Biomedical subjects

K G Rice

Publications and source records attributed to K G Rice.

At least 19 recordsLinked to original sources

PubMed1992

Quantitative mapping of the N-linked sialyloligosaccharides of recombinant erythropoietin: combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization.

A rapid quantitative analysis of the sialylated N-linked oligosaccharides of recombinant erythropoietin (EPO) expressed in Chinese hamster ovary (CHO) cells has been developed. The procedure utilizes a glycoamidase (glycopeptidase F) to release all of the N-linked oligosaccharides from the native glycoprotein, followed by direct chromatographic analysis using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. The eight sialyloligosaccharides isolated from HPAEC were characterized by derivatizing with 2-aminopyridine followed by two-dimensional HPLC mapping of the pyridylaminated asialooligosaccharides (Tomiya et al., 1988, Anal. Biochem. 171, 73-90). Seven kinds of complex-type asialooligosaccharides were identified ranging from a biantennary structure to N-acetyllactosamine-extended tetraantennary structure. Approximately 3% of the terminal galactose residues of the oligosaccharides released from EPO were not sialylated whereas 97% contained an alpha(2-->3)-linked sialic acid. Quantitative oligosaccharide mapping of four different lots of EPO from CHO cells was performed to quantify the molar balance and distribution of the N-linked oligosaccharides. The sialyloligosaccharides were distributed with approximately 5% disialylated (single type), 20% trisialylated (six types), and 75% tetrasialylated (four types) oligosaccharides with an average molar recovery of 85% starting from 750 pmol of EPO.

Amidohydrolases↗

PubMed1992

Comparison of N-glycosides of fetuins from different species and human alpha 2-HS-glycoprotein.

Complex type N-glycosides of commercial bovine fetuin preparations from pooled fetal calf serum have been shown to contain comparable amounts of Gal4,4,4TRI (see structure A below) and Gal4,4,3TRI (structure B) as major asialo-structures. To investigate whether there is a clear genetic specificity for synthesis of these oligosaccharides, N-glycosides from two preparations of bovine fetuin, each from a single calf, were examined. Both of these structures were present in each calf, and there was only a subtle quantitative difference in the ratio of these two structures between the calves. Thus, a specific galactosyltransferase, presumably required for the biosynthesis of the Gal4,4,3TRI structure, may exist in both of these individual calves. Comparison of fetuin N-glycosides was also extended to sheep, pig, and human alpha 2-HS-glycoprotein, the human counterpart of bovine fetuin, using high-pH anion-exchange chromatography of the reducing oligosaccharides as well as HPLC of their pyridinylamino derivatives. The N-glycosides of ovine fetuin also have both Gal4,4,4TRI and Gal4,4,3TRI structures in a ratio similar to that of bovine fetuin. However, the major N-glycoside of porcine fetuin is of a fucosyl biantennary complex type structure (structure C below) and human alpha 2-HS-glycoprotein has an N-glycoside which is almost exclusively a nonfucosylated biantennary structure (structure D). This species-specific presence of N-glycosides of fetuins and comparison with N-glycosides of other glycoproteins suggest that the polypeptide sequence of a glycoprotein may affect its N-glycan structure by regulating the activity of specific glycosyltransferases. [formula: see text]

Amidohydrolases↗

PubMed1991

Differential flexibilities in three branches of an N-linked triantennary glycopeptide.

