidase (type II) was purchased from Sigma Aldrich (N6514).…”

“…S-DIBO-biotin Labeling-Cells metabolically labeled by Ac 4 ManNAz or SEEL were biotinylated with S-DIBO-biotin. Typically, 30 M S-DIBO in DPBS containing 2% FBS was treated to cells either in Eppendorf tube (HEL cells) or 12-well dish (CHO or Lec2 cells) for 1 h at room temperature.…”

“…As an alternative approach, labeling methods have been developed that instead rely on the use of recombinant or purified glycosyltransferases to install functionalized sugars directly onto existing glycans (10 -12). This method, recently termed SEEL (selective exoenzymatic labeling), 4 leverages the inherent selectivity of the recombinant glycosyltransferases to label certain types (e.g. N-linked or O-linked) of glycans.…”

N-glycome of the Lysosomal Glycocalyx is Altered in Niemann-Pick Type C Disease (NPC) Model Cells

“…Moreover, it has been recently shown that modulation of glycosylation of APP, a key protein in the pathogenesis of AD, may represent a potential target for AD therapy (18). It has been previously shown that glycoproteins accumulate in NPC model (19), and that blocking the O-linked glycosylation lowers cholesterol levels and increases the number of lysosomes (20), thus rescuing the NPC cellular defects. In the present work, we studied N-glycosylation profile of the lysosomal membrane proteins in NPC1-null cells vs. wt-cells.…”