Factors Associated with Visceral Fat Loss in Response to a Multifaceted Weight Loss Intervention.

BACKGROUND: Visceral adipose tissue is more metabolically active than other fat depots and is more closely associated with obesity-related diseases, such as cardiovascular disease and type 2 diabetes, than indicators of obesity, such as body mass index. Across various strategies to promote weight loss, including energy-reduced diet and exercise, variable effects on VAT compared to loss of total body fat have been reported. METHODS: To examine the effect of a behavioral weight loss intervention using portion-controlled prepackaged entrées on VAT, we examined data and measurements from overweight/obese men and women (N=183) who were assigned to a weight loss intervention and prescribed a reduced-energy diet with either portion-controlled prepackaged entrées or self-selected meals in a randomized clinical trial. VAT was estimated with dual-energy X-ray absorptiometry at baseline and study end (12 weeks). RESULTS: VAT loss was greater for the prepackaged entrees group (p=0.02), with an average loss of 29% compared to an average loss of 19% among participants consuming self-selected meals. VAT (mean [SEM]) was 1651 (71) g and 1546 (157) g at baseline and 1234 (59) g and 1278 (118) g at study end in the prepackaged entrees and self-selected meal groups, respectively. Greater VAT loss was associated with higher baseline weight and VAT, and greater weight loss, but not associated with age or physical activity. CONCLUSION: Prescribing portion-controlled prepackaged entrees in a behavioral weight loss intervention promotes a reduction in VAT, which should promote improved metabolic profile and reduced cardiovascular disease risk.

High-performance liquid chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide.

The functional importance of glycolipids has emphasized the need for more sensitive methods of detection, characterization, and quantification than has often been possible using traditional thin-layer chromatographic techniques. We describe the use of ceramide glycanase and HPLC to identify and quantify gangliosides in which the carbohydrate is in Glcbeta1--> linkage with ceramide. Detection of released carbohydrate was by fluorescent labeling with 2-aminobenzamide at the reducing terminal prior to HPLC analysis. Under the conditions described, ceramide glycanase hydrolyzed all of the common gangliosides studied, offering a broad spectrum of specificity. Release and detection of carbohydrate were linear over a wide range (over two orders of magnitude) of micromolar glycolipid substrate concentrations. Use of an N-linked glycan as an internal standard allowed accurate quantification and a recovery of 93% was achieved. The method additionally maintained the sensitivity (chromatographic peaks containing 1 pmol were readily detected from tissue samples) and comparable resolution to related assays. This was shown by the separation, not only of isomeric carbohydrates from the "a" and "b" series, but also of ganglioside carbohydrate differing only by the presence of either N-acetyl- or N-glycolylneuraminic acid. Application of the method to neutral glycosphingolipids and to tissue samples, including 10-microl quantities of plasma, is illustrated. Glycan structures were confirmed by exoglycosidase digestion and/or matrix-assisted laser desorption/ionization mass spectrometry.

A family of novel, acidic N-glycans in Bowes melanoma tissue plasminogen activator have L2/HNK-1-bearing antennae, many with sulfation of the fucosylated chitobiose core.

A family of about 20 novel acidic bi- and tri-antennary N-glycans, amounting to almost half those expressed on Bowes melanoma tissue-plasminogen activator (t-PA) were found to possess Galbeta1-->4GlcNAcbeta1-->, sulfated and sialylated GalNAcbeta1-->4GlcNAcbeta1--> or sulfated GlcAbeta1--> 3Galbeta1-->4GlcNAcbeta1--> antennae, of which those containing sulfated GlcA, depicting the L2/HNK-1 carbohydrate epitope, were preferentially located on the 6 arm. A proportion of the glycans were highly charged, because of multiple and variously distributed sulfation, some of which was located on the fucosylated chitobiose core. Multiple expression of the L2/HNK-1 epitope on a single glycan was observed. The most abundant compound was a biantennary glycan carrying sulfated GlcA on the 6-branched antenna and an alpha2-->6 sialylated GalNAc on the other. The N-glycosylation sequon containing Asn448, which is known to express all of the sulfate-carrying N-glycans contains, unusually, an arginine residue. An electrostatic interaction between this cationic amino acid and the core-sulfate group of the N-glycan is proposed to reduce mobility of the carbohydrate in the region of the t-PA active site. Because of the 'brain-type' nature of the N-glycans described in this neuro-ectodermal cell line, the possibility of neural t-PA interacting with the L2/HNK-1-recognizing molecule, laminin, of the central nervous system extracellular matrix is discussed.