The solution conformation behavior of complex oligosaccharides was studied by resonance energy transfer, as measured by the time-resolved fluorescence method, to determine the conformational heterogeneity of a triantennary glycopeptide at various temperatures. Groups that acted as a fluorescence donor (naphthyl-2-acetyl, Nap) or acceptor (dansylethylenediamine, Dan) were selectively attached to the N terminus of the peptide and a Gal residue [either 6' (shown below), 6, or 8] of the oligosaccharide, respectively. [formula: see text] Time-resolved fluorescence energy-transfer measurements revealed two populations of conformers when Dan was attached to either Gal-6' or Gal-6. One conformer contained the antenna folded back toward the core region, and a second was in an extended conformation. The two conformations differed in donor-acceptor distance by about 10 A. Systematically increasing the temperature from 0 degrees C to 40 degrees C increased the ratio of extended to folded forms 2-fold for the Gal-6 isomer and 4-fold for the Gal-6' isomer, whereas the Gal-8 isomer showed only a single distance population throughout this temperature range. From these data, delta H and delta S for the reversible conformational change were calculated to be 3.1 kcal/mol and 10.8 cal/(mol.K) for the Gal-6 isomer and 7.1 kcal/mol and 25.8 cal/(mol.K) for the Gal-6' isomer. In addition to the structural microheterogeneity commonly associated with glycoproteins, the differential flexibilities of the different branches in the oligosaccharides contribute conformational heterogeneity and should be considered in conformational analysis. The data are discussed in terms of the most probable linkages that contribute to the observed flexibility of the individual triantennary branches, and the biological significance of flexible linkages in complex carbohydrates is considered.

Carbohydrate Conformation↗

PubMed1991

Interterminal distance and flexibility of a triantennary glycopeptide as measured by resonance energy transfer.

Three geometric isomers of a single triantennary glycopeptide, each containing two fluorophores attached to terminal positions in the molecule, were used to probe distance and flexibility of the oligosaccharide in solution. A dansyl group (energy acceptor) was attached to the C6 of Gal at either position 6', 6, or 8, and a naphthyl-2-acetyl group (energy donor) was coupled to the N terminus of the Ala-Asn peptide. (formula; see text) Resonance energy-transfer measurements revealed an average distance of approximately 22, 18, and 17 A between the donor and the acceptor attached to either the 6, 8, or 6' Gal residue, respectively. The lifetime of the donor's emission was nearly a single-exponential decay of 27 ns (96%), whereas the decay of the donor with proximally attached acceptor was fit by nonlinear least-squares analysis to a multiexponential for each glycopeptide probe. Fitting with a Lorentzian function revealed spatially distinct donor/acceptor distances presumably arising from glycopeptide branch flexibility. The results suggest that the acceptor located at Gal 8 is the most rigid relative to the donor with a single population of distances centered at 18.4 A. In contrast, the acceptor attached to either Gal 6' or 6 displayed two populations of different distances from the donor. The Gal 6 isomer contained a major population with average donor/acceptor separation distance of 21.7 A and a minor population with average separation distance of 9.7 A. Similarly, the Gal 6' isomer showed a major population with donor/acceptor separation distance of 18.3 A and a minor population with separation distance of 11.7 A. These data support the earlier conclusions that the Man alpha(1----6)Man linkage found in the core pentasaccharide of all branched N-linked oligosaccharides is flexible. In addition, the data suggest that the branch containing Gal 6 is also flexible in the triantennary glycopeptide.

Animals↗

PubMed1990

Modification of triantennary glycopeptide into probes for the asialoglycoprotein receptor of hepatocytes.

Triantennary glycopeptide was oxidized with galactose oxidase to convert the -CH2OH group on terminal galactose residues to the aldehyde group (oxo-form). Kinetic profiling by reverse phase high performance liquid chromatography allowed termination of the reaction when intermediate mono-oxo- and di-oxo-triantennary glycopeptides had been produced. The mixture of the oxo-glycopeptides was derivatized with 2,4-dinitrophenylhydrazine for efficient separation, and each isomeric triantennary hydrazone was separated by reverse phase high performance liquid chromatography. The purified hydrazones were reverted to three original isomeric mono-oxo- and di-oxo-glycopeptides, and a single tri-oxo-glycopeptide. Each of these isomers was characterized by proton NMR by a downfield shift in the anomeric signals of 6-oxo-Gal residue(s). The functionalized glycopeptides were successively modified with dansyl and naphthyl groups through the 6-oxo-Gal residue and the amino terminus of the peptide to prepare three isomeric glycopeptide probes suitable for conformation studies by fluorescence energy transfer measurements. Alternatively, glycopeptides were derivatized by attaching t-butyloxycarbonyl-L-tyrosine to the amino terminus of the peptide, and reductive amination of the 6-oxo-Gal residue, provided three isomeric triantennary photoaffinity probes which allow photolyzable groups to be attached to the newly introduced 6-amino-Gal residue.

