POMK regulates dystroglycan function via LARGE1-mediated elongation of matriglycan
- Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, United States
- Dubowitz Neuromuscular Centre, UCL Great Ormond Street Institute of Child Health & Great Ormond Street Hospital, United Kingdom
- Medical Nuclear Magnetic Resonance Facility, University of Iowa Roy J. and Lucille A. Carver College of Medicine, United States
- The State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking-Tsinghua Center for Life Sciences, Peking University, China
- Department of Pharmacology, Department of Cellular and Molecular Medicine, Department of Chemistry and Biochemistry, University of California, San Diego, United States
- National Institute for Health Research Great Ormond Street Hospital Biomedical Research Centre, UCL Great Ormond Street Institute of Child Health, United Kingdom
Figures
Figure 1
Synthesis of the α-DG Laminin-Binding Modification and Enzymes Involved.
Synthesis of the laminin-binding modification begins with the addition of the core M3 trisaccharide (GalNAc-β3-GlcNAc-β4-Man) on α-DG by the sequential actions of Protein O-Mannosyltransferase 1 and …
Figure 2 with 2 supplements
Characterization of a Patient with a Loss-of-Function Mutation in POMK.
(A) (above) Human POMK consists of a transmembrane domain (TM) and a kinase domain (N-lobe and C-lobe). The kinase domain contains the catalytic loop (orange) and activation segment (green). (below) …
Figure 2—figure supplement 1
Structural Modeling of POMK D204N Mutation.
This figure shows structural modeling of wild-type POMK and the POMK D204N mutation using human POMK protein sequence numbering, based on the crystal structure of zebrafish POMK. The green spheres …
Figure 2—figure supplement 2
Supplemental Analysis of POMK D204N Fibroblasts and Muscle.
(A) LARGE1 activity in control human fibroblasts and fibroblasts from patient NH13-284 (POMK D204N). Triple asterisks indicate statistical significance using Student’s unpaired t-test (three …
Figure 3 with 4 supplements
Mice with a Muscle-Specific Loss of Pomk Develop Hallmarks of a Mild Muscular Dystrophy.
(A) H&E and immunofluorescence analyses using IIH6 (anti-matriglycan) and an anti-β-DG antibody of quadriceps muscles of 4–6 week-old PomkLoxP/LoxP (Control) and MckCre; Pax7Cre; PomkLoxP/LoxP …
Figure 3—figure supplement 1
Schematic for Generation of Floxed Alleles of Pomk.
Map of 5’ and 3’ LoxP sites (orange). LoxP sites flanking exon 5 of Pomk (large black box), which encodes the majority of the kinase domain of POMK, were inserted using CRISPR/Cas9. Cre-mediated …
Figure 3—figure supplement 2
Results of PomkLoxP/LoxP Genotyping.
(A) Genotyping strategy for floxed Pomk Allele. PCR Primers were designed to flank the 5’ LoxP site. (B) The wild-type allele of Pomk is 197 bp, while the floxed allele is 235 base pairs.
Figure 3—figure supplement 3
Muscle-Specific Pomk Knockout Mice Have Reduced Grip Strength and Body Weight.
(A, B) 2-limb grip strength of 1-month-old (A) and 4-month-old (B) PomkLoxP/LoxP (Control) and MckCre; Pax7Cre; PomkLoxP/LoxP (M-POMK KO) mice. Triple asterisks indicate statistical significance …
Figure 3—figure supplement 4
Supplemental Biochemical Analysis of Pomk-null Skeletal Muscle.
(A, B) POMK (A) and LARGE1 (B) activity of PomkLoxP/LoxP (Control) and MckCre; Pax7Cre; PomkLoxP/LoxP (M-POMK KO) quadriceps muscle extracts (three replicates). Asterisks indicate statistical …
Figure 4
MckCre; Pax7Cre; PomkLoxP/LoxP Extensor Digitorum Longus (EDL) Muscle Demonstrates Eccentric Contraction-Induced Force Loss.
