Assessment of cellular cobalamin metabolism in Gaucher disease

Abstract

Background: Gaucher disease (GD) is a lysosomal disorder caused by biallelic pathogenic mutations in the GBA1 gene that encodes beta-glucosidase (GCase), and more rarely, by a deficiency in the GCase activator, saposin C. Clinically, GD manifests with heterogeneous multiorgan involvement mainly affecting hematological, hepatic and neurological axes. This disorder is divided into three types, based on the absence (type I) or presence and severity (types II and III) of involvement of the central nervous system. At the cellular level, deficiency of GBA1 disturbs lysosomal storage with buildup of glucocerebroside. The consequences of disturbed lysosomal metabolism on biochemical pathways that require lysosomal processing are unknown. Abnormal systemic markers of cobalamin (Cbl, B₁₂) metabolism have been reported in patients with GD, suggesting impairments in lysosomal handling of Cbl or in its downstream utilization events.

Methods: Cultured skin fibroblasts from control humans (n = 3), from patients with GD types I (n = 1), II (n = 1) and III (n = 1) and an asymptomatic carrier of GD were examined for their GCase enzymatic activity and lysosomal compartment intactness. Control human and GD fibroblasts were cultured in growth medium with and without 500 nM hydroxocobalamin supplementation. Cellular cobalamin status was examined via determination of metabolomic markers in cell lysate (intracellular) and conditioned culture medium (extracellular). The presence of transcobalamin (TC) in whole cell lysates was examined by Western blot.

Results: Cultured skin fibroblasts from GD patients exhibited reduced GCase activity compared to healthy individuals and an asymptomatic carrier of GD, demonstrating a preserved disease phenotype in this cell type. The concentrations of total homocysteine (tHcy), methylmalonic acid (MMA), cysteine (Cys) and methionine (Met) in GD cells were comparable to control levels, except in one patient with GD III. The response of these metabolomic markers to supplementation with hydroxocobalamin (HOCbl) yielded variable results. The content of transcobalamin in whole cell lysates was comparable in control human and GD patients.

Conclusions: Our results indicate that cobalamin transport and cellular processing pathways are overall protected from lysosomal storage damage in GD fibroblasts. Extending these studies to hepatocytes, macrophages and plasma will shed light on cell- and compartment-specific vitamin B₁₂ metabolism in Gaucher disease.

Keywords: Beta-glucosidase; Cobalamin; Gaucher disease; Homocysteine; Methylmalonic acid; Transcobalamin; Vitamin B12.

Fig. 1

α- and β-glucosidase activity in healthy human controls, Gaucher disease patients and an asymptomatic carrier of GD. a Gaucher patients (n = 3) had no detectable activity of β-glucosidase (nmol/mLxh), whereas the asymptomatic carrier of GD (n = 1) exhibited β-glucosidase activity comparable to those of human controls (n = 3); b All examined subjects presented comparable α-glucosidase activity (nmol/mLxh), suggesting preserved activity of lysosomal components not associated with the GBA1 mutation. Legend: without HOCbl – culture medium without hydroxocobalamin; with HOCbl – culture medium with hydroxocobalamin, GD – Gaucher disease, RecNciI/− − asymptomatic carrier of GD

Fig. 2

Content of transcobalamin in healthy human controls, Gaucher disease patients and an asymptomatic carrier of GD. a Whole cell lysates (30 μg) of human control and GD fibroblasts were examined for intracellular content of transcobalamin, with and without HOCbl supplementation. Under our experimental conditions, no differences were identified between control and GD cells, suggesting normal expression of transport protein transcobalamin. Left panel: Western blot results obtained by probing whole cell lysates with anti-TC (rabbit anti-human, dilution 1:500) and secondary goat anti-rabbit IgG-HRP (1:1000) antibody. Right panels: Ponceau staining of nitrocellulose membrane after semi-dry blot transfer as protein loading control. b Semi-quantitative analysis of TC content shown in panel (a) shows slightly reduced TC content in GD cells compared to healthy human controls, albeit without statistical significance. Values shown are mean normalized areas plus minus standard error of the mean