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D A Cumming

Publications and source records attributed to D A Cumming.

29 records · Page 2Linked to original sources
PubMed1988

On the utility of 13C-n.m.r. spectroscopy in the identification of the primary structures of manno-oligosaccharides and glycopeptides.

The utility of 13C-n.m.r. spectroscopy in the identification of the primary structures of mannose-containing glycans is investigated. Unlike 1H resonances where the chemical shifts reflect multiple short- and long-range effects, the chemical shifts of 13C resonances are dependent largely upon short-range effects classified as glycosylation (linkage) and substitution effects. These effects are parametized for glycans composed of mannose and encoded in a FORTRAN algorithm. Applications of this program to "unknown" sets of experimental chemical shifts for the resonances of anomeric carbons gave the following conclusions. (1) This program can be used to produce a sub-set of possible structures inclusive of the "known" structure. (2) For other than simple oligosaccharides, it is unlikely that a single structure is consistent with the data for anomeric carbons alone, even when the linkage composition of the glycan has been assessed from other spectral data. (3) When used in conjunction with other chemical techniques, this program can provide a powerful tool for primary analysis of the structure of mannose-containing glycans.

Algorithms↗
PubMed1988

Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human beta-hexosaminidase.

Human beta-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the beta b chain, and one non concanavalin A binding glycopeptide, localized to the beta a chain, were found associated with the beta-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the alpha subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the alpha and one of the beta b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the beta b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the beta a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the alpha and beta subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the alpha chain we found only one possible site for in vivo phosphorylation. In the beta it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an alpha 1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the beta b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.

Amino Acid Sequence↗
PubMed1987

Solution conformation of the branch points of N-linked glycans: synthetic model compounds for tri'-antennary and tetraantennary glycans.

The solution conformation of model compounds for the tri'-antennary and tetraantennary (six-arm) branch point of N-linked glycans has been determined through the use of chemical shift, relaxation, and nuclear Overhauser enhancement data. The object was to establish the conformation about the glycosidic linkages in the N-linked substructure GlcNAc(beta 1,6) [GlcNAc(beta 1,2)] Man(alpha)- by estimation of values for the appropriate glycosidic torsional angles. The GlcNAc(beta 1,6) linkage in a trisaccharide model compound was found to be constrained to a narrow rotameric subpopulation about the substituted Man C5-C6 bond (omega = -60 degrees) and a narrow range of possible phi - psi values. Free rotation about the Man C5-C6 bond was obstructed by unfavorable steric interactions between the GlcNAc(beta 1,6) and GlcNAc(beta 1,2) residues. A phi, psi value of 55 degrees, 190 degrees was found to be consistent with the NMR data for the GlcNAc(beta 1,6) linkage. However, the value of psi appears to be "virtual" in that the molecule is in equilibrium between two different values (90 degrees and 252 degrees). For the GlcNAc(beta 1,2) linkage, complete agreement between all the observed NMR parameters and all the calculated ensemble average values could only be obtained with a set of potential energy functions which included hydrogen bonding. Other choices of potentials yielded calculated values that disagreed with at least two of the observed quantities. As a result, we infer that an interresidue hydrogen bond is formed, and we find it to be between the GlcNAc(beta 1,2) ring oxygen and the Man C3 hydroxyl.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Conformation↗
PubMed1987

Virtual and solution conformations of oligosaccharides.