(A) Mass (milligrams) of PomkLoxP/LoxP (Control) and MckCre; Pax7Cre; PomkLoxP/LoxP (M-POMK KO) EDL muscles tested for force production. ***Statistical significance with Student’s unpaired t-test …
Figure 5 with 2 supplements
Mice with a Muscle-Specific Loss of Pomk Express Matriglycan.
(A) Biochemical analysis of Control and M-POMK KO skeletal muscle. Glycoproteins were enriched from quadriceps skeletal muscles of mice using wheat-germ agglutinin (WGA)-agarose. Immunoblotting was …
Figure 5—figure supplement 1
Solid-Phase Binding Analyses of Pomk-null Skeletal Muscle.
(A) Solid-phase binding analysis (relative Bmax for laminin-111) of PomkLoxP/LoxP (Control), MckCre; Pax7Cre; PomkLoxP/LoxP (M-POMK KO), MckCre; PomkLoxP/LoxP, and Largemyd skeletal muscle (three …
Figure 5—figure supplement 2
Pomk-null Muscle Expresses Matriglycan.
(A, B) Glycoproteins were enriched from skeletal muscles of Control, M-POMK KO, and Largemyd mice and treated in combination with α-xylosidase (Xylsa) and β-glucuronidase (Bgus). Immunoblotting was …
Figure 6 with 2 supplements
POMK D204N lacks Catalytic Activity.
(A) POMK or (B) LARGE1 activity in POMK KO HAP1 cells transduced with adenoviruses encoding POMK D204N, POMK D204A, or POMK WT. Triple asterisks: statistical significance (p-value<0.0001) compared …
Figure 6—figure supplement 1
Supplemental Biochemical Analysis of POMK D204N and POMK KO HAP1 Cells.
(A, B, C) POMK KO HAP1 cells were transduced with the indicated adenoviruses and immunoblotting was performed for: (A) POMK, (B) Core α-DG and β-DG, and (C) matriglycan (IIH6), (three replicates). (D…
Figure 6—figure supplement 2
Mass spectra of O-glycans carried by a DG mucin-like domain model (DG390) expressed in POMK KO (A) or Fukutin (FKTN) KO (B) HAP1 cells.
The glycans were reductively released from the protein backbone and permethylated prior to matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analyses. Mass spectrometry (MS) …
Figure 7 with 6 supplements
LARGE1 requires POMK to Elongate Matriglycan.
(A) WT, POMK KO, and POMK/LARGE1 KO HAP1 cells (left) or POMK/DAG1 KO HAP1 cells (right) (three replicates). (B) Solid-phase analysis of WT, POMK KO, POMK/DAG1 KO, and LARGE1 KO HAP1 cells (three …
Figure 7—figure supplement 1
Supplemental Biochemical Analysis of POMK/LARGE1 KO and POMK/DAG1 KO HAP1 Cells.
(A, B) Immunoblotting of WT, POMK KO and POMK/LARGE1 KO HAP1 cells with antibodies AF6868 (A) (Core α-DG and β-DG) or IIH6 (B). Glycoproteins were enriched using WGA-agarose as described in the …
Figure 7—figure supplement 2
Requirement for Ribitol-Phosphate in the Synthesis of the Non-Extended Form of Matriglycan.
(A, B, C) POMK KO HAP1 cells were transduced with an adenovirus encoding isoprenoid synthase domain-containing (Ad-ISPD). Immunoblotting was performed using antibodies AF6868 (A) or IIH6 (C). (B) A …
Figure 7—figure supplement 3
Fukutin Overexpression Enhances Synthesis of the Non-Extended Matriglycan.
(A, B, C) POMK KO HAP1 cells transduced with an adenovirus encoding Fukutin (FKTN), Ad-FKTN. Immunoblotting was performed using antibodies AF6868 (A) (Core α-DG and β-DG) or IIH6 (B) (three …
Figure 7—figure supplement 4
T317 is Required for Synthesis of the Non-Extended Matriglycan.
