Structural basis of laminin binding to the LARGE glycans on dystroglycan

Abstract

Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-β1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin-G-like (LG) domains 4 and 5 (LG4 and LG5) of laminin-α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-β1,3-xylose disaccharide repeat straddles a Ca2+ ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca2+-bound water molecules. The chelating binding mode accounts for the high affinity of this protein–carbohydrate interaction. These results reveal a previously uncharacterized mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Enzymatic digestion of oligosaccharide G5.
Figure 2: Enzymatic digestion of skeletal-muscle α-DG.
Figure 3: Crystal structure of laminin-α2 LG4–5 bound to oligosaccharide G6/7.
Figure 4: NMR analysis of binding of oligosaccharide G5 to laminin-α2 LG4–5.
Figure 5: Structural comparison of α-DG-binding LG domains.

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. 1

    Ervasti, J.M. & Campbell, K.P. A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J. Cell Biol. 122, 809–823 (1993).

    CAS  PubMed  Google Scholar 

  2. 2

    Ibraghimov-Beskrovnaya, O. et al. Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature 355, 696–702 (1992).

    CAS  PubMed  Google Scholar 

  3. 3

    Michele, D.E. et al. Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies. Nature 418, 417–422 (2002).

    CAS  PubMed  Google Scholar 

  4. 4

    Cohn, R.D. et al. Disruption of DAG1 in differentiated skeletal muscle reveals a role for dystroglycan in muscle regeneration. Cell 110, 639–648 (2002).

    CAS  PubMed  Google Scholar 

  5. 5

    Moore, S.A. et al. Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy. Nature 418, 422–425 (2002).

    CAS  PubMed  Google Scholar 

  6. 6

    Satz, J.S. et al. Distinct functions of glial and neuronal dystroglycan in the developing and adult mouse brain. J. Neurosci. 30, 14560–14572 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Barresi, R. & Campbell, K.P. Dystroglycan: from biosynthesis to pathogenesis of human disease. J. Cell Sci. 119, 199–207 (2006).

    CAS  PubMed  Google Scholar 

  8. 8

    Yurchenco, P.D. Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harb. Perspect. Biol. 3, a004911 (2011).

    PubMed  PubMed Central  Google Scholar 

  9. 9

    Godfrey, C., Foley, A.R., Clement, E. & Muntoni, F. Dystroglycanopathies: coming into focus. Curr. Opin. Genet. Dev. 21, 278–285 (2011).

    CAS  PubMed  Google Scholar 

  10. 10

    Sugita, S. et al. A stoichiometric complex of neurexins and dystroglycan in brain. J. Cell Biol. 154, 435–445 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11

    Sato, S. et al. Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation. Nat. Neurosci. 11, 923–931 (2008).

    CAS  PubMed  Google Scholar 

  12. 12

    Wright, K.M. et al. Dystroglycan organizes axon guidance cue localization and axonal pathfinding. Neuron 76, 931–944 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Cao, W. et al. Identification of α-dystroglycan as a receptor for lymphocytic choriomeningitis virus and Lassa fever virus. Science 282, 2079–2081 (1998).

    CAS  PubMed  Google Scholar 

  14. 14

    Jae, L.T. et al. Deciphering the glycosylome of dystroglycanopathies using haploid screens for lassa virus entry. Science 340, 479–483 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Yoshida-Moriguchi, T. & Campbell, K.P. Matriglycan: a novel polysaccharide that links dystroglycan to the basement membrane. Glycobiology 25, 702–713 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Yoshida-Moriguchi, T. et al. SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function. Science 341, 896–899 (2013).

    CAS  PubMed  Google Scholar 

  17. 17

    Yoshida-Moriguchi, T. et al. O-mannosyl phosphorylation of α-dystroglycan is required for laminin binding. Science 327, 88–92 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 18

    Kanagawa, M. et al. Identification of a post-translational modification with ribitol-phosphate and its defect in muscular dystrophy. Cell Rep. 14, 2209–2223 (2016).

    CAS  PubMed  Google Scholar 

  19. 19

    Inamori, K. et al. Dystroglycan function requires xylosyl- and glucuronyltransferase activities of LARGE. Science 335, 93–96 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 20

    Goddeeris, M.M. et al. LARGE glycans on dystroglycan function as a tunable matrix scaffold to prevent dystrophy. Nature 503, 136–140 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Grewal, P.K., Holzfeind, P.J., Bittner, R.E. & Hewitt, J.E. Mutant glycosyltransferase and altered glycosylation of alpha-dystroglycan in the myodystrophy mouse. Nat. Genet. 28, 151–154 (2001).

    CAS  PubMed  Google Scholar 

  22. 22

    Longman, C. et al. Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of α-dystroglycan. Hum. Mol. Genet. 12, 2853–2861 (2003).

