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Abstract: FR-PO611

ARL15 Regulates CNNM2-Dependent Mg2+ Transport by Modulating Its N-Linked Glycosylation

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic

Authors

  • Ma, Chao, Radboud University Medical Center, Nijmegen, Netherlands
  • Zolotarov, Yevgen, McGill University, Montreal, Quebec, Canada
  • Franken, Gijs A.C., Radboud Institute for Molecular Life Science, Nijmegen, Netherlands
  • Bindels, René J., Radboud University Medical Center, Nijmegen, Netherlands
  • Hoenderop, Joost, Radboud University Medical Center, Nijmegen, Netherlands
  • De Baaij, Jeroen H.F., Radboud University Medical Center, Nijmegen, Netherlands
Background

A large genome-wide association study identified that ARL15, a small GTP-binding protein, is associated with urinary Mg2+ excretion. Within the kidney, ARL15 is highly expressed in the thick ascending limb (TAL) and distal convoluted tubule (DCT), where Mg2+ reabsorption is tightly regulated. However, the exact function of ARL15 and the mechanism by which ARL15 regulates renal Mg2+ handling are still unknown.

Methods

To identify protein-interaction between ARL15 and Cyclin M (CNNM) proteins, proximity-dependent biotin identification (BioID) and co-immunoprecipitation were performed. Immunohistochemistry were used to investigate co-localization in mouse kidney and human embryonic kidney (HEK293) cells. Furthermore, cell surface biotinylation and 25Mg2+ uptake assays were used to assess cell surface expression of CNNM2 and Mg2+ transport activity. The glycosylation pattern of CNNMs was determined by far lectin Western blot and glycosidase assays.

Results

We identified members of the CNNM family as direct interaction partners of ARL15 by BioID. Immunoprecipitation with truncated CNNM2 proteins indicated that ARL15 interacts with CNNM2 at its carboxyl-(C)-terminal conserved CBS domain. CNNM2 and ARL15 co-localize in the DCT. Interestingly, overexpression of ARL15 in HEK293 cells showed subcellular localization in the Golgi-apparatus and resulted in an increased N-glycosylation of CNNM proteins. This ARL15-mediated glycosylation was Mg2+-sensitive and encompassed hybrid and complex glycosylation. The functional consequences of ARL15-dependent glycosylation were examined by 25Mg2+ uptake experiments. ARL15 increased 25Mg2+ uptake via CNNM2 by increasing its cell surface expression.

Conclusion

ARL15 increases CNNM2 plasma membrane expression by regulating its N-glycosylation pattern. Altogether, our results establish ARL15 as novel regulatory mechanism of Mg2+ transport within the DCT.

Funding

  • Government Support - Non-U.S.