The glucuronyltransferase B4GAT1 is required for initiation of LARGE-mediated α-dystroglycan functional glycosylation
- University of Iowa, Carver College of Medicine, United States
- Howard Hughes Medical Institute, University of Iowa, Carver College of Medicine, United States
- Tohoku Pharmaceutical University, Japan
- Graduate School of Engineering, Kyoto University, Japan
- Oregon Health and Science University, United States
Figures
Figure 1 with 1 supplement
Postulated α-DG modifying enzymes are involved in post-phosphorylation processes in the Golgi prior to LARGE.
(A) Phosphorylation of Fc-tagged DGFc340 in the context of α-DG glycosylation defects. Fc-tagged DGFc340 was produced in [32P] orthophosphate-labeled fibroblasts from control and …
Figure 1—figure supplement 1
α-DG functional glycosylation and known proteins contributing to its synthesis.
α-DG core M3 functional glycosylation can be divided in 2 major processing steps. O-mannosyl pre-phosphoryl modification which is carried out by enzymes in the endoplasmic reticulum (ER) …
Figure 2
β-GlcA serves as an acceptor sugar for LARGE modification starting with xylose.
(A) Schematic diagram showing the α-DG post-phosphoryl modification in the context of control and glycosylation defects. LARGE adds the ligand-binding glycan to α-DG via a proposed glucuronic acid …
Figure 3 with 4 supplements
B4GAT1 has xylose β1,4 glucuronyltransferase activity.
(A) Schematic representation of LARGE and B4GAT1 functional domains. GlcA-T (blue), Xyl-T (orange) and transmembrane domain (black) are indicated. (B) Representative HPLC profiles of the reaction …
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Figure 3—source data 1
Chemical shifts (ppm) of the signals in the 1H and 13C NMR spectra of the enzymatic reaction product of GlcA-β1,4-Xyl-β-MU of the glycosyltransferase B4GAT1.
- https://doi.org/10.7554/eLife.03941.008
Figure 3—figure supplement 1
Purification of B4GAT1dTM.
(A) Schematic representation of B4GAT1 and the B4GAT1dTM construct used in the enzymatic activity assay. The transmembrane (TM) sequence was replaced with a 3xFLAG-TEV tag sequence and the …
Figure 3—figure supplement 2
Basic characterization of the xylose β1,4-glucuronyltransferase activity of B4GAT1.
(A) Donor sugar specificity of B4GAT1dTM. Representative data from two independent assays, demonstrating relative activity (%) of B4GAT1dTM (enzyme) GlcA-T toward Xyl-α-MU and Xyl-β-MU (acceptor) …
Figure 3—figure supplement 3
NMR analysis reveals that B4GAT1 is a β1,4 glucuronyltransferase.
(A) HMQC spectrum (top) and overlay of HMQC (black) and HMBC (green) spectra (bottom) for the B4GAT1 enzymatic reaction product. The cross-peaks are labeled with a first letter representing the …
Figure 3—figure supplement 4
Test B4GAT1 for GlcNAc transferase activity with iGnT substrate Gal-β1,4-GlcNAc-β-MU.
(A) Using B4GALT1 we synthesized the hypothesized iGnT substrate Gal-β1,4-GlcNAc-β-MU by transferring a β1,4 Galactose to the acceptor GlcNAc-β-MU. The purified Gal-β1,4-GlcNAc-β-MU disaccharide was …
Figure 4 with 1 supplement
B4gat1-deficient MEFs have impaired α-DG functional glycosylation and endogenous B4GAT1 activity.
(A) Functional glycosylation and complementation analysis of α-DG in wild-type, Large- and B4gat1-deficient MEFs. Immunoblots and laminin overlay assay of WGA-enriched cell lysates extracted from …
Figure 4—figure supplement 1
B4gat1-deficient MEFs have impaired endogenous B4GAT1 activity.
Representative HPLC profiles of the reaction product are shown. (A/B) Endogenous B4GAT1 enzyme activity of cell lysates from wild-type (A) and B4gat1-deficient (B) MEFs . B4gat1-deficient MEFs …
Figure 5
Expression analysis and GlcA-T enzyme activity of B4GAT1 mutant constructs.
(A) Schematic presentation shows B4GAT1 enzyme product with functional domains and B4GAT1 mutations Mut1-Mut3 are indicated. (B) Expression analysis of B4GAT1-Myc control and mutant constructs in …
Figure 6
β-xylose is the endogenous acceptor for B4GAT1.
(A) B4GAT1dTM enzymatic transfer of [14C] radiolabeled GlcA to DGFc340. Fc-tagged DGFc340 (acceptor) was produced in control, Largemyd (Large-deficient) and B4gat1-deficient MEFs and isolated from …
Figure 7
NMR analyses of the tetrasacharide generated from B4GAT1dTM and LARGEdTM enzymatic reactions.
(A) Schematic depiction of the tetrasaccharide structure produced by the sequential reactions of B4GAT1dTM followed by LARGEdTM with the sugar units labeled A-D to indicate the order of their …
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Figure 7—source data 1
Chemical shifts (ppm) of the signals in the 1H and 13C NMR spectra of the tetrasaccharide of GlcA-β1,3-Xyl-α1,3-GlcA-β1,4-Xyl-β-MU produced by B4GAT1 and LARGE.
- https://doi.org/10.7554/eLife.03941.018
Figure 8 with 1 supplement
Model of proposed α-DG O-mannosyl laminin-binding glycan structure and the enzymes that contribute to its synthesis.
Post-phosphoryl modification of α-DG requires B4GAT1 (β1,4 glucuronyltransferase); this enzyme generates the acceptor glycan, which serves as a primer for the glycosyltransferase LARGE to initiate …
Figure 8—figure supplement 1
B4GAT1 and LARGE expression in human tissues.
qPCR revealing ubiquitous B4GAT1 and LARGE expression in all tissues analyzed, with highest expression of LARGE in brain and heart. cDNA was synthesized using random primers and oligo(dT) on …
Author response image 1
[3H] Xylose metabolic labeling of DGFc340. DGFc340 and DGFc340mut fusion proteins were enriched by Protein A-agarose from culture medium of [3H] Xylose labeled control and B4gat-deficient MEF cells. …
Tables
Table 1
Summary of features of control and glycosylation-deficient cell lines
| Mutant gene | Clinical phenotype | Cell type | Nucleotide variant | Amino acid | reference |
|---|---|---|---|---|---|
| Control (human) | none | Human skin fibroblast | CRL-2127 (ATCC) | ||
| POMK | WWS/MEB | Human skin fibroblast | 14bp homozygous deletion (c.720_733delGCTGGTG AGTGCG)], homozygous | p.Leu241Profs*26 | (Yoshida-Moriguchi et al., 2013) |
| FKTN | WWS | Human skin fibroblast | c.385delA | p.I129fsX1 | GM16192 |
| c.1176C > A, heterozygous | p.Y392X | (Coriell Cell Repository) | |||
| FKRP | WWS | Human skin fibroblast | c.1A > G, homozygous | p.M1V | (Van Reeuwijk et al., 2010) |
| TMEM5 | WWS | Human skin fibroblast | c.1101 G > A, homozygous | p.G333R | unpublished |
| B4gat1 (B3gnt1) | CMD | MEF | c.464T > C, compound het with LacZ null allele, B4gat1LacZ/M155T | p.M155T | (Wright et al., 2012) |
| Largemyd | CMD | MEF | deletion of exons 5−7, homozygous | (Grewal et al., 2001) | |
| Control (mouse) | none | MEF |
