Lysosomal N-acetyltransferase interacts with ALIX and is detected in extracellular vesicles

Anthony O. Fedele, Sandra Isenmann, Makoto Kamei, Marten Snel, Paul Trim, Christopher Proud, John Hopwood

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Heparan acetyl CoA: α-glucosaminide N-acetyltransferase (HGSNAT) is a lysosomal multi-pass transmembrane protein whose deficiency may lead to an accumulation of heparan sulphate and the neurodegenerative lysosomal storage disorder mucopolysaccharidosis (MPS) IIIC. In this study, HGSNAT activity was detected in extracellular vesicles isolated from both human urine and culture medium conditioned with HEK 293T cells. We also demonstrate that HGSNAT co-immunoprecipitates with antibodies to ALIX, which is associated with the endosomal sorting complexes required for transport (ESCRT) proteins, and is implicated in the targeting of proteins to intraluminal vesicles of multivesicular bodies, the origin of exosomes. Furthermore, mutation of a putative LYPXnL-based binding site within HGSNAT for the V-domain of ALIX ablated association of HGSNAT with ALIX, post-translational maturation, and transport through the endo-lysosomal network. Unexpectedly, however, a mutation within the V-domain of ALIX demonstrated enhanced HGSNAT association, perhaps due to the actual involvement of other binding sites in this interaction. Indeed, HGSNAT still co-immunoprecipitates with truncations of ALIX lacking the V-domain. Interestingly, CRISPR/Cas9 mediated knock-down of ALIX did not inhibit HGSNAT trafficking through the endo-lysosomal network, suggesting that there is an alternative pathway for trafficking HGSNAT that does not require ALIX. Nonetheless, the targeting of HGSNAT to extracellular vesicles may provide a mechanism to subsequently transfer this enzyme extracellularly to provide a foundation for a therapy for MPS IIIC patients.

Original languageEnglish
Pages (from-to)1451-1464
Number of pages14
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1865
Issue number10
DOIs
Publication statusPublished - 1 Oct 2018

Keywords

  • ALIX
  • Endosomal sorting complexes required for transport (ESCRT)
  • Extracellular vesicle
  • Heparan acetyl CoA: α-glucosaminide N-acetyltransferase
  • Heparan sulfate
  • Lysosome

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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