Probing the stability of the “naked” mucin-like domain of human α-dystroglycan
Tóm tắt
α-Dystroglycan (α-DG) is heavily glycosylated within its central mucin-like domain. The glycosylation shell of α-dystroglycan is known to largely influence its functional properties toward extracellular ligands. The structural features of this α-dystroglycan domain have been poorly studied so far. For the first time, we have attempted a recombinant expression approach in E. coli cells, in order to analyze by biochemical and biophysical techniques this important domain of the α-dystroglycan core protein. We expressed the recombinant mucin-like domain of human α-dystroglycan in E. coli cells, and purified it as a soluble peptide of 174 aa. A cleavage event, that progressively emerges under repeated cycles of freeze/thaw, occurs at the carboxy side of Arg461, liberating a 151 aa fragment as revealed by mass spectrometry analysis. The mucin-like peptide lacks any particular fold, as confirmed by its hydrodynamic properties and its fluorescence behavior under guanidine hydrochloride denaturation. Dynamic light scattering has been used to demonstrate that this mucin-like peptide is arranged in a conformation that is prone to aggregation at room temperature, with a melting temperature of ~40°C, which indicates a pronounced instability. Such a conclusion has been corroborated by trypsin limited proteolysis, upon which the protein has been fully degraded in less than 60 min. Our analysis indirectly confirms the idea that the mucin-like domain of α-dystroglycan needs to be extensively glycosylated in order to reach a stable conformation. The absence/reduction of glycosylation by itself may greatly reduce the stability of the dystroglycan complex. Although an altered pattern of α-dystroglycan O-mannosylation, that is not significantly changing its overall glycosylation fraction, represents the primary molecular clue behind currently known dystroglycanopathies, it cannot be ruled out that still unidentified forms of αDG-related dystrophy might originate by a more substantial reduction of α-dystroglycan glycosylation and by its consequent destabilization.
Tài liệu tham khảo
Bozzi M, Morlacchi S, Bigotti MG, Sciandra F, Brancaccio A: Functional diversity of dystroglycan. Matrix Biol. 2009, 28: 179-187. 10.1016/j.matbio.2009.03.003.
Brancaccio A, Schulthess T, Gesemann M, Engel J: Electron microscopic evidence for a mucin-like region in chick muscle α-dystroglycan. FEBS Lett. 1995, 368: 139-142. 10.1016/0014-5793(95)00628-M.
Ibraghimov-Beskrovnaya O, Ervasti JM, Leveille CJ, Slaughter CA, Sernett SW, Campbell KP: Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature. 1992, 355: 696-702. 10.1038/355696a0.
Brancaccio A: DAG1, no gene for RNA regulation?. Gene. 2012, 497: 79-82. 10.1016/j.gene.2012.01.046.
Godfrey C, Foley AR, Clement E, Muntoni F: Dystroglycanopathies: coming into focus. Curr Opin Genet Dev. 2011, 21: 278-285. 10.1016/j.gde.2011.02.001.
Zhang P, Hu H: Differential glycosylation of α-dystroglycan and proteins other than α-dystroglycan by like-glycosyltransferase. Glycobiology. 2012, 22: 235-247. 10.1093/glycob/cwr131.
Yoshida-Moriguchi T, Yu L, Stalnaker SH, Davis S, Kunz S, Madson M, Oldstone MB, Schachter H, Wells L, Campbell KP: O-mannosyl phosphorylation of α-dystroglycan is required for laminin binding. Science. 2010, 327: 88-92. 10.1126/science.1180512.
Nilsson J, Nilsson J, Larson G, Grahn A: Characterization of site-specific O-glycan structures within the mucin-like domain of α-dystroglycan from human skeletal muscle. Glycobiology. 2010, 20: 1160-1169. 10.1093/glycob/cwq082.
Hara Y, Kanagawa M, Kunz S, Yoshida-Moriguchi T, Satz JS, Kobayashi YM, Zhu Z, Burden SJ, Oldstone MB, Campbell KP: Like-acetylglucosaminyltransferase (LARGE)-dependent modification of dystroglycan at Thr-317/319 is required for laminin binding and arenavirus infection. Proc Natl Acad Sci USA. 2011, 108: 17426-17431. 10.1073/pnas.1114836108.
Harrison R, Hitchen PG, Panico M, Morris HR, Mekhaiel D, Pleass RJ, Dell A, Hewitt JE, Haslam SM: Glycoproteomic characterization of recombinant mouse α-dystroglycan. Glycobiology. 2012, 22: 662-675. 10.1093/glycob/cws002.
Tran DT, Lim JM, Liu M, Stalnaker SH, Wells L, Ten Hagen KG, Live D: Glycosylation of α-dystroglycan: O-mannosylation influences the subsequent addition of GalNAc by UDP-GalNAc polypeptide N-acetylgalactosaminyltransferases. J Biol Chem. 2012, 287: 20967-20974. 10.1074/jbc.M112.370387.
Gomez Toledo A, Raducu M, Cruces J, Nilsson J, Halim A, Larson G, Rüetschi U, Grahn A: O-Mannose and O-N-acetyl galactosamine glycosylation of mammalian α-dystroglycan is conserved in a region-specific manner. Glycobiology. 2012, 22: 1413-1423. 10.1093/glycob/cws109.
Bozzi M, Sciandra F, Ferri L, Torreri P, Pavoni E, Petrucci TC, Giardina B, Brancaccio A: Concerted mutation of Phe residues belonging to the β-dystroglycan ectodomain strongly inhibits the interaction with α-dystroglycan in vitro. FEBS J. 2006, 273: 4929-4943. 10.1111/j.1742-4658.2006.05492.x.
Uversky VN: Natively unfolded proteins: a point where biology waits for physics. Protein Sci. 2002, 11: 739-756. 10.1110/ps.4210102.
De Rosa MC, Pirolli D, Bozzi M, Sciandra F, Giardina B, Brancaccio A: A second Ig-like domain identified in dystroglycan by molecular modelling and dynamics. J Mol Graph Model. 2011, 29: 1015-1024. 10.1016/j.jmgm.2011.04.008.
Shogren R, Gerken TA, Jentoft N: Role of glycosylation on the conformation and chain dimensions of O-linked glycoproteins: light-scattering studies of ovine submaxillary mucin. Biochemistry. 1989, 28: 5525-5536. 10.1021/bi00439a029.
Di Stasio E, Sciandra F, Maras B, Di Tommaso F, Petrucci TC, Giardina B, Brancaccio A: Structural and functional analysis of the N-terminal extracellular region of β-dystroglycan. Biochem Biophys Res Commun. 1999, 266: 274-278. 10.1006/bbrc.1999.1803.
Michele DE, Barresi R, Kanagawa M, Saito F, Cohn RD, Satz JS, Dollar J, Nishino I, Kelley RI, Somer H, Straub V, Mathews KD, Moore SA, Campbell KP: Post-translational disruption of dystroglycan-ligand interactions in congenital muscular dystrophies. Nature. 2002, 418: 417-422. 10.1038/nature00837.
Satoyoshi E: Therapeutic trials on progressive muscular dystrophy. Intern Med. 1992, 31: 841-846. 10.2169/internalmedicine.31.841.
Kumar A, Bhatnagar S, Kumar A: Matrix metalloproteinase inhibitor batimastat alleviates pathology and improves skeletal muscle function in dystrophin-deficient mdx mice. Am J Pathol. 2010, 177: 248-260. 10.2353/ajpath.2010.091176.
Kanagawa M, Saito F, Kunz S, Yoshida-Moriguchi T, Barresi R, Kobayashi YM, Muschler J, Dumanski JP, Michele DE, Oldstone MB, Campbell KP: Molecular recognition by LARGE is essential for expression of functional dystroglycan. Cell. 2004, 117: 953-964. 10.1016/j.cell.2004.06.003.
Lommel M, Willer T, Cruces J, Strahl S: POMT1 is essential for protein O-mannosylation in mammals. Methods Enzymol. 2010, 479: 323-342.
Nakamura N, Stalnaker SH, Lyalin D, Lavrova O, Wells L, Panin VM: Drosophila Dystroglycan is a target of O-mannosyltransferase activity of two protein O-mannosyltransferases, Rotated Abdomen and Twisted. Glycobiology. 2010, 20: 381-394. 10.1093/glycob/cwp189.
Weir ML, Oppizzi ML, Henry MD, Onishi A, Campbell KP, Bissell MJ, Muschler JL: Dystroglycan loss disrupts polarity and β-casein induction in mammary epithelial cells by perturbing laminin anchoring. J Cell Sci. 2006, 119: 4047-4058. 10.1242/jcs.03103.
Griffon N, Di Stasio E: Thermodynamics of Na+ binding to coagulation serine proteases. Biophys Chem. 2001, 90: 89-96. 10.1016/S0301-4622(01)00129-6.
Di Stasio E, Bizzarri P, Misiti F, Pavoni E, Brancaccio A: A fast and accurate procedure to collect and analyze unfolding fluorescence signal: the case of dystroglycan domains. Biophys Chem. 2004, 107: 197-211. 10.1016/j.bpc.2003.09.005.