Prion glycoprotein: structure, dynamics, and roles for the sugars.

The prion protein contains two N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The large size of the N-linked sugars, together with their dynamic properties, enables them to shield two orthogonal faces of the protein almost completely. Thus, the sugars can protect large regions of the protein surface from proteases and from nonspecific protein-protein interactions. Immunoprecipitation of prion protein with calnexin suggests that in the ER the oligosaccharides may provide a route for protein folding via the calnexin pathway. Major questions relate to the relevance of the glycoform distribution (as defined by glycan site occupancy) to strain type and disease transmission. Glycan analysis has shown that prion protein contains at least 52 different sugars, that these consist of a subset of brain sugars, and that there is site specific glycan processing. PrP(Sc) from the brains of Syrian hamsters contains the same set of glycans as PrP(C), but a higher proportion of tri- and tetra-antennary sugars. This may be attributed to a decrease in the activity of GnTIII. The GPI anchor, which is modified with sialic acid, may allow the prion protein to be mobile in the lipid bilayer. Potentially, this provides a possible means for translocating the prions from one cell to another.

Composition of N-linked carbohydrates from ovalbumin and co-purified glycoproteins.

Analysis of commercial samples of chicken ovalbumin by reversed-phase high performance liquid chromatography and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) showed the presence of several other co-purifying glycoproteins. These were isolated, subjected to tryptic digestion, and two of them were identified as ovomucoid and chicken riboflavin binding-protein following database matching of the peptide masses obtained by MALDI. The N-linked glycans were released from the glycoproteins and their structures were examined by MALDI-MS in combination with exoglycosidase digestion. Ovalbumin was found to be glycosylated mainly with high-mannose and hybrid structures, consistent with profiles obtained on the intact glycoprotein by electrospray. The other glycoproteins contained mainly larger, complex glycans with up to five antennae, many of which had earlier been associated with ovalbumin.

Glycosylation of a CNS-specific extracellular matrix glycoprotein, tenascin-R, is dominated by O-linked sialylated glycans and "brain-type" neutral N-glycans.

As a member of the tenascin family of extracellular matrix glycoproteins, tenascin-R is located exclusively in the CNS. It is believed to play a role in myelination and axonal stabilization and, through repulsive properties, may contribute to the lack of regeneration of CNS axons following damage. The contrary functions of the tenascins have been localized to the different structural domains of the protein. However, little is known concerning the influence of the carbohydrate conjugated to the many potential sites for N - and O -glycosylation (10-20% by weight). As a first analytical requirement, we show that >80% of the N -glycans in tenascin-R are neutral and dominated by complex biantennary structures. These display the "brain-type" characteristics of outer-arm- and core-fucosylation, a bisecting N -acetylglucosamine and, significantly, an abundance of antennae truncation. In some structures, truncation resulted in only a single mannose residue remaining on the 3-arm, a particularly unusual consequence of the N -glycan processing pathway. In contrast to brain tissue, hybrid and oligomannosidic N -glycans were either absent or in low abundance. A high relative abundance of O -linked sialylated glycans was found. This was associated with a significant potential for O -linked glycosylation sites and multivalent display of the sialic acid residues. These O -glycans were dominated by the disialylated structure, NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAc. The possibility that these O -glycans enable tenascin-R to interact in the CNS either with the myelin associated glycoprotein or with sialoadhesin on activated microglia is discussed.

Characterisation of tissue-specific oligosaccharides from rat brain and kidney membrane preparations enriched in Na+,K+-ATPase.

