Visualizing alterations in the trafficking of glycoconjugates within niemann-pick type c cells using cu-free click technology

Eric Ngalle Mbua, Steven Johnson, Heather Flanagan-Steet, Margreet Wolfert, Geert-Jan Boons

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

The bioorthogonal chemical reporter strategy is emerging as a versatile methodology for labeling biomolecules such as nucleic acids, lipids, carbohydrates and proteins. In particular, a biomolecule modified by an azido group can be tagged by Staudinger ligation using modified phosphines, by a copper (I)-catalyzed cycloaddition with terminal alkynes (CuAAC), or by a strainpromoted alkyne-azide cycloaddition (SPAAC). Using the compound, 4-dibenzocyclooctynol (DIBO) and its derivatives, we previously demonstrated differences in cell surface sialylation within a group of established CHO glycosylation mutant cells (Lec2, Lec 13, Lec 32, Cog-1, and Cog-2). Here, we show that this technology can be used to investigate the trafficking defects associated with Niemann-Pick type C (NPC), a lysosomal storage disorder characterized by impaired cholesterol efflux from late endosomes and lysosomes due to deficiency in the integral membrane protein NPC1. Consistent with the endocytic transport defect in these cells, accumulation of sialylated molecules was observed within intracellular vesicles in NPC-null fibroblasts. These vesicles exhibited partial co-localization with markers of early and late enodsomes. Interestingly, unlike mucolipidosis type II fibroblasts, no detectable labeling was observed in cholesterol-laden lysosomes in the NPC cells, suggesting that the accumulation of cholesterol renders these lysosomes incapable of receiving glycosylated molecules for degradation. Our preliminary data indicated that treatment of NPC fibroblasts with cyclodextrin effectively reduced cholesterol accumulation but did not appear to impact the accumulation of labeled vesicles. This finding implies that NPC1 may have additional functions within the endocytic network. Together, our results provide a novel perspective on the trafficking defects in NPC cells and further highlight the utility of this methodology to analyze cells with altered biosynthesis, trafficking and turnover of glycoproteins.
Original languageEnglish
Pages (from-to)1526
Number of pages1
JournalGlycobiology
Volume21
Issue number11
DOIs
Publication statusPublished - 1 Nov 2011
Externally publishedYes

Keywords

  • glycoconjugate
  • cholesterol
  • alkyne
  • phosphine derivative
  • carbohydrate
  • marker
  • cyclodextrin
  • cuprous ion
  • azide
  • membrane protein
  • nucleic acid
  • lipid
  • protein
  • glycoprotein
  • glycobiology
  • society
  • technology
  • click
  • lysosome
  • fibroblast
  • cycloaddition
  • methodology
  • ligation
  • cell surface
  • effusion
  • mucolipidosis type 2
  • biosynthesis
  • turnover rate
  • sialylation
  • glycosylation
  • cell mutant
  • lysosome storage disease
  • storage disease
  • endosome

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