POMK regulates dystroglycan function via LARGE-mediated elongation of matriglycan
- Howard Hughes Medical Institute, University of Iowa Roy J. and Lucille A. Carver College of Medicine, United States
- Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health & Great Ormond Street Hospital, United Kingdom
- University of Iowa, United States
- Peking University, China
- University of California, San Diego, United States
- UCL Great Ormond Street Institute of Child Health, United Kingdom
Abstract
Matriglycan [-GlcA-β1,3-Xyl-α1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetylglucosaminyltransferase-1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNac-β1,3-GlcNac-β1,4-Man) preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.
Data availability
All data generated or analysed during this study are included in the manuscript.
Article and author information
Author details
Funding
Paul D. Wellstone Muscular Dystrophy Specialized Research Center grant (1U54NS053672)
- Kevin P Campbell
Great Ormond Street Hospital for Children NHS Foundation Trust and University College
- Silvia Torelli
- Francesco Muntoni
Medical Scientist Training Program Grant by the National Institute of General Medical Sciences (5 T32 GM007337)
- Ameya S Walimbe
Howard Hughes Medical Institute
- Kevin P Campbell
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) protocols of the University of Iowa (#0081122).
Human subjects: All procedures performed in this study involving human participants were in accordance with the ethical standards of NHS Health Research Authority (REC ref: 06/Q0406/33). We acknowledge and thank the BRC/MRC Centre for Neuromuscular Diseases Biobank for providing patients' serum and biopsy samples. We confirm that informed consent was provided to the patient and family regarding the nature of the genetic studies to be performed upon collection of samples and is available for our patient.
Copyright
© 2020, Walimbe et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 2,123
- views
-
- 296
- downloads
-
- 23
- citations
Views, downloads and citations are aggregated across all versions of this paper published by eLife.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
POMK regulates dystroglycan function via LARGE-mediated elongation of matriglycan
eLife 9:e61388.
https://doi.org/10.7554/eLife.61388
Further reading
-
- Biochemistry and Chemical Biology
The protein ligase Connectase can be used to fuse proteins to small molecules, solid carriers, or other proteins. Compared to other protein ligases, it offers greater substrate specificity, higher catalytic efficiency, and catalyzes no side reactions. However, its reaction is reversible, resulting in only 50% fusion product from two equally abundant educts. Here, we present a simple method to reliably obtain 100% fusion product in 1:1 conjugation reactions. This method is efficient for protein-protein or protein-peptide fusions at the N- or C-termini. It enables the generation of defined and completely labeled antibody conjugates with one fusion partner on each chain. The reaction requires short incubation times with small amounts of enzyme and is effective even at low substrate concentrations and at low temperatures. With these characteristics, it presents a valuable new tool for bioengineering.
-
- Biochemistry and Chemical Biology
N 6,2’-O-dimethyladenosine (m6Am) is a modified nucleotide located at the first transcribed position in mRNA and snRNA that is essential for diverse physiological processes. m6Am mapping methods assume each gene uses a single start nucleotide. However, gene transcription usually involves multiple start sites, generating numerous 5’ isoforms. Thus, gene-level annotations cannot capture the diversity of m6Am modification in the transcriptome. Here, we describe CROWN-seq, which simultaneously identifies transcription-start nucleotides and quantifies m6Am stoichiometry for each 5’ isoform that initiates with adenosine. Using CROWN-seq, we map the m6Am landscape in nine human cell lines. Our findings reveal that m6Am is nearly always a high stoichiometry modification, with only a small subset of cellular mRNAs showing lower m6Am stoichiometry. We find that m6Am is associated with increased transcript expression and provide evidence that m6Am may be linked to transcription initiation associated with specific promoter sequences and initiation mechanisms. These data suggest a potential new function for m6Am in influencing transcription.