The organ-specific nature of the glycosylation of Na+,K+-ATPase-enriched preparations from kidney and brain tissues has earlier been indicated by the use of lectin-staining techniques. Na+,K+-ATPase is ubiquitous and abundant, and subject to upregulation during cell-division and in certain pathological conditions. Lectins specific for the different carbohydrates displayed by the Na+,K+-ATPases may, therefore, be useful carriers/mediators in tissue-specific targeting. N-linked oligosaccharides purified from Na+,K+-ATPase-enriched preparations from rat brain and kidney were consequently characterised in detail in this study using weak anion exchange and normal phase HPLC (combined with serial glycosidase digestions) and matrix-assisted laser desorption/ionisation mass spectrometry. The oligomannose series of glycans were most abundant in the brain tissue preparation and this contrasted with the renal-associated oligosaccharides that were dominated by families of tetra-antennary glycans (with/without a core fucose) with up to four lactosaminylglycan residues in either branched or linear formation.

Sialylated N-glycans in adult rat brain tissue--a widespread distribution of disialylated antennae in complex and hybrid structures.

This paper extends our earlier work on the analysis of neutral N-glycans from adult rat brain to glycans carrying NeuAc residues as their sole charged groups. These structures comprised at least 40% of the total (acidic and neutral) N-glycan pool. Compounds were identified by a combination of endoglycosidase and exoglycosidase digestions, anion-exchange chromatography, normal and reverse-phase high-performance liquid chromatography, matrix-assisted laser desorption/ionisation-mass spectrometry and combined gas chromatography/mass spectrometry. Mono-, di- and trisialylated components, together with components substituted with four (or more) NeuAc residues, showed abundances of approximately 12, 10, 7 and 7%, respectively, relative to the total N-glycan pool. In addition, neuraminidase digestion resulted in the neutralisation of a fraction of highly charged species, possibly indicating the presence of N-glycans substituted with short chains of polysialic acid. Sialylated bi-, tri- [mainly the (2,4)-branched isomer], tetraantennary complex, polylactosamine and hybrid structures were detected. Typically, for 'brain-type' N-glycosylation, these sialylated structures were variously modified by the presence of core alpha1-6-linked and outer-arm alpha1-3-linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewis(x) and NeuAc alpha2-3 substituted Galbeta1-4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal beta1-3-linked galactose residues was observed. The presence of beta1-3-linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAc alpha2-3Galbeta1-3(NeuAc alpha2-6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N-glycans with more than one beta1-3galactose residue and (2,4)-branched triantennary oligosaccharides containing three such residues. Disialylated hybrid glycans containing beta1-3-linked galactose and 'polylactosamine' N-glycans with one to three terminal beta1-3galactose residues were additional novel features. The N-glycans modified by polysialylation lacked outer-arm fucose and bisecting GlcNAc residues but all contained one or more terminal beta1-3-linked galactose residues. These may be representative, therefore, of the polysialylated N-glycans expressed mainly on neural cell-adhesion molecules and known to be present in adult rat brain. The diversity of presentation of terminal sialylated groups in rat brain implies potential specificity for possible charge or lectin-mediated interactions. The distinguishing sets of sialylated structures described here are indicative of differences in the natural glycosylation processing pathways in different cell types within the central nervous system, a specificity that may be further magnified on the individual glycoproteins.

Identification of lectin-purified neural glycoproteins, GPs 180, 116, and 110, with NMDA and AMPA receptor subunits: conservation of glycosylation at the synapse.

The postsynaptic apparatus is associated with a number of glycoproteins with apparent molecular masses of 180, 116, and 110 kDa, which are highly concentrated in and may be uniquely associated with this structure. These glycoproteins, purified by concanavalin A lectin-affinity chromatography, showed immunoreactivity in the present study with subunit-specific antibodies to glutamate receptors as follows: GP 180, NMDA receptor subunits NR2A/NR2B; GP 116, NMDA receptor NR1 (1a); and GP 110, pan-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (pan-AMPA) receptors. Sensitivities to the glycosidases peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H on both western blots and silver-stained gels suggested that the glutamate receptors were at least major constituents of the glycoprotein bands. Similar detailed glycosylation was observed for all three glycoproteins, with neutral oligosaccharides being dominant. Oligomannosidic glycans (with from five to nine mannoses) accounted for approximately 50% of the neutral sugars, with Man 5 (at almost 20% of the neutral sugars) always the major glycan. Other abundant neutral oligosaccharides were of the complex type. Similar sensitivities to peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase H were observed for cell line-expressed NMDA receptor subunits, suggesting that irrespective of the glycosylation processing available, the least highly processed oligosaccharides will be expressed. This may be indicative of glycosylation sites in these receptors that are inaccessible to the later processing enzymes and favours the oligomannosidic class of glycans in functional roles.

