David Live

Glycomic Analyses of Mouse Models of Congenital Muscular Dystrophy

Dystroglycanopathies are a subset of congenital muscular dystrophies wherein α-dystroglycan (α-DG) is hypoglycosylated. α-DG is an extensively O-glycosylated extracellular matrix-binding protein and a key component of the dystrophin-glycoprotein complex. Previous studies have shown α-DG to be post-translationally modified by both O-GalNAc- and O-mannose-initiated glycan structures. Mutations in defined or putative glycosyltransferase genes involved in O-mannosylation are associated with a loss of ligand-binding activity of α-DG and are causal for various forms of congenital muscular dystrophy. In this study, we sought to perform glycomic analysis on brain O-linked glycan structures released from proteins of three different knock-out mouse models associated with O-mannosylation (POMGnT1, LARGE (Myd), and DAG1−/−). Using mass spectrometry approaches, we were able to identify nine O-mannose-initiated and 25 O-GalNAc-initiated glycan structures in wild-type littermate control mouse brains. Through our analysis, we were able to confirm that POMGnT1 is essential for the extension of all observed O-mannose glycan structures with β1,2-linked GlcNAc. Loss of LARGE expression in the Myd mouse had no observable effect on the O-mannose-initiated glycan structures characterized here. Interestingly, we also determined that similar amounts of O-mannose-initiated glycan structures are present on brain proteins from α-DG-lacking mice (DAG1) compared with wild-type mice, indicating that there must be additional proteins that are O-mannosylated in the mammalian brain. Our findings illustrate that classical β1,2-elongation and β1,6-GlcNAc branching of O-mannose glycan structures are dependent upon the POMGnT1 enzyme and that O-mannosylation is not limited solely to α-DG in the brain.

The Catalytic and Lectin Domains of UDP-GalNAc:Polypeptide α-N-Acetylgalactosaminyltransferase Function in Concert to Direct Glycosylation Site Selection

UDP-GalNAc:polypeptide α-N-Acetylgalactosaminyltransferases (ppGalNAcTs), a family (EC 2.4.1.41) of enzymes that initiate mucin-type O-glycosylation, are structurally composed of a catalytic domain and a lectin domain. Previous studies have suggested that the lectin domain modulates the glycosylation of glycopeptide substrates and may underlie the strict glycopeptide specificity of some isoforms (ppGalNAcT-7 and -10). Using a set of synthetic peptides and glycopeptides based upon the sequence of the mucin, MUC5AC, we have examined the activity and glycosylation site preference of lectin domain deletion and exchange constructs of the peptide/glycopeptide transferase ppGalNAcT-2 (hT2) and the glycopeptide transferase ppGalNAcT-10 (hT10). We demonstrate that the lectin domain of hT2 directs glycosylation site selection for glycopeptide substrates. Pre-steady-state kinetic measurements show that this effect is attributable to two mechanisms, either lectin domain-aided substrate binding or lectin domain-aided product release following glycosylation. We find that glycosylation of peptide substrates by hT10 requires binding of existing GalNAcs on the substrate to either its catalytic or lectin domain, thereby resulting in its apparent strict glycopeptide specificity. These results highlight the existence of two modes of site selection used by these ppGalNAcTs: local sequence recognition by the catalytic domain and the concerted recognition of distal sites of prior glycosylation together with local sequence binding mediated, respectively, by the lectin and catalytic domains. The latter mode may facilitate the glycosylation of serine or threonine residues, which occur in sequence contexts that would not be efficiently glycosylated by the catalytic domain alone. Local sequence recognition by the catalytic domain differs between hT2 and hT10 in that hT10 requires a pre-existing GalNAc residue while hT2 does not.