The possibility that observed nuclear Overhauser enhancements and bulk longitudinal relaxation times, parameters measured by 1H NMR and often employed in determining the preferred solution conformation of biologically important molecules, are the result of averaging over many conformational states is quantitatively evaluated. Of particular interest was to ascertain whether certain 1H NMR determined conformations are "virtual" in nature; i.e., the fraction of the population of molecules actually found at any time within the subset of conformational space defined as the "solution conformation" is vanishingly small. A statistical mechanics approach was utilized to calculate an ensemble average relaxation matrix from which (NOE)'s and (T1)'s are calculated. Model glycosidic linkages in four oligosaccharides were studied. The solution conformation at any glycosidic linkage is properly represented by a normalized, Boltzmann distribution of conformers generated from an appropriate potential energy surface. The nature of the resultant population distributions is such that 50% of the molecular population is found within 1% of available microstates, while 99% of the molecular population occupies about 10% of the ensemble microstates, a number roughly equal to that sterically allowed. From this analysis we conclude that in many cases quantitative interpretation of NMR relaxation data, which attempts to define a single set of allowable torsion angle values consistent with the observed data, will lead to solution conformations that are either virtual or reflect torsion angle values possessed by a minority of the molecular population. On the other hand, calculation of ensemble average NMR relaxation data yields values in agreement with experimental results. Observed values of NMR relaxation data are the result of the complex interdependence of the population distribution and NOE (or T1) surfaces in conformational space. In conformational analyses, NMR data can therefore be used to test different population distributions calculated from empirical potential energy functions.

Algorithms↗
PubMed1987

Reevaluation of rotamer populations for 1,6 linkages: reconciliation with potential energy calculations.

Applications of ensemble averaging to the solution conformation of model compounds for N-linked glycans are further investigated. Specifically, the interpretation usually applied to observed values of J5,6', a parameter reflecting the rotameric distribution about C5-C6 bonds (torsion angle omega in 1,6 glycosidic linkages) in 6-O-substituted hexopyranosides, was found to be inconsistent with populations derived from potential energy calculations. However, agreement between observed and calculated, ensemble-averaged values of J5,6' was obtained and the distribution of omega rotamers reinterpreted. Values of J5,6' that were previously interpreted as indicative of equipartition between two rotamers in fact reflect a marked preference for one of them. Additional potential energy terms, previously absent from energy calculations, are introduced and shown to be without effect on interpretations of omega rotamer distributions. From comparisons with both NMR relaxation and scalar coupling constant data, it is concluded that a simple empirical algorithm, HSEA, calculating van der Waals, exo-anomeric, and (as appropriate) hydrogen-bonding terms, is best suited for describing the population distributions in solution for oligosaccharides and N-linked glycans.

Carbohydrate Conformation↗
PubMed1986

Purification of oligosaccharides having a free reducing-end from glycopeptide sources.

A hydrazinolysis-N-reacetylation procedure, modified by the inclusion of a mild acid-hydrolysis step after N-acetylation, was used to prepare, in overall yields of 60-70%, pure oligosaccharides containing a reducing D-GlcNAc residue from glycopeptide sources. Three types of asparagine-linked glycopeptides were treated: a high-mannose type, a complex-type not containing sialic acid, and a complex-type containing sialic acid, linked both alpha-(2----3) and alpha-(2----6) to beta-D-Galp residues. After the hydrazinolysis-N-reacetylation procedure, there was often contamination of the reducing oligosaccharides with glycopeptide that remained intact through the procedure, as well as minor oligosaccharide products, altered in the nature of the residue at the reducing end. Oligosaccharides having a reducing D-GlcNAc residue were purified by standard liquid chromatography and high-pressure liquid chromatography (l.c.) 360-MHz 1H-n.m.r. was valuable in establishing common structural reporter signals which enabled major products to be identified at stages during the production of free reducing oligosaccharides, and their purity to be assessed.

Animals↗
PubMed1986

Specific deuteration of a trimannoside confirms the existence of a disputed interresidue nuclear Overhauser enhancement.

The relative orientation of the 3-arm of N-linked glycans in solution can be determined, in part, by two interresidue nuclear Overhauser enhancements. The existence of one of these enhancements, that observed to the H5 proton of the (alpha 1,3)-linked mannose (attached to the core beta-linked mannose) upon irradiation of the beta-linked mannose H2 proton, has been disputed by other investigators (Homans, S. W., Dwek, R. A., Fernandes, D. L., and Rademacher, T. W. (1983) FEBS Lett. 164, 231-235). To demonstrate unequivocally the existence of this interresidue enhancement, we have synthesized a mannotrioside to model the corresponding moiety in N-linked glycans. Just as in N-linked glycans, the disputed enhancement cannot be directly observed due to spectral overlap with other enhancements but is detectable by careful quantitative analysis. By employing specifically deuterated derivatives of the mannotrioside, however, the disputed enhancement can be directly visualized.