Neutral N-glycans in adult rat brain tissue--complete characterisation reveals fucosylated hybrid and complex structures.

Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potentially of crucial importance in determining many cell interactions. The complexity of cellular organisation of the brain and suggested involvement of N-glycosylation in neural development, make this an ideal system to study the potential role of glycosylation in tissue development, maintenance and function. Neural tissues are known to contain some highly unusual glycan structures but the structures expressed in neural tissue have not as yet been studied systematically. As a first initiative to assess the type of N-glycosylation occurring in neural tissue, we have characterised all of the major neutral N-linked oligosaccharides expressed in adult rat using a combination of matrix-assisted laser-desorption ionisation mass spectrometry, exoglycosidase sequencing combined with normal-phase HPLC, and two-dimensional HPLC mapping. Oligomannosidic glycans, Man(9-5)GlcNAc2, constituted approximately 15% of the total brain N-glycan pool. The other neutral N-glycan components consisted of a series of diantennary structures (6.5%), (2,6)-branched triantennary glycans (1%) and hybrid structures (3%). Both the complex and hybrid N-glycans were characterised by the presence of outer-arm alpha(1,3)-fucosylation (forming the Lewis[x] determinant), alpha(1,6)-core fucosylation and a bisecting GlcNAc residue. Some of these are unusual or novel structures not having been reported elsewhere. A large proportion of the diantennary N-glycans either lacked Gal residues entirely or were unsubstituted on one Man residue of the trimannosyl core, notably the Man alpha(1,3)-arm. This isomeric form is indicative of the action of a novel beta-hexosaminidase activity and suggests a modification in the classical biosynthetic pathway for N-linked oligosaccharides. Furthermore, expression of large amounts of oligomannosidic glycans is not usually associated with tissue glycoproteins and suggests a possible involvement of these structures in neural cell interactions.

Identification of highly fucosylated N-linked oligosaccharides from the human parotid gland.

The glycosylation of a number of constituents of human saliva is known to modify its biological roles, such as its lubricating properties and binding of microbial flora. Gillece-Castro et al. [Gillece-Castro, B. L., Prakobphol, A., Burlingame, A. L., Leffler, H. & Fisher, S. J. (1991) J. Biol. Chem. 266, 17358-17368] have proposed that the major glycan on the salivary proline-rich glycoproteins is a trifucosylated biantennary sugar with one difucosylated and one unfucosylated antenna. Furthermore, they proposed that the non-fucosylated antenna mediated adherence to a peridontal pathogen, Fusobacterium nucleatum. The detailed structures and roles of other highly fucosylated glycans that co-exist in the parotid gland are not fully known. In view of the influence of outer-arm fucosylation on carbohydrate recognition processes in general, this paper reports the use of a combination of HPLC (normal and reversed phase), matrix-assisted laser-desorption/ionisation (MALDI) mass spectrometry and exoglycosidase digestions to dissect the detailed structures of the most abundant of these polyfucosylated glycans. For measurement of reversed-phase HPLC retention times, new calibration units were used which paralleled the glucose units used for normal-phase HPLC. These differed in that the difference in retention times were compared with those derived from a ladder of 2-aminobenzamide-labelled arabinose oligomers instead of the corresponding oligomers from partially hydrolysed dextran. Over sixty neutral sugars were identified from the parotid gland and many of these were additionally found substituted with sialic acid (both alpha2-3-linked and alpha2-6-linked) and sulphate. These glycans were mainly bi- and tri-antennary sugars with up to five and seven fucose residues respectively, containing fucose alpha1-3-linked to the outer-arm GlcNAc residues and alpha1-2-linked to the galactose. All fucosylated structures contained a core (alpha1-6-linked) fucose. The detailed structure of the trifucosylated biantennary glycan was confirmed, together with the structures of another 12 fucosylated biantennary glycans. Smaller amounts of hybrid and tetraantennary structures were also found and bisected glycans were shown to be constituents of parotid glycoproteins for the first time. Acidic glycans were mainly substituted with sialic acid. Most were monosialylated as the presence of fucose on the antennae was found to suppress the addition of extra sialic acid moieties. The possible functional significance of highly fucosylated N-glycans is discussed in relation to their modification of the availability of other non-reducing terminal monosaccharides for recognition processes.

