Involvement of thapsigargin– and cyclopiazonic acid–sensitive pumps in the rescue of TMEM165‐associated glycosylation defects by Mn²⁺

Abstract

Congenital disorders of glycosylation are severe inherited diseases in which aberrant protein glycosylation is a hallmark. Transmembrane protein 165 (TMEM165) is a novel Golgi transmembrane protein involved in type II congenital disorders of glycosylation. Although its biologic function is still a controversial issue, we have demonstrated that the Golgi glycosylation defect due to TMEM165 deficiency resulted from a Golgi Mn2+ homeostasis defect. The goal of this study was to delineate the cellular pathway by which extracellular Mn2+ rescues N‐glycosylation in TMEM165 knockout (KO) cells. We first demonstrated that after extracellular exposure, Mn2+ uptake by HEK293 cells at the plasma membrane did not rely on endocytosis but was likely done by plasma membrane transporters. Second, we showed that the secretory pathway Ca2+‐ATPase 1, also known to mediate the influx of cytosolic Mn2+ into the lumen of the Golgi apparatus, is not crucial for the Mn2+‐induced rescue glycosylation of lysosomal‐associated membrane protein 2 (LAMP2). In contrast, our results demonstrate the involvement of cyclopiazonic acid—and thapsigargin (Tg)‐sensitive pumps in the rescue of TMEM165‐associated glycosylation defects by Mn2+. Interestingly, overexpression of sarco/endoplasmic reticulum Ca2+‐ATPase (SERCA) 2b isoform in TMEM165 KO cells partially rescues the observed LAMP2 glycosylation defect. Overall, this study indicates that the rescue of Golgi N‐glycosylation defects in TMEM165 KO cells by extracellular Mn2+ involves the activity of Tg and cyclopiazonic acid–sensitive pumps, probably the SERCA pumps.—Houdou, M., Lebredonchel, E., Garat, A., Duvet, S., Legrand, D., Decool, V., Klein, A., Ouzzine, M., Gasnier, B., Potelle, S., Foulquier, F. Involvement of thapsigargin—and cyclopiazonic acid–sensitive pumps in the rescue of TMEM165‐associated glycosylation defects by Mn2+. FASEB J. 33, 2669–2679 (2019). http://www.fasebj.org

Fetal bovine serum impacts the observed N‐glycosylation defects in TMEM165 KO HEK cells

“…We previously reported that LAMP2 glycosylation defects found in TMEM165 KO HEK cells were totally suppressed by the addition of exogenous MnCl 2 in the culture medium. This was observed from 8 hours of incubation with 1 μM MnCl 2 . We recently observed that this suppression could appear without any supplementation of MnCl 2 probably due to cell culture conditions (data not shown).…”

“…This was observed from 8 hours of incubation with 1 μM MnCl 2 . 18,19 We recently observed that this suppression could appear without any supplementation of MnCl 2 probably due to cell culture conditions (data not shown). This urged us to investigate the effects of different sources of FBS on the appearance and/or rescue of the N-glycosylation defects in TMEM165 KO HEK cells.…”

“…18,20 The hypothesis that TMEM165 is crucial in regulating Golgi manganese homeostasis, a major cofactor of many glycosylation enzymes, 21,22 came from the observation that low Mn 2+ concentrations (100 nM-1 μM) in the culture medium were sufficient to suppress the observed Golgi glycosylation defects. 18,19 In the present study, we examined the contribution of fetal bovine serum on these observed Golgi glycosylation defects. This came from the observation that Golgi glycosylation defects could be suppressed depending on culture conditions.…”

“…Our previous results unambiguously demonstrated a link between TMEM165 and Golgi Mn 2+ homeostasis 18 through the rescue of Golgi glycosylation defects observed in TMEM165 Knock Out Human Embryonic Kidney (KO HEK) cells by MnCl 2 supplementation. 18,19 Recently, we noticed that suppression of these glycosylation defects depends on cell culture conditions. In this study, we investigate the effects of different fetal bovine sera (FBS) on Golgi glycosylation defects in TMEM165 KO HEK cells.…”

ZIP14 is degraded in response to manganese exposure

“…The Golgi apparatus appears to play an important role in cellular Mn homeostasis and effects on its function often occur over the Mn concentration range used for our studies (Carmona et al 2019;Houdou et al 2019). In fact, previous studies examining Mnregulated proteins included treatments of cells with 1-500 lM Mn over time courses ranging from 1 to 24 h (Masuda et al 2013;Mukhopadhyay et al 2010;Potelle et al 2017;Tewari et al 2015;Towler et al 2000).…”