Characterization of glycan substrates accumulating in GM1 Gangliosidosis

Abstract

IntroductionGM1 gangliosidosis is a rare autosomal recessive genetic disorder caused by the disruption of the GLB1 gene that encodes β-galactosidase, a lysosomal hydrolase that removes β-linked galactose from the non-reducing end of glycans. Deficiency of this catabolic enzyme leads to the lysosomal accumulation of GM1 and its asialo derivative GA1 in β-galactosidase deficient patients and animal models. In addition to GM1 and GA1, there are other glycoconjugates that contain β-linked galactose whose metabolites are substrates for β-galactosidase. For example, a number of N-linked glycan structures that have galactose at their non-reducing end have been shown to accumulate in GM1 gangliosidosis patient tissues and biological fluids.ObjectiveIn this study, we attempt to fully characterize the broad array of GLB1 substrates that require GLB1 for their lysosomal turnover.ResultsUsing tandem mass spectrometry and glycan reductive isotope labeling with data-dependent mass spectrometry, we have confirmed the accumulation of glycolipids (GM1 and GA1) and N-linked glycans with terminal beta-linked galactose. We have also discovered a novel set of core 1 and 2 O-linked glycan metabolites, many of which are part of structurally-related isobaric series that accumulate in disease. In the brain of GLB1 null mice, the levels of these glycan metabolites increased along with those of both GM1 and GA1 as a function of age. In addition to brain tissue, we found elevated levels of both N-linked and O-linked glycan metabolites in a number of peripheral tissues and in urine. Both brain and urine samples from human GM1 gangliosidosis patients exhibited large increases in steady state levels for the same glycan metabolites, demonstrating their correlation with this disease in humans as well.ConclusionsOur studies illustrate that GLB1 deficiency is not purely a ganglioside accumulation disorder, but instead a broad oligosaccharidosis that include representatives of many β-linked galactose containing glycans and glycoconjugates including glycolipids, N-linked glycans, and various O-linked glycans. Accounting for all β-galactosidase substrates that accumulate when this enzyme is deficient increases our understanding of this severe disorder by identifying metabolites that may drive certain aspects of the disease and may also serve as informative disease biomarkers to fully evaluate the efficacy of future therapies.

Intracerebroventricular enzyme replacement therapy with β-galactosidase reverses brain pathologies due to GM1 gangliosidosis in mice

“…In our biomarker analyses of GM1 gangliosidosis mice, we observed elevated levels of the A2G2' glycan substrate in brain as well as several systemic tissues, including liver, spleen, kidney and urine, when compared with wild-type littermate control mice (3). A2G2' glycan substrate was also elevated in brain tissue and urine from GM1 gangliosidosis patients, when compared with samples from normal individuals (3), raising the possibility of utilizing urinary A2G2' levels as an indicator of systemic exposure of rhBeta-Gal following ICV-ERT.…”

“…GLB1 encodes lysosomal β-gal, which catalyzes the stepwise lysosomal degradation of multiple galactose-containing substrates ( 2 ). Three pathogenic and biochemically distinct classes of galactose-containing substrates accumulate in lysosomes of β-gal–deficient cells; GM1 and GA1 gangliosides, the glycosaminoglycan, keratan sulfate (KS), and oligosaccharides derived from glycoprotein metabolism (glycan substrates) ( 3 , 4 , 39 ). A wide spectrum of clinical phenotypes have been described for GM1 gangliosidosis.…”

Mechanism of Secondary Ganglioside and Lipid Accumulation in Lysosomal Disease

“…As many other lysosomal hydrolases, the GM1-β-galactosidase is a promiscuous glycosidase, which cleaves many oligosaccharides. Its deficiency triggers the accumulation of many other glycan substrates in GM1 gangliosidosis [40].…”

Biomarkers for Lysosomal Storage Disorders with an Emphasis on Mass Spectrometry

“…Due to a relatively larger molecular weight, which might be beyond the typical working range of a tandem mass spectrometer, these oligosaccharides are often identified using time-of-flight mass spectrometry (TOF-MS). The biological source of oligosaccharides may be derived from either Oor N-linked glycans or glycosphingolipids [38]. Usually, N-linked oligosaccharides need to be treated with peptide N-glycosidase, while O-linked oligosaccharides are treated with either acid or alkali.…”

“…A recent study by Lawrence and colleagues provides a comprehensive profile of glycans in individuals with β-galactosidase (GLB) deficiency [38]. In this paper, the authors use tandem mass spectrometry and glycan reductive isotope labeling to provide evidence that GLB deficiency is not a simple ganglioside accumulation disorder, but rather it elevates many oligosaccharides including many β-linked galactoses.…”