Ti^(3+) in meteoritic and synthetic hibonite

Electron spin resonance has been used to make the first direct determination of Ti^(3+) in synthetic hibonite and hibonite from inclusion SH-7 of the Murchison C2 chondrite. Ti^(3+) concentrations range from 0.02 to 0.64 wt% in synthetic blue hibonite and 0.35–0.44 wt% in hibonite from SH-7. No Ti^(3+) could be detected in orange hibonite, supporting the earlier conclusion that the orange-to-blue transition is associated with the presence of Ti^(3+). At constant temperature and oxygen fugacity, Ti^(3+)/Ti^(4+) in synthetic hibonite increases with decreasing V but is not strongly dependent on bulk Ti. At the concentration levels encountered in meteoritic hibonite, Fe and Cr contents do not have a significant effect on the amount of Ti^(3+). In both synthetic and meteoritic hibonite, Ti^(3+) occupies a 5-coordinated crystallographic site, which is consistent with the formation of doubly ionized oxygen vacancies. At low oxygen fugacities, essentially all Ti^(4+) on the five-fold Al-site has been reduced to Ti^(3+). Hibonite from SH-7 equilibrated with a gas that could have been as reducing as a gas of solar composition. This is consistent with other estimates based on mineral equilibria of high temperature oxygen fugacities in Ca-Al-rich inclusions. With the possible exception of Mo-W depletions, indicators based on bulk trace element concentrations in CAIs are inconclusive. There is considerable evidence that as CAIs cooled to lower temperatures, they experienced conditions significantly more oxidizing than those of a solar gas, perhaps in planetary environments.

Deciphering structural elements of mucin glycoprotein recognition.

Mucin glycoproteins present a complex structural landscape arising from the multiplicity of glycosylation patterns afforded by their numerous serine and threonine glycosylation sites, often in clusters, and with variations in respective glycans. To explore the structural complexities in such glycoconjugates, we used NMR to systematically analyze the conformational effects of glycosylation density within a cluster of sites. This allows correlation with molecular recognition through analysis of interactions between these and other glycopeptides, with antibodies, lectins, and sera, using a glycopeptide microarray. Selective antibody interactions with discrete conformational elements, reflecting aspects of the peptide and disposition of GalNAc residues, are observed. Our results help bridge the gap between conformational properties and molecular recognition of these molecules, with implications for their physiological roles. Features of the native mucin motifs impact their relative immunogenicity and are accurately encoded in the antibody binding site, with the conformational integrity being preserved in isolated glycopeptides, as reflected in the antibody binding profile to array components.

B4GAT1 is the priming enzyme for the LARGE-dependent functional glycosylation of α-dystroglycan

Recent studies demonstrated that mutations in B3GNT1, an enzyme proposed to be involved in poly-N-acetyllactosamine synthesis, were causal for congenital muscular dystrophy with hypoglycosylation of α-dystroglycan (secondary dystroglycanopathies). Since defects in the O-mannosylation protein glycosylation pathway are primarily responsible for dystroglycanopathies and with no established O-mannose initiated structures containing a β3 linked GlcNAc known, we biochemically interrogated this human enzyme. Here we report this enzyme is not a β-1,3-N-acetylglucosaminyltransferase with catalytic activity towards β-galactose but rather a β-1,4-glucuronyltransferase, designated B4GAT1, towards both α- and β-anomers of xylose. The dual-activity LARGE enzyme is capable of extending products of B4GAT1 and we provide experimental evidence that B4GAT1 is the priming enzyme for LARGE. Our results further define the functional O-mannosylated glycan structure and indicate that B4GAT1 is involved in the initiation of the LARGE-dependent repeating disaccharide that is necessary for extracellular matrix protein binding to O-mannosylated α-dystroglycan that is lacking in secondary dystroglycanopathies. DOI: http://dx.doi.org/10.7554/eLife.03943.001

Synthetic, Structural, and Biosynthetic Studies of an Unusual Phospho-Glycopeptide Derived from α-Dystroglycan

Aberrant glycosylation of α-dystroglycan (α-DG) results in loss of interactions with the extracellular matrix and is central to the pathogenesis of several disorders. To examine protein glycosylation of α-DG, a facile synthetic approach has been developed for the preparation of unusual phosphorylated O-mannosyl glycopeptides derived from α-DG by a strategy in which properly protected phospho-mannosides are coupled with a Fmoc protected threonine derivative, followed by the use of the resulting derivatives in automated solid-phase glycopeptide synthesis using hyper-acid-sensitive Sieber amide resin. Synthetic efforts also provided a reduced phospho-trisaccharide, and the NMR data of this derivative confirmed the proper structural assignment of the unusual phospho-glycan structure. The glycopeptides made it possible to explore factors that regulate the elaboration of critical glycans. It was established that a glycopeptide having a 6-phospho-O-mannosyl residue is not an acceptor for action by the enzyme POMGnT1, which attaches β(1,2)-GlcNAc to O-mannosyl moietes, whereas the unphosphorylated derivate was readily extended by the enzyme. This finding implies a specific sequence of events in determining the structural fate of the O-glycan. It has also been found that the activity of POMGnT1 is dependent on the location of the acceptor site in the context of the underlying polypeptide/glycopeptide sequence. Conformational analysis by NMR has shown that the O-mannosyl modification does not exert major conformational effect on the peptide backbone. It is, however, proposed that these residues, introduced at the early stages of glycoprotein glycosylation, have an ability to regulate the loci of subsequent O-GalNAc additions, which do exert conformational effects. The studies show that through access to discrete glycopeptide structures, it is possible to reveal complex regulation of O-glycan processing on α-DG that has significant implications both for its normal post-translational maturation, and the mechanisms of the pathologies associated with hypoglycosylated α-DG.

Synthesis and characterization of nido-carborane-cobalamin conjugates

Three vitamin B12 (cyanocobalamin) conjugates bearing one nido-carborane molecule or two nido-carborane molecules linked to the propionamide side chains via a four carbon linker have been synthesized. Reaction of o-carboranoylchloride with 1,4-diaminobutane in pyridine produced nido-carboranoyl(4-amidobutyl)amine, which was linked to the b- and d-monocarboxylic acids and the b,d-dicarboxylic acid of cyanocobalamin. Mass spectrometry analysis as well as 11B nuclear magnetic resonance demonstrated that during the reaction of o-carboranonylchloride with diaminobutane one of the boron atoms was eliminated. In vitro biological activity of the cyanocobalamin-nido-carborane conjugates was assessed by the unsaturated vitamin B12 binding capacity assay. When compared with 57Co cyanocobalamin, the biological activity of cyanocobalamin-b-nido-carborane, cyanocobalamin-d-nido-carborane, and cyanocobalamin-b-d-bis-nido-carborane conjugates were 92.93%, 35.75%, and 37.02%, respectively. These findings suggest that the 10B cobalamin conjugates might be useful agents in treating malignant tumors via neutron capture therapy.

Natural-abundance 15N NMR studies of turkey ovomucoid third domain. Assignment of peptide 15N resonances to the residues at the reactive site region via proton-detected multiple-quantum coherence

Abstract Heteronuclear two-dimensional 1 H{ 15 N} multiple-quantum (MQ) spectroscopy has been applied to a protein sample at natural abundance: ovomucoid third domain from turkey ( Meleagris gallopavo ), a serine proteinase inhibitor of 56 amino acid residues. Peptide amide 1 H NMR assignments obtained by two-dimensional 1 H{ 1 H} NMR methods (R. Krishnamoorthi and J. L. Markley, unpublished data) led to identification of the corresponding 1 H{ 15 N} MQ coherence cross peaks. From these, 15 N NMR chemical shifts were determined for several specific backbone amide groups of amino acid residues located around the reactive site region of the inhibitor. The results suggest that amide 15 N chemical shifts, which are readily obtained in this way, may serve as sensitive probes for conformational studies of proteins.

Dissecting the Molecular Basis of the Role of the O-Mannosylation Pathway in Disease: α-Dystroglycan and Forms of Muscular Dystrophy

Dystroglycanopathies form a subgroup of muscular dystrophies that arise from defects in enzymes that are implicated in the recently elucidated O‐mannosylation pathway, thereby resulting in underglycosylation of α‐dystroglycan. The emerging identification of additional brain proteins modified by O‐mannosylation provides a broader context for interpreting the range of neurological consequences associated with dystroglycanopathies. This form of glycosylation is associated with protein mucin‐like domains that present numerous serine and threonine residues as possible sites for modification. Furthermore, the O‐Man glycans coexist in this region with O‐GalNAc glycans (conventionally associated with such protein sequences), thus resulting in a complex glycoconjugate landscape. Sorting out the relationships between the various molecular defects in glycosylation and the modes of disease presentation, as well as the regulatory interplay among the O‐Man glycans and the effects on other modes of glycosylation in the same domain, is challenging. Here we provide a perspective on chemical biology approaches employing synthetic and analytical methods to address these questions.

Nuclear magnetic resonance spectroscopy. Concentration dependence of the T1 relaxation time for 13C in dioxane-D2O. Some experimental problems with T1 measurements

The ^(13)C T_1 relaxation times determined for dioxane-D_2O solutions by the progressive saturation method have been found to range from 5 sec for 1:1 (v/v) dioxane-D_2O to 12 sec for neat dioxane. The concentration dependence roughly parallels the inverse of the viscosity and is likely to be associated with changes in correlation times because of hydrogen bonding. A 2:1 (v/v) dioxane-D_2O mixture with a T_1 of 6.2 ± 0.6 sec at 30°C has been found to be a useful standard for checking instrument performance in the determination of T_1 values. A number of experimental problems associated with measuring T_1 are discussed.