(A, B, C) Biochemical analysis of POMK/DAG1 KO HAP1 cells expressing the indicated adenoviruses (three replicates). DGE is for viral expression of α-DG that lacks the Dystroglycan N-terminal domain …
Figure 7—figure supplement 5
POMK Enables LARGE1-mediated Elongation of Matriglycan.
(A, B, C) Immunoblots of the following HAP1 cells: (A) LARGE1 KO, overexpressing Ad-LARGE1; (B, C) POMK KO, overexpressing Ad-LARGE1; (D) POMK/LARGE1 KO, overexpressing Ad-LARGE1 with or without …
Figure 7—figure supplement 6
Supplemental Characterization of POMK-null Matriglycan Synthesis.
(A, B, C) POMK KO HAP1 cells were transduced with an adenovirus encoding DG WT (Ad-DG) and immunoblotting was performed with antibodies IIH6 (A) and AF6868 (C) (three replicates). A laminin overlay …
Figure 8 with 3 supplements
NMR Analyses of POMK and LARGE1 Binding to GGM-MU and GGMp-MU.
(A, B) 1D 1H NMR spectra of the anomeric region of GGM-MU (A) and GGMp-MU (B) were acquired for the glycan concentration of 10.0 µM in the presence of various concentrations of LARGE1 as indicated. …
Figure 8—figure supplement 1
NMR Spectra of POMK Binding to GGMp-MU and Structure of GGMp-MU.
(A) 1D 1H NMR spectra of the glycan sample (10.0 µM GGMp-MU) were acquired in the presence of various concentrations of zebrafish POMK as indicated. The 13C and 1H resonances of GGMp-MU have been …
Figure 8—figure supplement 2
Model of Full-Length and Non-extended Matriglycan Synthesis.
(A) Mature matriglycan is a long polysaccharide that is synthesized by LARGE1. (B) In the absence of the core M3 phosphate added by POMK, LARGE1 generates a shorter, non-extended form of matriglycan.
Figure 8—figure supplement 3
Biochemical and Histologic Analysis of MckCre; PomkLoxP/LoxP Quadriceps Muscle.
(A, B, C) Representative biochemical analysis of glycoproteins enriched from quadriceps skeletal muscles of PomkLoxP/LoxP, MckCre; PomkLoxP/LoxP, and Largemyd mice using WGA-agarose (three …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent Mus musculus | PomkLoxP/LoxP ICR | This paper | Campbell Lab | Materials and Methods ‘Generation of PomkLoxP/LoxP Mice’ |
| Genetic reagent Mus musculus | Pax7Cre C57BL/6J | The Jackson Laboratory, Bar Harbor ME. | JAX:010530, RRID:IMSRJAX:010530 | Pax7tm1(cre)Mrc |
| Genetic reagent Mus musculus | MckCre C57BL/6J | The Jackson Laboratory, Bar Harbor ME. | JAX:006475, RRID:IMSR_JAX:006475 | B6.FVB(129S4)-Tg(Ckmm-cre)5Khn/J |
| Genetic reagent Mus musculus | Largemyd C57BL/6J | The Jackson Laboratory, Bar Harbor ME. | JAX:000300, RRID:IMSR_JAX:000300 | MYD/Le-Os +/+ Largemyd/J myd |
| Antibody | anti-DG (Sheep polyclonal) | R and D Systems | Cat# AF6868, RRID:AB_10891298 | WB (1:500) |
| Antibody | anti-α-DG (IIH6C4) (Mouse monoclonal) | DSHB Campbell Lab | Cat# IIH6 C4, RRID:AB_2617216 | WB (1:10-1:100) |
| Antibody | anti-myc clone 4A6 (Mouse monoclonal) | Millipore | Cat# 05–724, RRID:AB_309938 | WB (1:2000) |