Affinity Labels↗

PubMed1990

Defined geometry of binding between triantennary glycopeptide and the asialoglycoprotein receptor of rat heptocytes.

Three derivatives of a triantennary glycopeptide, each containing a single uniquely located 6-amino-galactose residue at either position 6', 6, or 8, were modified at the 6-amino group by attachment of a photolyzable reagent and radiolabeled by iodination of tyrosine. These were allowed to bind to the asialoglycoprotein receptor of isolated rat hepatocytes and photolyzed for affinity labeling. (formula; see text) Each probe specifically labeled either the major (RHL1) or minor (RHL2/3) subunits which comprise the receptor. A photolyzable group attached to galactose residue 6 6' specifically radiolabeled RHL1, whereas a photolyzable group attached to galactose 8 specifically labeled RHL2/3. Photoaffinity labeling of a soluble rat hepatic lectin preparation demonstrated that the minor subunits (RHL2/3) were no longer labeled by the triantennary probe with a photolyzable group at galactose 8. The inhibitory potency of a variety of complex glycopeptides against radiolabeled ligand binding to both rat hepatocytes and soluble lectin are in agreement with photoaffinity results that galactose 8 of triantennary glycopeptide is of unique importance by binding solely to the minor subunits (RHL2/3) of the asialoglycoprotein receptor on hepatocytes. Conversely, galactose residues 6 and 6' bind specifically to the major subunit (RHL1), indicating a precise binding geometry between the trivalent ligand and lectin.

Affinity Labels↗

PubMed1990

Large-scale preparation and characterization of N-linked glycopeptides from bovine fetuin.

A preparative scheme has been developed to purify asialo-glycopeptides from each of the three N-linkage sites of bovine fetuin, allowing the isolation of 100-mumols quantities of asialo-glycopeptides from 20 g of fetuin. The procedure yields seven asialo-glycopeptides which were determined to be 95% homogenous in peptide and oligosaccharide structure. The isolation scheme uses two high-capacity reverse-phase eluant systems. The primary RP-HPLC purification performed with boric acid buffered to pH 7 with triethylamine resolved sialylated tryptic glycopeptides simultaneously on the basis of glycosylation site and degree of sialylation. A second RP-HPLC purification was performed eluting isocratically with dilute phosphoric acid which resolved residual peptide and oligosaccharide heterogeneity from asialo-glycopeptides containing short peptides. Structural characterization of the products was performed utilizing 400-MHz proton NMR spectroscopy and amino acid and monosaccharide analysis. The glycopeptides contain two previously identified variant triantennary oligosaccharides which possess either Gal beta(1----4) or Gal beta(1----3) linkages to N-acetylglucosamine at one terminal branch or a biantennary oligosaccharide. These compounds should prove to be invaluable in studying carbohydrate-protein interactions, such as binding by the Gal/GalNAc lectin of mammalian hepatocytes, in the detailed three-dimensional structural analysis of complex oligosaccharides, and as purified substrates for the study of the action of glycoconjugate-modifying enzymes.

Animals↗

PubMed1990

Identity status and self-construct systems: process X structure interactions.

This study investigated the relationship between identity status and structural dimensions of individuals' self-theories or personal construct systems. The sample was composed of 31 Achievers, 31 Moratoriums, 30 Foreclosures, and 26 Diffusions. Participants completed a modified version of Kelly's (1955) Role Construct Repertory test designed to assess their personal sense of integrative continuity over time. Status differences in self-construct differentiation, integration, and self-certainty were analyzed. Results indicated that Moratoriums and Diffusions had the highest self-construct differentiation. Achievers were the most self-certain in their ratings, Diffusions the least. Status differences in self-theory integration were found, but only when a qualitative, structural analysis of levels of self-construct differentiation and integration was performed. No sex effects were found. Results are discussed as replicating and extending previous work in the area, and future research directions are noted.