    CAS  PubMed  Google Scholar 

  23. 23

    Rudenko, G., Hohenester, E. & Muller, Y.A. LG/LNS domains: multiple functions -- one business end? Trends Biochem. Sci. 26, 363–368 (2001).

    CAS  PubMed  Google Scholar 

  24. 24

    Hohenester, E., Tisi, D., Talts, J.F. & Timpl, R. The crystal structure of a laminin G-like module reveals the molecular basis of α-dystroglycan binding to laminins, perlecan, and agrin. Mol. Cell 4, 783–792 (1999).

    CAS  PubMed  Google Scholar 

  25. 25

    Wizemann, H. et al. Distinct requirements for heparin and α-dystroglycan binding revealed by structure-based mutagenesis of the laminin α2 LG4-LG5 domain pair. J. Mol. Biol. 332, 635–642 (2003).

    CAS  PubMed  Google Scholar 

  26. 26

    Salleh, H.M. et al. Cloning and characterization of Thermotoga maritima β-glucuronidase. Carbohydr. Res. 341, 49–59 (2006).

    CAS  PubMed  Google Scholar 

  27. 27

    Moracci, M. et al. Identification and molecular characterization of the first α -xylosidase from an archaeon. J. Biol. Chem. 275, 22082–22089 (2000).

    CAS  PubMed  Google Scholar 

  28. 28

    Smirnov, S.P. et al. Contributions of the LG modules and furin processing to laminin-2 functions. J. Biol. Chem. 277, 18928–18937 (2002).

    CAS  PubMed  Google Scholar 

  29. 29

    Talts, J.F., Andac, Z., Göhring, W., Brancaccio, A. & Timpl, R. Binding of the G domains of laminin α1 and α2 chains and perlecan to heparin, sulfatides, α-dystroglycan and several extracellular matrix proteins. EMBO J. 18, 863–870 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    DeLucas, L. & Bugg, C.E. Calcium binding to D-glucuronate residues: crystal structure of a hydrated calcium bromide salt of D-glucuronic acid. Carbohydr. Res. 41, 18–29 (1975).

    CAS  PubMed  Google Scholar 

  31. 31

    Feinberg, H., Castelli, R., Drickamer, K., Seeberger, P.H. & Weis, W.I. Multiple modes of binding enhance the affinity of DC-SIGN for high mannose N-linked glycans found on viral glycoproteins. J. Biol. Chem. 282, 4202–4209 (2007).

    CAS  Google Scholar 

  32. 32

    Nagae, M. & Yamaguchi, Y. Three-dimensional structural aspects of protein-polysaccharide interactions. Int. J. Mol. Sci. 15, 3768–3783 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33

    Timpl, R. et al. Structure and function of laminin LG modules. Matrix Biol. 19, 309–317 (2000).

    CAS  PubMed  Google Scholar 

  34. 34

    Reissner, C. et al. Dystroglycan binding to α-neurexin competes with neurexophilin-1 and neuroligin in the brain. J. Biol. Chem. 289, 27585–27603 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Harrison, D. et al. Crystal structure and cell surface anchorage sites of laminin α1LG4-5. J. Biol. Chem. 282, 11573–11581 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Weis, W.I. & Drickamer, K. Structural basis of lectin-carbohydrate recognition. Annu. Rev. Biochem. 65, 441–473 (1996).

    CAS  PubMed  Google Scholar 

  37. 37

    Somers, W.S., Tang, J., Shaw, G.D. & Camphausen, R.T. Insights into the molecular basis of leukocyte tethering and rolling revealed by structures of P- and E-selectin bound to SLe(X) and PSGL-1. Cell 103, 467–479 (2000).

    CAS  PubMed  Google Scholar 

  38. 38

    Gesemann, M. et al. Alternative splicing of agrin alters its binding to heparin, dystroglycan, and the putative agrin receptor. Neuron 16, 755–767 (1996).

    CAS  PubMed  Google Scholar 

  39. 39

    Hara, Y. et al. Like-acetylglucosaminyltransferase (LARGE)-dependent modification of dystroglycan at Thr-317/319 is required for laminin binding and arenavirus infection. Proc. Natl. Acad. Sci. USA 108, 17426–17431 (2011).

    CAS  PubMed  Google Scholar 

  40. 40

    Vester-Christensen, M.B. et al. Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins. Proc. Natl. Acad. Sci. USA 110, 21018–21023 (2013).

    CAS  PubMed  Google Scholar 

  41. 41

    Kanagawa, M. et al. Molecular recognition by LARGE is essential for expression of functional dystroglycan. Cell 117, 953–964 (2004).

    CAS  PubMed  Google Scholar 

  42. 42

    Stetefeld, J. et al. Modulation of agrin function by alternative splicing and Ca2+ binding. Structure 12, 503–515 (2004).