| Antibody | anti-α-DG (IIH6C4) (Mouse monoclonal) | Millipore Campbell Lab | Cat# 05–593, RRID:AB_309828 | IF (1:1000-1:2000) |
| Antibody | anti-Laminin (Rabbit polyclonal) | Sigma-Aldrich | Cat# L9393, RRID:AB_477163 | WB (1:1000), Solid-Phase Assay (1:5000) |
| Antibody | anti-β-DG (Rabbit polyclonal) | Campbell Lab PMID:1741056 DOI:10.1038/355696a0 | AP83 | IF (1:50) |
| Antibody | anti-β-DG mouse IgM (Mouse monoclonal) | Leica Biosystems | Cat# NCL-b-DG, RRID:AB_442043 | IF (1:50 to 1:200) |
| Antibody | anti-Na+,K+ ATPase (Mouse monoclonal) | BD Biosciences | Cat# 610993 RRID:AB_398306 | WB (1:1000) |
| Antibody | anti-sheep IgG (Donkey polyclonal) | Rockland | Cat# 613-731-168, RRID:AB_220181 | WB (1:2000) |
| Antibody | anti-mouse IgG (H + L) (Donkey polyclonal) | LI-COR Biosciences | Cat# 926–32212, RRID:AB_621847 | WB (1:15,000), IF (1:800) |
| Antibody | anti-rabbit IgG (H + L) (Donkey polyclonal) | LI-COR Biosciences | Cat# 926–32213, RRID:AB_621848 | WB (1:15,000), IF (1:800) |
| Antibody | anti-mouse IgM (Goat polyclonal) | LI-COR Biosciences | Cat# 926–32280, RRID:AB_2814919 | WB (1:2500) |
| Antibody | anti-mouse IgG1 (Goat polyclonal) | LI-COR Biosciences | Cat# 926–32350, RRID:AB_2782997 | WB (1:2000, 1:10,000) |
| Antibody | anti-rabbit IgG (H+L) (Goat polyclonal) | Thermo Fisher Scientific | Cat# A-11034, RRID:AB_2576217 | IF (1:1000 to 1:2000) |
| Antibody | anti-mouse IgM (Goat polyclonal) | Thermo Fisher Scientific | Cat# A-21042, RRID:AB_2535711 | IF (1:1000 to 1:2000) |
| Antibody | anti-human FLJ23356 (Mouse monoclonal) | Novus | Cat# H00084197-M03, RRID:AB_2188284 | WB (1:500) |
| Commercial assay or kit | Creatine Kinase (CK) Liqui-UV Test | Fisher Scientific/Stanbio | Cat# 22-022-630 | |
| Cell line (Homo-sapiens) | Parental cell line C631 | Horizon Discovery | Cat# C631 | Mycoplasma testing passed |
| Cell line (Homo-sapiens) | POMK/DAG1 KO | Horizon Discovery | HZGHC001338c004, RRID:CVCL_TF19 | Authenticated by Sanger sequencing. Mycoplasma testing passed. |
| Cell line (Homo-sapiens) | POMK/LARGE1 KO | Horizon Discovery | HZGHC007364c011 | Authenticated by Sanger sequencing. Mycoplasma testing passed. |
| Cell line (Homo-sapiens) | POMK KO | Horizon Discovery | HZGHC001338c004, RRID:CVCL_TF19 | Authenticated by Sanger sequencing. Mycoplasma testing passed. |
| Cell line (Homo-sapiens) | POMK/ISPD KO | Horizon Discovery | HZGHC001338c001, RRID:CVCL_TF18 | Authenticated by Sanger sequencing. Mycoplasma testing passed. |
| Cell line (Homo-sapiens) | FKTN KO | Horizon Discovery | HZGHC000721c010, RRID:CVCL_SN68 | Authenticated by Sanger sequencing. Mycoplasma testing passed. |
| Cell line (Homo-sapiens) | LARGE1 KO | Horizon Discovery | HZGHC000122c007, RRID:CVCL_SV31 | Authenticated by Sanger sequencing. Mycoplasma testing passed. |
| Cell line (Homo-sapiens) | Primary dermal fibroblasts, human | ATCC | PCS-201–012 | |
| Cell line (Homo-sapiens) | Human fibroblasts (POMK D204N) | This paper | NH13-284 | Dubowitz Neuromuscular Center, Campbell Lab |