A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles.

A sensitive and reproducible HPLC technology has been developed, capable of resolving sub-picomolar quantities of mixtures of fluorescently labeled neutral and acidic glycans simultaneously and in their correct molar proportions. The elution positions of standard glycans were determined in glucose units with reference to a dextran ladder, and incremental values for the addition of monosaccharides to oligosaccharide cores were calculated. This information was used to interpret the full oligosaccharide profiles of glycoproteins in a predictive manner based on arm specificity, linkage, and monosaccharide composition. The technique was applied to several systems. For example, a family of glycans isolated from the human parotid gland was extensively resolved on the basis of type and extent of outer arm fucosylation. Second, a serum IgG glycan pool was resolved into 20 peaks which were analyzed simultaneously by sequentially digesting the pool of sugars with exoglycosidase enzymes. In addition, alterations in the glycosylation of IgG associated with rheumatoid arthritis were directly monitored. The reproducibility of the separation system, the predictability of glucose unit values, and the quantitative response of the detection system for individual fluorescently labeled glycans also allowed the automatic analysis of neutral sugars using combinations of enzymes as in the reagent array analysis method (RAAM). In addition, the simultaneous resolution of both acidic (sialylated) and neutral products from the RAAM digestion allowed direct analysis of sialylated glycans, eliminating the previous need to remove sialic acid residues in a preliminary step. Overall, the technologies described here represent a significant advance toward faster, more automated, and more detailed glycan analysis.

Glycosylation of CD4 and Thy-1.

The site-specific glycosylation of soluble recombinant variants of human and rat CD4 (sCD4) expressed in Chinese hamster ovary (CHO) cells has been characterized. The presence of identical oligosaccharides at the conserved glycosylation site in domain 3 of rat and human sCD4 and the greater abundance of oligomannose and hybrid type glycans at the non-conserved glycosylation site of rat sCD4 clearly indicate that the protein structure influences oligosaccharide processing. Comparisons of rat sCD4 glycopeptides with mutant molecules with only single glycosylation sites and with a truncated form containing only the two NH2-terminal domains, indicate that independent processing occurs at each glycosylation site and that domain interactions can also affect oligosaccharide processing. These and other analyses of sCD2 expressed in CHO cells and Thy-1 purified from various tissues suggest that the diversity of oligosaccharide structures on a protein is regulated by the location of the glycosylation sites and the nature of the target protein, cell and tissue. The functional significance of this control remains to be determined.

Comparative analysis of the N-glycans of rat, mouse and human Thy-1. Site-specific oligosaccharide patterns of neural Thy-1, a member of the immunoglobulin superfamily.