    CAS  PubMed  Google Scholar 

  43. 43

    Le, B.V. et al. Crystal structure of the LG3 domain of endorepellin, an angiogenesis inhibitor. J. Mol. Biol. 414, 231–242 (2011).

    CAS  PubMed  Google Scholar 

  44. 44

    Sheckler, L.R., Henry, L., Sugita, S., Südhof, T.C. & Rudenko, G. Crystal structure of the second LNS/LG domain from neurexin 1α: Ca2+ binding and the effects of alternative splicing. J. Biol. Chem. 281, 22896–22905 (2006).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Chen, F., Venugopal, V., Murray, B. & Rudenko, G. The structure of neurexin 1α reveals features promoting a role as synaptic organizer. Structure 19, 779–789 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46

    Combs, A.C. & Ervasti, J.M. Enhanced laminin binding by α-dystroglycan after enzymatic deglycosylation. Biochem. J. 390, 303–309 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Beedle, A.M., Nienaber, P.M. & Campbell, K.P. Fukutin-related protein associates with the sarcolemmal dystrophin-glycoprotein complex. J. Biol. Chem. 282, 16713–16717 (2007).

    CAS  PubMed  Google Scholar 

  48. 48

    Kohfeldt, E., Maurer, P., Vannahme, C. & Timpl, R. Properties of the extracellular calcium binding module of the proteoglycan testican. FEBS Lett. 414, 557–561 (1997).

    CAS  PubMed  Google Scholar 

  49. 49

    Paracuellos, P., Briggs, D.C., Carafoli, F., Lončar, T. & Hohenester, E. Insights into collagen uptake by C-type mannose receptors from the crystal structure of Endo180 domains 1-4. Structure 23, 2133–2142 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Winter, G., Lobley, C.M. & Prince, S.M. Decision making in xia2. Acta Crystallogr. D Biol. Crystallogr. 69, 1260–1273 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51

    Karplus, P.A. & Diederichs, K. Linking crystallographic model and data quality. Science 336, 1030–1033 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52

    McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53

    Tisi, D., Talts, J.F., Timpl, R. & Hohenester, E. Structure of the C-terminal laminin G-like domain pair of the laminin α2 chain harbouring binding sites for α-dystroglycan and heparin. EMBO J. 19, 1432–1440 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  54. 54

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 60, 2126–2132 (2004).

    PubMed  PubMed Central  Google Scholar 

  55. 55

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57

    Mayer, M. & Meyer, B. Group epitope mapping by saturation transfer difference NMR to identify segments of a ligand in direct contact with a protein receptor. J. Am. Chem. Soc. 123, 6108–6117 (2001).

    CAS  PubMed  Google Scholar 

  58. 58

    Nyberg, N.T., Duus, J.O. & Sørensen, O.W. Editing of H2BC NMR spectra. Magn. Reson. Chem. 43, 971–974 (2005).

    CAS  PubMed  Google Scholar 

  59. 59

    Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  Google Scholar 

  60. 60

    Johnson, B.A. & Blevins, R.A. NMR View: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank S.G. Withers (University of British Columbia) for a gift of T. maritima β-glucuronidase and C.M. Blaumueller for critical reading of the manuscript. The IIH6 antibody was obtained from the Developmental Studies Hybridoma Bank, University of Iowa. We acknowledge Diamond Light Source for time on beamlines I02 and I04-1 under proposal MX9424. This work was funded by a Wellcome Trust Senior Investigator Award to E.H. (101748/Z/13/Z) and a Paul D. Wellstone Muscular Dystrophy Cooperative Research Center grant to K.P.C. (1U54NS053672).

Author information

Affiliations

Authors

Contributions

D.C.B. co-designed the project, carried out the crystallographic experiments, analyzed the data and co-wrote the manuscript. T.Y.-M. produced xylosidase and glucuronidase, and performed enzyme digestions and binding assays. T.Z. generated the reagent X2, produced xylosidase and glucuronidase, and performed the G5 digestion experiment. D.V. generated reagents G3, G5 and G6/7. M.A. performed enzyme digestions and western blotting. A.S. and M.M. provided well-characterized xylosidase. L.Y. carried out the NMR experiments and analyzed the data. E.H. and K.P.C. co-designed the project, co-wrote the manuscript and supervised the research. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Erhard Hohenester or Kevin P Campbell.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1 and 2 and Supplementary Figures 1–7. (PDF 5119 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Briggs, D., Yoshida-Moriguchi, T., Zheng, T. et al. Structural basis of laminin binding to the LARGE glycans on dystroglycan. Nat Chem Biol 12, 810–814 (2016). https://doi.org/10.1038/nchembio.2146

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing