POMT1 is essential for protein O-mannosylation in mammals
ABSTRACT Over the past decade it has emerged that O-mannosyl glycans are not restricted to yeast and fungi but are also present in higher eukaryotes up to humans. In mammals, the protein O-mannosyltransferases POMT1 and POMT2 act as a heteromeric complex to initiate O-mannosylation in the endoplasmic reticulum. In humans, mutations in POMT1 and POMT2 result in hypoglycosylation of alpha-dystroglycan (alpha-DG) thereby abolishing its binding to extracellular matrix ligands such as laminin. As a consequence, POMT mutations cause a heterogeneous group of severe recessive congenital muscular dystrophies in humans. However, little is known about the function of O-mannosyl glycans in mammals apart from its crucial role for the ligand binding abilities of alpha-DG. In this chapter we discuss the methods used to analyze the expression of Pomt1 in adult mouse organs and during embryo development. Further, we describe the generation and immunohistochemical analysis of Pomt1 knockout mice.
- SourceAvailable from: Andrea Brancaccio
[Show abstract] [Hide abstract]
- "Last but not least, from an exquisitely experimental point of view, our novel recombinant peptide may represent an interesting target to be employed to test in vitro the enzymatic action of POMT1 and POMT2, the first known players along the α-DG glycosylation cascade [22,23]. "
ABSTRACT: Background 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. Results 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. Conclusions 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.BMC Biochemistry 07/2013; 14(1). DOI:10.1186/1471-2091-14-15 · 1.44 Impact Factor
[Show abstract] [Hide abstract]
- "Deficiencies in genes controlling the initiation of other types of protein glycosylation generally cause severe phenotypes . Thus, deficiencies in the oligosaccharyltransferase complex are lethal in eukaryotes (Heesen et al. 1993; Kelleher and Gilmore 2006); deficiency in the O-mannosyltransferases ( protein-O-mannosyltransferase T1 and T2 function in a heteromeric complex) leads to severe muscular dystrophies (Reeuwijk et al. 2006) and targeted disruption leads to embryonic lethality in mice (Lommel et al. 2010); deficiency in either of the two fucosyltransferases initiating O-fucosylation [ protein O-fucosyltransferases 1 and 2 function with different substrates] is incompatible with life (Shi and Stanley 2003; Du et al., 2010); deficiency in the enzyme initiating O-glucosylation (POGLUT) is lethal (Stanley 2008; Acar et al. 2008; Fernandez-Valdivia et al. 2011); deficiency in protein O-xylosyltransferase 2 (XYLT2; one of the two xylosyltransferases XYLT1 and XYLT2) initiating proteoglycan chains leads to polycystic liver and kidney disease (Condac et al. 2007); and inactivation of lysyl hydroxylase 3 (one of three isoenzymes that precedes core Hyl galactosylation) causes early lethality in mice (Rautavuoma et al. 2004). In striking contrast, as will be discussed in the " GalNAcTs and disease " section, deficiencies in some of the vertebrate GalNAc-Ts produce only subtle phenotypes, suggesting a considerable degree of redundancy as well as unique functions of individual isoforms. "
ABSTRACT: Glycosylation of proteins is an essential process in all eukaryotes and a great diversity in types of protein glycosylation exists in animals, plants and microorganisms. Mucin-type O-glycosylation, consisting of glycans attached via O-linked N-acetylgalactosamine (GalNAc) to serine and threonine residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 22.214.171.124). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The GalNAc-T family is the largest glycosyltransferase enzyme family covering a single known glycosidic linkage and it is highly conserved throughout animal evolution, although absent in bacteria, yeast and plants. Emerging studies have shown that the large number of genes (GALNTs) in the GalNAc-T family do not provide full functional redundancy and single GalNAc-T genes have been shown to be important in both animals and human. Here, we present an overview of the GalNAc-T gene family in animals and propose a classification of the genes into subfamilies, which appear to be conserved in evolution structurally as well as functionally.Glycobiology 12/2011; 22(6):736-56. DOI:10.1093/glycob/cwr182 · 3.15 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Protein O-mannosylation is an essential modification in fungi and mammals. It is initiated at the endoplasmic reticulum by a conserved family of dolichyl phosphate mannose-dependent protein O-mannosyltransferases (PMTs). PMTs are integral membrane proteins with two hydrophilic loops (loops 1 and 5) facing the endoplasmic reticulum lumen. Formation of dimeric PMT complexes is crucial for mannosyltransferase activity, but the direct cause is not known to date. In bakers' yeast, O-mannosylation is catalyzed largely by heterodimeric Pmt1p-Pmt2p and homodimeric Pmt4p complexes. To further characterize Pmt1p-Pmt2p complexes, we developed a photoaffinity probe based on the artificial mannosyl acceptor substrate Tyr-Ala-Thr-Ala-Val. The photoreactive probe was preferentially cross-linked to Pmt1p, and deletion of the loop 1 (but not loop 5) region abolished this interaction. Analysis of Pmt1p loop 1 mutants revealed that especially Glu-78 is crucial for binding of the photoreactive probe. Glu-78 belongs to an Asp-Glu motif that is highly conserved among PMTs. We further demonstrate that single amino acid substitutions in this motif completely abolish activity of Pmt4p complexes. In contrast, both acidic residues need to be exchanged to eliminate activity of Pmt1p-Pmt2p complexes. On the basis of our data, we propose that the loop 1 regions of dimeric complexes form part of the catalytic site.Journal of Biological Chemistry 09/2011; 286(46):39768-75. DOI:10.1074/jbc.M111.281196 · 4.57 Impact Factor