Protein structure and tissue type are known to influence glycosylation of proteins. We have previously investigated the N-glycans at each of the three glycosylation sites of the cell surface glycoprotein Thy-1 when isolated from rat brain and thymocytes. Here we report a comparative analysis of the site-specific N-glycosylation patterns from rat (Asn 23, 74, 98), mouse (Asn 23, 75, 99) and human (Asn 23, 60, 100) neural Thy-1. Despite considerable differences in amino acid sequence, the results show a remarkable conservation of the pattern of N-glycans at corresponding sites between the three species, as judged by chromatographic comparisons and glycosidase susceptibility. This is particularly marked for sites at Asn 74/75 in rat/mouse and the equivalent site at 60 in human Thy-1, as well as for sites at Asn 98/99 and 100, respectively. The sites at Asn 23 in rat/mouse also contained almost identical glycosylation patterns, but at this site human Thy-1 showed significantly different glycosylation patterns. These site glycosylation patterns are discussed in relation to the likely accessibility of the oligosaccharides for processing. It is known that within a species, the glycosylation of Thy-1 is tissue specific; therefore, this degree of conservation of glycosylation of Thy-1 expressed in the same tissue in different species is all the more striking, given the known variation between species in the amino acid sequence of Thy-1. It is therefore proposed that neural cells have a particular requirement for specific surface carbohydrates and that the Thy-1 polypeptide serves as an appropriate carrier for these structures.

Use of large-scale hydrazinolysis in the preparation of N-linked oligosaccharide libraries: application to brain tissue.

In this report, we describe the preparation of a library of N-linked glycans from whole murine brain obtained by the large-scale hydrazinolysis of an acetone powder of the tissue followed by chromatographic procedures. 84% of the characterized oligosaccharides were found to be anionic, the remainder neutral. The anionic species were successively neutralized by neuraminidase (29%), aq. hydrofluoric acid (30%), and methanolysis (26%), indicating that approximately equal portions were sensitive to desialylation, dephosphorylation and desulfation, respectively. The presence of the sulfated fraction was confirmed by direct 35SO4 metabolic labelling. A residual partially characterized fraction was found to be anionic through possession of carboxylic acid groups, unrelated to sialic acid. The purified oligosaccharides, in the absence of their original protein conjugates, were shown to retain those immunological characteristics essential for recognition by a specific monoclonal antibody, LS (412), that is known to recognize a carbohydrate epitope present on a number of neural adhesion molecules and functional in neural cell adhesion. These properties confirm the viability of scaling up the size of the hydrazinolysis procedure and adapting it to whole tissue for the production of glycan libraries and for the probing of structures of interest.

Fast sequencing of oligosaccharides: the reagent-array analysis method.

A method of oligosaccharide analysis involving controlled fragmentation resulting from enzymatic digestion is presented. The principle involves generating a set of fragments from the original oligosaccharides, characterizing them in terms of their hydrodynamic volumes, determining their molar proportions, and identifying the oligosaccharides by comparison with a computer-generated data base. Experimentally, this technique involves incubation of aliquots of a sample with a set of defined mixtures of exoglycosidases followed by pooling of the products and a single analysis on the product pool. This method has several practical advantages over current techniques, including speed and the ability to use smaller amounts of starting material. The detection of the intensity-versus-hydrodynamic volume profile is limited only by the specific activity of the labeling method. The ability to perform the enzyme digestions is limited by the individual Km values of the enzymes.

Detection of multisulphated N-linked glycans in the L2/HNK-1 carbohydrate epitope expressing neural adhesion molecule P0.

P0, the most abundant glycoprotein of PNS myelin, is a homophilic and heterophilic adhesion molecule. P0 is known to contain a glycoform population that expresses the L2/HNK-1 carbohydrate epitope found on other neural adhesion molecules, and to be functionally implicated centrally in neural cell adhesion and neurite outgrowth. This carbohydrate epitope has been characterized previously from glycolipid structures and contains a sulphated glucuronic acid residue. However, the L2/HNK-1 carbohydrate epitope has not been characterized in glycoproteins. Because P0 possesses only one glycosylation sequon, the number of P0 glycoforms is equal to the heterogeneity of the glycan species. Here we report that the carbohydrate analysis of L2/HNK-1-reactive P0 showed the presence of anionic structures containing sialic acid and sulphate in various combinations. At least one sulphate residue was present in 80% of the monosaccharide sequences, and 20% contained three sulphates. High-resolution P4 gel chromatography of the desialylated and desulphated oligosaccharides showed substantial heterogeneity of monosaccharide sequences. Sequential exoglycosidase digestions indicated that the majority of the structures were of the hybrid class, although the sulphated structures were found to be endoglycosidase H-resistant.