Magnetic Resonance Spectroscopy↗
PubMed1985

Hydrazinolysis-N-reacetylation of glycopeptides and glycoproteins. Model studies using 2-acetamido-1-N-(L-aspart-4-oyl)-2-deoxy-beta-D-glucopyranosy lamine.

2-Acetamido-1-N-(L-aspart-4-oyl)-2-deoxy-beta-D-glucopyranosyla mine (1) was used as a model glycopeptide to study the hydrazinolysis-N-reacetylation procedure. The major, initial product was the beta-acetohydrazide derivative of 2-acetamido-2-deoxy-D-glucose (2) which gave 2-acetamido-2-deoxy-D-glucose (5) after exposure to acidic conditions. Very mild conditions of hydrolysis of 2 gave a 75-80% overall yield of 5 from 1 after the hydrazinolysis-N-reacetylation procedure. Several other minor compounds were detected which were not converted into 5 upon mild acid hydrolysis, indicating that 20-25% of product cannot be recovered as 5 at the reducing end of oligosaccharides.

Acetylation↗
PubMed1981

Identification of a 2-aminotetrose in a lipid-soluble fraction of rat liver.

Chromatography on DEAE-cellulose of chloroform:methanol:water (10:10:3) extracts obtained from the livers of rats pulsed 50 min with [32P]Pi and D-[1-3H]-mannose yielded two radioactive components which eluted at salt concentrations greater than 0.1 M. One of the components yielded, upon mild acid treatment (20 mM HCl, 100 degrees C, 20 min), a low molecular weight water-soluble component (PO), which contained most of the 32P label associated with the parental lipid-oligosaccharide. PO was periodate-negative, ninhydrin-negative, and permanganate-positive. The phosphate group was stable to strong acid, which converted PO to a single, 32P-labeled product, but the phosphate was readily released by incubation under mild alkaline conditions. Strong alkali produced a product reactive with periodate, ninhydrin, and fluorescamine. When dephosphorylated PO was hydrolyzed with acid and the products were converted to the alditol acetate derivatives, gas chromatography revealed a single major component. Gas chromatography/mass spectroscopy of this compound showed it to be the derivative of a 2-acetamido-2-deoxytetrose. That 2-acetamido-2-deoxytetrose is glycosidically bound in PO is indicated by the fact that PO must be hydrolyzed by strong acid before reduction with NaBD4 is able to incorporate deuterium into the tetrose.

Amino Sugars↗
PubMed1981

Fructose 2,6-bisphosphate. A new activator of phosphofructokinase.

A new activator of rat liver phosphofructokinase was partially purified from rat hepatocyte extracts by DEAE-Sephadex chromatography. The activator, which eluted in the sugar diphosphate region, was sensitive to acid treatment but resistant to heating in alkali. Mild acid hydrolysis resulted in the appearance of a sugar monophosphate which was identified as fructose 6-phosphate by gas chromatography/mass spectroscopy. These observations suggest that the activator is fructose 2,6-bisphosphate. This compound was synthesized by first reacting fructose 1,6-bisphosphate with dicyclohexylcarbodiimide and then treating the cyclic intermediate with alkali. The structure of the synthetic compound was definitively identified as fructose 2,6-bisphosphate by 13C NMR spectroscopy. Fructose 2,6-bisphosphate had properties identical with those of the activator purified from hepatocyte extracts. It activated both the rat liver and rabbit skeletal muscle enzyme in the 0.1 microM range and was several orders of magnitude more effective than fructose 1,6-bisphosphate. Fructose 2,6-bisphosphate was not a substrate for aldolase or fructose 1,6-bisphosphatase. It is likely that this new activator is an important physiologic factor of phosphofructokinase in vivo.

Animals↗