Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation

Abstract

The stage differentiation from trophozoite to cyst (i.e., encystation) is an essential step for Giardia to survive outside its human host and spread the infection via the fecal-oral route. We have previously shown that Giardia expresses glucosylceramide transferase 1 (GlcT1) enzyme, the activity of which is elevated during encystation. We have also reported that blocking the activity of gGlcT1 interferes with the biogenesis of encystation-specific vesicles (ESVs) and cyst viability in Giardia. To further understand the role of this enzyme and how it regulates encystation, we overexpressed, knocked down, and rescued the giardial GlcT1 (gGlcT1) gene and measured its enzymatic activity in live parasites as well as in isolated membrane fractions using NBD-ceramide and UDP-glucose or UDP-galactose. We observed that gGlcT1 is able to catalyze the synthesis of both glucosylceramide (GlcCer) and galactosylceramide (GalCer), however the synthesis of GalCer is 2–3 fold higher than of GlcCer. Although both activities follow Michaelis–Menten kinetics, the bindings of UDP-glucose and UDP-galactose with the enzyme appear to be non-competitive and independent of each other. The modulation of gGlcT1 synthesis concomitantly influenced the expression cyst-wall protein (CWP) and overall encystation. We propose that gGlcT1 is a unique enzyme and that Giardia uses this enzyme to synthesize both GlcCer and GalCer to facilitate the process of encystation/cyst production.

A Targeted Mass Spectrometric Analysis Reveals the Presence of a Reduced but Dynamic Sphingolipid Metabolic Pathway in an Ancient Protozoan, Giardia lamblia

“…The induction of encystation triggers the biogenesis of cyst walls that covers the plasma membrane of the entire trophozoites (Lauwaet et al, 2007), as depicted in Figure 4A. Previously, it has been demonstrated that glucosylceramide transferase 1 (GlcT1)—an enzyme of the SL metabolic pathway—regulated the encystation and cyst production by Giardia (Mendez et al, 2013; Robles-Martinez et al, 2017), suggesting the importance of SLs in this process. Therefore, to gain an in-depth understanding regarding the abundance of SLs in encysting cells and cysts, we carried out in vitro encystation (Gillin et al, 1989) followed by extracting lipids from non-encysting trophozoites, encysting cells (12-h and 24-h post-induction of encystation or 12-h and 24-h PIE) and cysts and analyzed as described in the Methods section.…”

“…These differences were clear in variations in the abundance of individual species and their contribution to the class as a whole. For GSLs, the differences could be attributed to the appearance of new GSLs during encystation (Mendez et al, 2013; Robles-Martinez et al, 2017) (Table S4; Figure 4B) and characteristic separation of encysting SLs from trophozoites and cysts (Figure 5). Specifically, as encystation progressed to form cysts, there was an increase in the species of Hex 2 Cer from ~1.0 pmol in trophozoites to ~3.0 pmol in cysts, while the total Hex 3 Cer abundance in cysts was only ~2 fold higher than trophozoites (Figure 4B, “glycosphingolipids”).…”

“…Stage-specific expression of SLs may have long-term biological implications in Giardia . Increased Cer (Figure 4B) is likely to be involved in synthesizing new GSLs facilitated by giardial GlcT1 (Mendez et al, 2013; Robles-Martinez et al, 2017). Although GSLs are acquired by Giardia from the medium, it remains unclear how this parasite selects and utilizes both endogenous and exogenous SLs for the purpose of cell signaling and encystation (Figure 4B).…”

“…The abundance of GSL species, especially mono-hexosyl and di-hexosylceramides are elevated during early encysting cells (12-h PIE) and declines during late encystation (24-h PIE) and in cysts (Figure 4). This can be attributed to the fact that gGlcT1 activity is upregulated in encysting cells and could therefore be linked to the increased synthesis of new GSLs (Mendez et al, 2013; Robles-Martinez et al, 2017). Interestingly, in an earlier report, Stefanic et al (2010) demonstrated that Hex3Cer level in Giardia increases during encystation and its abundance in encysting cells is several fold higher than non-encysting trophozoites or other GSLs.…”

“…Interestingly, the normalization of gGlcT1 expression restores ESV biogenesis and cyst viability, further establishing the regulatory role of gGlcT1 in encystation and cyst production. More recently, it was demonstrated that gGlcT1 is a dual-substrate enzyme and can catalyze the synthesis of both GlcCer and galactosylceramide (GalCer) (Robles-Martinez et al, 2017). These results prompted us to analyze SLs in Giardia and the culture medium to elucidate the influence of environmental SLs in maintaining overall SL homeostasis in this parasite.…”