Adolescent↗

PubMed1989

Binding characteristics of N-acetylglucosamine-specific lectin of the isolated chicken hepatocytes: similarities to mammalian hepatic galactose/N-acetylgalactosamine-specific lectin.

Binding characteristics of N-acetylglucosamine- (GlcNAc) specific lectin on the chicken hepatocyte surface were probed by an inhibition assay using various sugars and glycosides as inhibitors. Results indicated that the binding area of the lectin is small, interacting only with GlcNAc residues whose 3- and 4-OH's are open. The combining site is probably of trough-type, since substitution with as large a group as monosaccharide is permitted on the C-6 side of GlcNAc, and on the C-1 side, the aglycon of GlcNAc can be very large (e.g., a glycoprotein). These binding characteristics are shared with the homologous mammalian lectin specific for galactose/N-acetylgalactosamine, suggesting that tertiary structure of the combining area of these two lectins is similar. This is understandable, since there is approximately 40% amino acid sequence identity in the carbohydrate recognition domain of these two lectins [Drickamer, K., Mannon, J. F., Binns, G., & Leung, J. O. (1984) J. Biol. Chem. 259, 770-778]. A series of glycosides, each containing two GlcNAc residues separated by different distances (from 0.8 to 4.7 nm), were synthesized. Inhibition assay with these and other cluster glycosides indicated that clustering of two or more GlcNAc residues increased the affinity toward the chicken lectin tremendously. Among the ligands containing two GlcNAc residues, the structure which allows a maximal inter-GlcNAc distance of 3.3 nm had the strongest affinity, its affinity increase over GlcNAc (monosaccharide) amounting to 100-fold. Longer distances slightly diminished the affinity, while shortening the distance caused substantial decrease in the affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylgalactosamine↗

PubMed1989

Study of structurally defined oligosaccharide substrates of heparin and heparan monosulfate lyases.

The rapid preparation of multimilligram quantities of five heparin-derived oligosaccharides (1-5) is described. These oligosaccharides are the final products obtained from the action of heparin lyase (heparinase, E.C. 4.2.2.7) at its primary sites in the heparin polymer. Five oligosaccharides comprise from 75-85 wt% of commercial porcine mucosal heparins and are recovered in good yield and high purity. Four of these five oligosaccharides were further acted upon at much lower rates by prolonged treatment with heparin lyase or heparan monosulfate lyase (heparitinase, E.C. 4.2.2.8), revealing the subspecificities of these enzymes. These oligosaccharides were used as defined substrates for heparin lyase and heparan monosulfate lyase and their kinetic constants were obtained. Potential applications for these oligosaccharides include their use as defined substrates for purification of heparin monosulfate lyases, and for establishing the catalytic purity of enzyme preparations.

Carbohydrate Sequence↗

PubMed1989

Nonrandom structural features in the heparin polymer.

Computer simulation studies were used to prepare an ensemble of heparin number chains. The polydispersity of these chains was simulated by introducing a specific "fraction of terminators", and it closely resembled the experimentally observed polydispersity of a porcine mucosal, glycosaminoglycan heparin. The same percentage of simulated chains contained antithrombin III (ATIII) binding site sequences as are typically found to contain ATIII binding sites using affinity chromatography. Heparin lyase action was then simulated by using Michaelis-Menten kinetics. In one model, heparin chains were constructed from the random assembly of monosaccharide units using the observed mole percentage of each. After simulated depolymerization, the final oligosaccharides formed were compared to the observed oligosaccharide products. The simulation which assumed a random distribution of monosaccharide units in heparin did not agree with experimental observations. In particular, no ATIII binding site sequences were found in the simulated number chains. The results of this simulation indicate that heparin is not simply a random assembly of monosaccharide units. These results are consistent with the known, ordered biosynthesis of heparin. In a second model, heparin chains were constructed from randomly assembled oligosaccharides at the mole percentage in which each is found in the final product mixture. The action of heparin lyase was then simulated, and the distribution of the oligosaccharide products was measured throughout the simulated time course of the depolymerization reaction. The simulated rate of formation and final concentration of a particular oligosaccharide which contains a portion of heparin's ATIII binding site were similar to those observed experimentally. These results are consistent with the random distribution of ATIII binding sites within glycosaminoglycan heparin.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Conformation↗

PubMed1989

Resistance of heparinase-derived heparin fragments to biotransformation.

