Introduction

GM1 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.

Objective

In this study, we attempt to fully characterize the broad array of GLB1 substrates that require GLB1 for their lysosomal turnover.

Results

Using 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.

Conclusions

Our 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.

中文翻译：

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在 GM1 神经节苷脂沉积症中积累的聚糖底物的表征。

介绍

GM1 神经节苷脂沉积症是一种罕见的常染色体隐性遗传疾病，由编码 β-半乳糖苷酶的GLB1基因破坏引起，β-半乳糖苷酶是一种溶酶体水解酶，可从聚糖的非还原端去除 β-连接的半乳​​糖。这种分解代谢酶的缺乏导致 GM1 及其去唾液酸衍生物 GA1 在 β-半乳糖苷酶缺陷患者和动物模型中的溶酶体积累。除 GM1 和 GA1 外，还有其他含有 β-连接半乳糖的糖缀合物，其代谢物是 β-半乳糖苷酶的底物。例如，许多在其非还原端具有半乳糖的 N-连接聚糖结构已显示在 GM1 神经节苷脂沉积症患者组织和生物体液中积累。

客观的

在这项研究中，我们试图全面表征需要 GLB1 进行溶酶体转换的广泛 GLB1 底物。

结果

使用串联质谱和聚糖还原同位素标记与数据相关的质谱，我们已经确认糖脂（GM1 和 GA1）和 N 连接聚糖与末端 β 连接半乳糖的积累。我们还发现了一组新的核心 1 和 2 O-连接聚糖代谢物，其中许多是在疾病中积累的结构相关的同量异位素系列的一部分。在GLB1的大脑中在缺失小鼠的情况下，这些聚糖代谢物的水平随着 GM1 和 GA1 的水平随着年龄的增长而增加。除了脑组织外，我们还发现许多外周组织和尿液中 N 连接和 O 连接的聚糖代谢物水平升高。来自人类 GM1 神经节苷脂沉着症患者的大脑和尿液样本都表现出相同聚糖代谢物的稳态水平大幅增加，这也证明了它们与人类这种疾病的相关性。

结论

我们的研究表明，GLB1 缺乏症不仅仅是一种神经节苷脂积累障碍，而是一种广泛的寡糖贮积症，包括许多含有 β-连接半乳糖的聚糖和糖缀合物的代表，包括糖脂、N-连接聚糖和各种 O-连接聚糖。考虑到当这种酶缺乏时积累的所有 β-半乳糖苷酶底物，我们可以通过识别可能驱动疾病某些方面的代谢物来增加我们对这种严重疾病的理解，并且还可以作为信息丰富的疾病生物标志物，以全面评估未来疗法的功效。