| Peptide, recombinant protein | β-Glucuronidase | PMID:16303119 DOI:10.1016/j.carres.2005.10.005 | ||
| Peptide, recombinant protein | α-Xylosidase | PMID:10801892 DOI:10.1074/jbc.M910392199 | ||
| Biological sample (Homo-sapiens) | Control human skeletal muscle | This paper | Dubowitz Neuromuscular Center, Campbell Lab | |
| Biological sample (Homo-sapiens) | Human skeletal muscle | This paper | (NH13-284, POMK D204N) | Dubowitz Neuromuscular Center, Campbell Lab |
| Chemical compound, drug | Purified Danio rerio POMK | PMID:27879205 DOI:10.7554/eLife.22238 | ||
| Chemical compound, drug | Purified mammalian dTMLARGE1 | PMID:22223806 DOI:10.1126/science.1214115 | ||
| Chemical compound, drug | GGM-MU and GGMp-MU | PMID:23929950 DOI:10.1126/science.1239951 | ||
| Chemical compound, drug | UDP-Xylose | CarboSource | https://www.ccrc.uga.edu/~carbosource/CSS_substrates.html | |
| Chemical compound, drug | 4-Methylumbelliferyl-β-D-xylopyranoside | Sigma/Millipore | Cat# M7008 | |
| Chemical compound, drug | UDP-Glucuronic acid | Sigma/Millipore | Cat# U6751 | |
| Chemical compound, drug | Uridine 5′-diphospho-N-acetylgalactosamine disodium salt | Sigma/Millipore | Cat# U5252 | |
| Chemical compound, drug | Uridine 5′-diphospho-N-acetylglucosamine sodium salt | Sigma/Millipore | Cat# U4375 | |
| Chemical compound, drug | 4-methylumbelliferyl α-(GlcNAc-β(1-4)Man) GM-MU | Sussex Research | https://www.sussex-research.com/ | |
| Chemical compound, drug | Xylose-α1,3-GlcA-β-MU | Sussex Research | https://www.sussex-research.com/ | |
| Chemical compound, drug | Pepstatin A | Sigma/Millipore | Cat# 516481 | |
| Chemical compound, drug | Calpain Inhibitor I (25 mg) | Sigma/Millipore | Cat# A6185 | |
| Chemical compound, drug | Aprotinin from bovine lung | Sigma/Millipore | Cat# A1153 | |
| Chemical compound, drug | Leupeptin (25 mg) | Sigma/Millipore | Cat# 108975 | |
| Chemical compound, drug | PMSF | Sigma/Millipore | Cat# P7626-25G | |
| Chemical compound, drug | Immobilon-FL PVDF | Sigma/Millipore | Cat# IPFL00010 | |
| Chemical compound, drug | Calpeptin | Fisher Scientific | Cat# 03-340-05125M | |
| Chemical compound, drug | Bis-acrylamide solution-30% (37:1) | Fisher Scientific/Hoefer | Cat# HBGR337500X | |
| Chemical compound, drug | Benzamidine Hydrochloride Hydrate | MP Biochemicals | Cat# 195068 | |
| Chemical compound, drug | WGA agarose bound | Vector Labs | Cat# AL-1023, RRID:AB_2336862 | |
| Chemical compound, drug | Precision Plus Protein All Blue Standards-500ul | Bio-Rad | Cat# 161–0373 | |
| Software, algorithm | SigmaPlot | SigmaPlot | RRID:SCR_003210 | |
| Software, algorithm | Excel | Microsoft | RRID:SCR_016137 | |
| Software, algorithm | GraphPad Prism | https://www.graphpad.com/scientific-software/prism/ | RRID:SCR_002798 | Version 8.3 |
| Software, algorithm | FlowJo | https://www.flowjo.com/solutions/flowjo/downloads | RRID:SCR_008520 | Version 7.6.5 |