The biotransformation of heparinase-derived heparin fragments was examined via a combined approach using 35S-labeled heparin fragments as well as unlabeled chemically defined heparin fragments. Rats dosed with either [35S]di-, tetra-, hexa-, or octasaccharide fragments (2 mg/kg body weight, intravenously) excreted 63-69% of the injected radioactivity into the urine within 24 h with two-thirds being excreted during the first 6 h. Gel permeation chromatography of the urinary material shows that the tetra- and octasaccharides have undergone minor (approximately 5%) depolymerization whereas no change was observed for the di- and hexasaccharides. No N-desulfation was demonstrated for any of the substances. The hexa- and octasaccharide metabolites present in the urine 24 h after dosing exhibited the same antifactor Xa activity as that of the injected material. A chemically defined trisulfated disaccharide and a hexasulfated tetrasaccharide were prepared and dosed in a similar manner. Only one metabolite was recovered from animals dosed with disaccharide. This compound was characterized by anion exchange chromatography, proton nuclear magnetic resonance spectroscopy, Fourier transform infrared spectrometry, and mass spectrometry and shown to be identical to the injected disaccharide. Five metabolites were isolated from the urine of rats dosed with the hexasulfated tetrasaccharide. The major metabolite, consisting of at least 65% of the total, was characterized as described for the disaccharide and shown to be identical to the injected compound. The remaining material appeared to be disaccharides and, possibly, a tetrasaccharide conjugate. Taken together, our results show that the heparinase-derived heparin fragments are very resistant to biotransformation compared with heparin and endogenous heparin fragments. These fragments may therefore be useful in defining structure activity relationships in vivo.

Animals↗

PubMed1988

Mapping and quantification of the major oligosaccharide components of heparin.

A new method of determining the oligosaccharide composition of commercial glycosaminoglycan heparin is described in which heparin was first depolymerized using heparin lyase (EC 4.2.2.7), and then analysed by a single h.p.l.c. step. All 20 of the porcine and bovine heparins examined were found to contain a small number of major oligosaccharide components, which on average comprised 86% of their mass. The five most abundant oligosaccharides have defined chemical structures. Although the relative abundance of oligosaccharides varied, the heparins examined were surprisingly similar. Porcine, bovine, low-Mr, and high and low antithrombin III (ATIII)-affinity heparins, however, each had distinctly different proportions of these major oligosaccharide components. The concentrations of one of these five oligosaccharides, containing a portion of the ATIII binding site, correlated with the anticoagulant activity of the ATIII-affinity-fractionated porcine-mucosal heparins from which it was derived. An additional oligosaccharide of undetermined structure was found in significant quantities in both bovine heparin and high ATIII-affinity porcine-mucosal heparin. The correlation between oligosaccharide concentration and anticoagulant activity suggests that the oligosaccharide is derived from a structural variant of the ATIII-binding site. Finally, for the heparins examined chondroitin/dermatan sulphate formed 0.6-7.4% of their mass.

Animals↗

PubMed1988

Homogeneous, structurally defined heparin-oligosaccharides with low anticoagulant activity inhibit the generation of the amplification pathway C3 convertase in vitro.

