Involvement of endoplasmic reticulum stress in a novel Classic Galactosemia model

Abstract

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) activity in humans leads to a potentially lethal disorder called Classic Galactosemia. It is well known that patients often accumulate high levels of galactose metabolites such as galactose-1-phosphate (gal-1-p) in their tissues. However, specific targets of gal-1-p and other accumulated metabolites remain uncertain. In this study, we developed a new model system to study this toxicity using primary fibroblasts derived from galactosemic patients. GALT activity was reconstituted in these primary cells through lentivirus-mediated gene transfer. Gene expression profiling showed that GALT-deficient cells, but not normal cells, responded to galactose challenge by activating a set of genes characteristic of endoplasmic reticulum (ER) stress. Western blot analysis showed that the master regulator of ER stress, BiP, was up-regulated at least threefold in these cells upon galactose challenge. We also found that treatment of these cells with galactose, but not glucose or hexose-free media reduced Ca²⁺ mobilization in response to activation of Gq-coupled receptors. To explore whether the muted Ca²⁺ mobilization is related to reduced inositol turnover, we discovered that gal-1-p competitively inhibited human inositol monophosphatase (hIMPase1). We hypothesize that galactose intoxication under GALT-deficiency resulted from accumulation of toxic galactose metabolite products, which led to the accumulation of unfolded proteins, altered calcium homeostasis, and subsequently ER stress.

Introduction

Classic Galactosemia (G/G) is an autosomal recessive, multi-system disorder caused by deleterious mutations of the human galactose-1-phosphate uridyltransferase (GALT) gene, which results in inability to metabolize galactose [1], [2], [3] (Fig. 1). Although a galactose-restricted diet prevents the neonatal lethality of this disorder, many well-treated patients continue to develop debilitating complications such as premature ovarian failure (POF), mental retardation, and some neurological defects [4], [5]. The causes of these unsatisfactory outcomes remain unclear. Recent studies, however, showed that patients placed on a galactose-restricted diet are never truly free of galactose insult, as a significant amount of galactose is found in non-dairy foodstuffs such as vegetables and fruits [6], [7]. More importantly, galactose moieties can be produced endogenously from UDP-glucose via the UDP-4-galactose epimerase (GALE) reaction (Fig. 1), and natural turnover of glycoproteins/glycolipids. In fact, using isotopic labeling, Berry et al. elegantly demonstrated that a 50 kg adult male could produce up to 2 g of galactose per day [8], [9]. Once the galactose is formed intracellularly, it will be converted to galactose-1-phosphate (gal-1-p) by galactokinase (GALK).

Paradoxically, patients with an inherited deficiency of galactokinase (GALK) do not manifest either the acute toxicity syndrome or chronic complications seen in galactosemic patients [3]. Since GALK-deficient patients do not accumulate gal-1-p in their tissues [3], [10], researchers have long proposed that gal-1-p plays a significant role in the pathogenesis of Classic Galactosemia [11], [12], [13], [14]. Yet the in vivo targets of this presumably toxic intermediate have never been identified. Concurrently, the lack of overt galactose toxicity in the GALT-knockout mouse model has continued to hamper the efforts to delineate the molecular mechanism of this disease [15], [16], [17].

In this study, we developed a novel isogenic human cell model of GALT-deficiency, and showed that galactose challenge of these cells resulted in accumulation of gal-1-p and distinct signs of endoplasmic reticulum (ER) stress. Thus, our findings not only shed light on the pathogenic mechanisms of Classic Galactosemia, but also expanded the contemporary metabolomics model that depicted the fundamental genetic/physical interactions between galactose utilization and other metabolic pathways [18].