Sialic acid catabolism by N-acetylneuraminate pyruvate lyase is essential for muscle function
Xiao-Yan Wen, … , Clara D.M. van Karnebeek, Dirk J. Lefeber
Xiao-Yan Wen, … , Clara D.M. van Karnebeek, Dirk J. Lefeber
Published December 20, 2018
Citation Information: JCI Insight. 2018;3(24):e122373. https://doi.org/10.1172/jci.insight.122373.
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Categories: Research Article Genetics Metabolism

Sialic acid catabolism by N-acetylneuraminate pyruvate lyase is essential for muscle function

Abstract

Sialic acids are important components of glycoproteins and glycolipids essential for cellular communication, infection, and metastasis. The importance of sialic acid biosynthesis in human physiology is well illustrated by the severe metabolic disorders in this pathway. However, the biological role of sialic acid catabolism in humans remains unclear. Here, we present evidence that sialic acid catabolism is important for heart and skeletal muscle function and development in humans and zebrafish. In two siblings, presenting with sialuria, exercise intolerance/muscle wasting, and cardiac symptoms in the brother, compound heterozygous mutations [chr1:182775324C>T (c.187C>T; p.Arg63Cys) and chr1:182772897A>G (c.133A>G; p.Asn45Asp)] were found in the N-acetylneuraminate pyruvate lyase gene (NPL). In vitro, NPL activity and sialic acid catabolism were affected, with a cell-type-specific reduction of N-acetyl mannosamine (ManNAc). A knockdown of NPL in zebrafish resulted in severe skeletal myopathy and cardiac edema, mimicking the human phenotype. The phenotype was rescued by expression of wild-type human NPL but not by the p.Arg63Cys or p.Asn45Asp mutants. Importantly, the myopathy phenotype in zebrafish embryos was rescued by treatment with the catabolic products of NPL: N-acetyl glucosamine (GlcNAc) and ManNAc; the latter also rescuing the cardiac phenotype. In conclusion, we provide the first report to our knowledge of a human defect in sialic acid catabolism, which implicates an important role of the sialic acid catabolic pathway in mammalian muscle physiology, and suggests opportunities for monosaccharide replacement therapy in human patients.

Authors

Xiao-Yan Wen, Maja Tarailo-Graovac, Koroboshka Brand-Arzamendi, Anke Willems, Bojana Rakic, Karin Huijben, Afitz Da Silva, Xuefang Pan, Suzan El-Rass, Robin Ng, Katheryn Selby, Anju Mary Philip, Junghwa Yun, X. Cynthia Ye, Colin J. Ross, Anna M. Lehman, Fokje Zijlstra, N. Abu Bakar, Britt Drögemöller, Jacqueline Moreland, Wyeth W. Wasserman, Hilary Vallance, Monique van Scherpenzeel, Farhad Karbassi, Martin Hoskings, Udo Engelke, Arjan de Brouwer, Ron A. Wevers, Alexey V. Pshezhetsky, Clara D.M. van Karnebeek, Dirk J. Lefeber

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Figure 2

p.Asn45Asp and p.Arg63Cys mutations affect enzymatic activity and expression and/or stability of the NPL protein.

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p.Asn45Asp and p.Arg63Cys mutations affect enzymatic activity and expres...
(A) Cultured HEK293T cells cotransfected with plasmids encoding cDNA of the wild-type (WT) human NPL or its p.Arg63Cys and p.Asn45Asp mutants containing a C-terminal DYK tag and pCMV-HGSNAT-GFP plasmid encoding human acetyl-CoA: α-glucosaminide N-acetyltransferase (HGSNAT). Forty-eight hours after transfection, the cells were harvested and the enzymatic NPL and HGSNAT activities were measured in cell homogenates. (B) The reaction rate of Neu5Ac hydrolysis by the WT NPL and the p.Asn45Asp NPL mutant were measured at different initial concentrations of the substrate to calculate the Km and Vmax values for the WT and mutant enzyme. (C) The protein levels of WT NPL and its mutants were measured by Western blot using antibodies against the DYK tag. Panels show representative images of 3 independent experiments. The bar graph shows intensities of the DYK cross-reactive protein bands (means ± SD of the values from 3 independent experiments) measured with ImageJ software and normalized to the intensities of the control HGSNAT protein band stained with anti-HGSNAT antibody. (D) Stability of WT NPL protein and its mutants was measured by nonradioactive pulse-chase experiment. HEK293T cells expressing WT or mutant NPL were treated with 7 μM cycloheximide to inhibit de novo protein synthesis, chased 2, 4, 6, 18, and 24 hours, and analyzed by Western blot to measure intensity of the NPL protein band (DYK tag). Panels show representative images of 3 independent experiments; 20 and 60 μg of protein was loaded per well for the cells transfected with the WT and mutant plasmids, respectively. Bands representing mature NPL (NPLm) and its precursor (NPLp) are marked. The graph shows intensities of the DYK cross-reactive protein bands (means ± SD of the values from 3 independent experiments) measured with ImageJ software. Table shows half-life of WT and mutant NPL protein (hours) calculated using 1-phase decay nonlinear regression. *P < 0.05, **P < 0.01, ***P < 0.001 versus WT by 1- and 2-way ANOVA.