This paper demonstrates that heparin-oligosaccharides with low anticoagulant activity have a high capacity to inhibit activation of the amplification pathway of complement in vitro. We prepared heparin-oligosaccharides by partial depolymerization of heparin using purified flavobacterial heparinase. The resulting oligosaccharide mixture was then fractionated using strong anion exchange-high pressure liquid chromatography to produce individual oligosaccharide components of this mixture, with degree of polymerization ranging from 2 to 16. These heparin-oligosaccharides were examined for both their anticoagulant activity and capacity to inhibit activation of the amplification pathway of complement. Although there was little difference among commercial heparins, a correlation between molecular weight and activity to inhibit convertase generation was clearly established for heparin-oligosaccharides between degree of polymerization 2 through 16. Heparin-oligosaccharides of degree of polymerization 10-16 (Mr 3888-5320) demonstrated up to 54% of heparin's activity on a molar basis (and up to 163% of heparin's activity on a weight basis) in inhibiting the amplification pathway of complement in vitro while showing almost no anticoagulant activity. These studies, for the first time, completely separate heparin's ability to inhibit complement activation from its anticoagulant activity.

Animals↗

PubMed1987

Fractionation of heparin-derived oligosaccharides by gradient polyacrylamide-gel electrophoresis.

Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed.

Animals↗

PubMed1986

Structure and activity of a unique heparin-derived hexasaccharide.

A hexasaccharide representing a major sequence in porcine mucosal heparin has been enzymatically prepared from heparin. Its structure was determined by an integrated approach using chemical, enzymatic, and spectroscopic methods. Two-dimensional 1H homonuclear COSY, C-H correlation NMR, and selective irradiation were used to assign many of the NMR resonances. In addition, new techniques including sulfate determination by ion chromatography and Fourier transform IR and californium plasma desorption mass spectroscopy have been applied, resulting in an unambiguous structural assignment of delta IdoAp2S(1----4)-alpha-D-GlcNp2S6S(1----4)-alpha-L-IdoAp++ +(1----4)-alpha-D-GlcNA cp6S-(1----4)-beta-D-GlcAp(1----4)-alpha-D-GlcNp2S3S6S (where delta IdoA represents 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, p represents pyranose, and GlcA and IdoA represent glucuronic and iduronic acid). This hexasaccharide contains a portion of the antithrombin III-binding site and has a Kd of 4 X 10(-5) M. Unlike other small heparin oligosaccharides, which are specific for coagulation factor Xa, it inhibits both factors IIa and Xa equally through antithrombin III. This hexasaccharide may have the unique capacity to act primarily through heparin cofactor II to inhibit thrombin (factor IIa) and shows over half of heparin's heparin cofactor II-mediated anti-factor IIa activity. These studies suggest the occurrence of contiguous binding sites on heparin for Xa, antithrombin III, and heparin cofactor II.

Animals↗

PubMed1985

Evidence of random structural features in the heparin polymer.

The first use of computer-simulation studies to examine heparin's structure has been reported. The product distributions obtained when porcine mucosal heparins were depolymerized with heparinase have been compared to computer-simulated distributions. The modeled distribution was relatively unaffected by the polydispersity and molecular weight of heparin. However, the percent of heparinase-cleavable glycosidic linkages and their distribution throughout the polymer resulted in a marked change in the simulated product distribution. The similarity between experimentally observed and computer-simulated product distributions is consistent with the random distribution of heparinase-cleavable sites in porcine mucosal heparin. Finally, a random distribution of N-acetyl residues with respect to heparinase-cleavable sites was experimentally observed.

Animals↗

PubMed1985

High-performance liquid chromatographic separation of heparin-derived oligosaccharides.

Heparin has been enzymatically depolymerized with heparinase (heparin lyase (EC 4.2.2.7)) and then separated into di-, tetra-, hexa-, octa-, and decasaccharide mixtures by low-pressure gel-permeation chromatography (GPC). These sized mixtures were resolved by strong anion-exchange (SAX) HPLC into multiple components. The fractions from the SAX-HPLC were collected and characterized for size by GPC-HPLC and sulfate content by ion chromatography. This study provides detailed methodology for the separation of larger and more highly sulfated oligosaccharides than previously reported. It describes the first use of ion chromatography for the accurate determination of the sulfate content of heparin oligosaccharides, a method which can also be applied to heparin and other glycosaminoglycans.

Chromatography, Gel↗