Human TMEM165 Knockout Cell Line-HEK293T

TMEM165 is associated with a rare human genetic disease named TMEM165-CDG (congenital disorder of glycosylation). It is localized to the Golgi apparatus, is highly conserved during evolution, and belongs to uncharacterized protein family 0016 (UPF0016). The use of homologous TMEM165 knockout HEK293 cells was crucial to reveal the role of TMEM165 in Golgi manganese homeostasis. Manganese is a major cofactor for many glycosylation enzymes. Severe Golgi glycosylation defects were observed in TMEM165 knockout human embryonic kidney (KO HEK) 293 cells that could be rescued by exogenous manganese supplementation. Interestingly, here the researchers demonstrate that the observed Golgi glycosylation defects are critically dependent on fetal bovine serum, especially its manganese levels. These results also suggest that iron and/or galactose can modulate the glycosylation defects observed in TMEM165 knockout HEK293 cells.

Previous studies have shown that supplementation with galactose can suppress some of the glycosylation defects of TMEM165-CDG. Here, the researchers wondered whether this suppression also depended on the source of the FBS. To tackle this point, TMEM165 KO HEK293 cells were cultured in two different FBS (FBS 2 or FBS 4) and supplemented with 1 μM MnCl₂, 1 mM galactose or 1 μM MnCl2 + 1 mM galactose. Intriguingly, all treatments resulted in fully glycosylated forms of LAMP2 in TMEM165 KO HEK293 cells cultured in FBS 2 (Figure 1A). The result was completely different in cells grown in FBS 4 (Figure 1B). In the latter condition, galactose or Mn²⁺ supplementation poorly rescued LAMP2 glycosylation (Figure 1B). However, the combination of these two factors rescued the fully glycosylated form of LAMP2 (Figure 1B). This result suggests that, depending on the source of FBS, Mn²⁺ can enhance the effect of galactose on suppressing LAMP2 glycosylation defects (and vice versa). These results emphasize the critical importance of serum content on glycomic results.

Figure 1. The suppression of LAMP2 glycosylation defect by galactose supplementation depends on the FBS used for cell culture. (Vicogne D, et al., 2020)