| Software, algorithm | Li-Cor Image Studio Software | https://www.licor.com/bio/image-studio-lite/download | RRID:SCR_015795 | |
| Other | Streptavidin, Alexa Fluor 594 conjugate | Thermo Fisher Scientific | Cat# S11227 | IF (1:1000 to 1:2000) |
| Other | Adenovirus: DGC (DG, delta H30-A316) | PMID:21987822 DOI:10.1073/pnas.1114836108 | ||
| Other | Adenovirus: DG T317A | PMID:21987822 DOI:10.1073/pnas.1114836108 | ||
| Other | Adenovirus: DG T319A | PMID:21987822 DOI:10.1073/pnas.1114836108 | ||
| Other | Adenovirus: DG T317A/319A | PMID:21987822 DOI:10.1073/pnas.1114836108 | ||
| Other | Adenovirus: DG Wild-Type (WT) | PMID:21987822 DOI:10.1073/pnas.1114836108 | ||
| Other | Adenovirus: POMK WT | PMID:27879205 DOI:10.7554/eLife.22238 | ||
| Other | Adenovirus: POMK D204A | PMID:27879205 DOI:10.7554/eLife.22238 | ||
| Other | Adenovirus: POMK D204N | This paper | Campbell Lab | Materials and methods ‘Adenovirus Production’ |
| Other | Adenovirus: DG390TEVHis | This paper | Campbell Lab | Materials and methods ‘Adenovirus Production’ |
| Other | Adenovirus: Fukutin | PMID:22522420 DOI:10.1038/ng.2252 | ||
| Other | Adenovirus: Isoprenoid Synthase Domain-Containing (ISPD) | PMID:22522420 DOI:10.1038/ng.2252 | ||
| Other | Adenovirus: LARGE1 | PMID:22522420 DOI:10.1038/ng.2252 | ||
| Other | NMR spectrometer | Bruker | Avance II 800 MHz | |
| Other | Rodent Treadmill | Columbus Instruments | Exer 3/6 Treadmill | |
| Other | Western Blot Imager | Li-Cor | Odyssey CLx | |
| Other | Mouse treadmill | Omnitech Electronics | Accupacer Treadmill | |
| Other | Isolated Mouse Muscle System | Aurora Scientific | 1200A | |
| Other | Mouse Grip Strength Meter | Columbus Instruments | 1027 Mouse | |
| Other | Prominence HPLC | Shimadzu | LC-20 system | |
| Other | Tabletop ultracentrifuge | Beckman Coulter | Optima max, 130K | |
| Other | Ultracentrifuge | Beckman Coulter | Optima-L-100 XP | |
| Other | Centrifuge | Beckman Coulter | Avanti J-E HPC | |
| Other | HPLC LC18 column | Supelco | 58368 |
Additional files
-
Source data 1
Raw MALDI-TOF data of DG390 expressed in FKTN KO HAP1 cells (Figure 6—figure supplement 2B).
Data were exported to the TXT format by FlexAnalysis 3.3 (Bruker Daltonics). The mass spectra in the article were zoomed into the range of m/z 750–950 to better present MS signals corresponding to core M3 glycan structures.
- https://cdn.elifesciences.org/articles/61388/elife-61388-data1-v3.txt.zip
-
Source data 2
Raw MALDI-TOF data of DG390 expressed in POMK KO HAP1 cells (Figure 6—figure supplement 2A).
Data were exported to the TXT format by FlexAnalysis 3.3 (Bruker Daltonics). The mass spectra in the article were zoomed into the range of m/z 750–950 to better present MS signals corresponding to core M3 glycan structures.
- https://cdn.elifesciences.org/articles/61388/elife-61388-data2-v3.txt.zip
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/61388/elife-61388-transrepform-v3.docx
