Oligosaccharide Chains Of Glycoproteins Research Articles

The Intrinsic Relationship Between Structure and Function of the Sialyltransferase ST8Sia Family Members.

As a subset of glycosyltransferases, the family of sialyltransferases catalyze transfer of sialic acid (Sia) residues to terminal non-reducing positions on oligosaccharide chains of glycoproteins and glycolipids, utilizing CMP-Neu5Ac as the activated sugar nucleotide donor. In the four known sialyltransferase families (ST3Gal, ST6Gal, ST6GalNAc and ST8Sia), the ST8Sia family catalyzes synthesis of α2, 8-linked sialic/polysialic acid (polySia) chains according to their acceptor specificity. We have determined the 3D structural models of the ST8Sia family members, designated ST8Sia I (1), II(2), IV(4), V(5), and VI(6) using the Phyre2 server. Accuracy of these predicted models are based on the ST8Sia III crystal structure as the calculated template. The common structural features of these models are: (1) Their parallel templates and disulfide bonds are buried within the enzymes and are predominately surrounded by helices; (2) The anti-parallel β-sheets are located at the N-terminal region of the enzymes; (3) The mono-sialytransferases (mono-STs), ST8Sia I and ST8Sia VI, contain only a single pair of disulfide bonds, and there are no anti-parallel β-sheets in ST8Sia VI; (4) The Nterminal region of all of the mono-STs are located some distant away from their core structure; (5) These conformational features show that the 3D structures of the mono-STs are less compact than the two polySTs, ST8Sia II and ST8Sia IV, and the oligo-ST, ST8Sia III. These structural features relate to the catalytic specificity of the monoSTs; (6) In contrast, the more compact structural features of ST8Sia II, ST8Sia IV and ST8Sia III relate to their ability to catalyze the processive synthesis of oligo- (ST8Sia III) and polySia chains (ST8Sia II & ST8Sia IV); (7) Although ST8Sia II, III and IV have similar conformations in their corresponding polysialyltransferase domain (PSTD) and polybasic region (PBR) motifs, the structure of ST8Sia III is less compact than ST8Sia II and ST8Sia IV, and the amino acid components of the several three-residue-loops in the two motifs of ST8Sia III are different from that in ST8Sia II and ST8Sia IV. This is likely the structural basis for why ST8Sia III is an oligoST and not able to polysialylate and; (8) In contrast, essentially all amino acids within the threeresidue- loops in the PSTD of ST8Sia II and ST8Sia IV are highly conserved, and many amino acids in the loops and the helices of these two motifs are critical for NCAM polysialylation, as determined by mutational analysis and confirmed by our recent NMR results. In summary, these new findings provide further insights into the molecular mechanisms underlying polyST-NCAM recognition, polySTpolySia/ oligoSia interactions, and polysialylation of NCAM.

Release of Flavonoids from Lupin Globulin Proteins during Digestion in a Model System

Lupin seed globulin proteins form complexes with flavonoids, predominantly apigenin C-glycosides. Enzymes typical for the gastrointestinal tract were used to hydrolyze lupin seed globulins. Release of native flavonoids as a result of the proteolysis reaction was observed. Different analytical methods such as size exclusion chromatography, HPLC-MS, and fluorescence spectroscopy (steady-state fluorescence, fluorescence anisotropy, fluorescence lifetimes) were used for a detailed characterization of this phenomenon. Flavonoids liberated from lupin globulin proteins as a result of pancreatin-catalyzed digestion were bound by γ-conglutin resistant to this enzyme. Two possible mechanisms of this interaction may be suggested: hydrogen bonding between oligosaccharide chains of glycoproteins and the sugar moieties of the flavonoid glycosides or electrostatic attraction between positively charged γ-conglutin and flavonoids partially ionized at pH 7.5.

The sialyl-alpha2,6-lactosaminyl-structure: biosynthesis and functional role.

Sialylation represents one of the most frequently occurring terminations of the oligosaccharide chains of glycoproteins and glycolipids. Sialic acid is commonly found alpha2,3- or alpha2,6-linked to galactose (Gal), alpha2,6-linked to N-acetylgalactosamine (GalNAc) or alpha2,8-linked to another sialic acid. The biosynthesis of the various linkages is mediated by the different members of the sialyltransferase family. The addition of sialic acid in alpha2,6-linkage to the galactose residue of lactosamine (type 2 chains) is catalyzed by beta-galactoside alpha2,6-sialyltransferase (ST6Gal.I). Although expressed by a single gene, this enzyme shows a complex pattern of regulation which allows its tissue- and stage-specific modulation. The cognate oligosaccharide structure, NeuAcalpha2,6Galbeta1,4GlcNAc, is widely distributed among tissues and is involved in biological processes such as the regulation of the immune response and the progression of colon cancer. This review summarizes the current knowledge on the biochemistry of ST6Gal.I and on the functional role of the sialyl-alpha2,6-lactosaminyl structure.

Characterization of a lectin from the leaves of great northern bean, Phaseolus vulgaris L.

A novel lectin (GNLL) was isolated from the leaves of the Great Northern bean, Phaseolus vulgaris. GNLL was purified by affinity chromatography on ovomucoid-Sepharose 4B. GNLL had a molecular mass of 135 kDa on gel filtration and gave two bands on SDS-polyacrylamide gel electrophoresis (PAGE)(band A of 34.0 kDa and band B of 34.2 kDa). Binding assay of horseradish peroxidase (HRP)-glycoproteins to the bands electroblotted onto polyvinylidene difluoride (PVDF) membrane showed that both bands could bind to complex-type N-linked oligosaccharide chains in glycoproteins. The N-terminal amino acid sequences of both bands were identical through the 10 residues and identical to that of alpha-subunit of a pod lectin (pod-alpha-subunit) from the same bean. On the other hand, band B cross-reacted with monoclonal antibody against a seed lectin from the same bean, but band A did not.

The resolution of oligosaccharides by high pH anion exchange chromatography

The natural heterogeneity of the oligosaccharide chains of glycoproteins requires that chromatography be an essential part of structural elucidation. Until recently, the rapid analysis of underivatised carbohydrates by HPLC was hampered, due to the extensive structural heterogeneity, by the absence of a chromatographic medium for satisfactory separation and by the lack of a sufficiently sensitive detection system. This problem has been overcome by the introduction of high pH anion exchange chromatography (HPAEC) with pulsed electrochemical detection (PED) which separates oligosaccharides as their oxyanions on micropellicular quaternary ammonium resins [1-10]. This technique resolves the linkage and branch isomers of neutral and anionic oligosaccharides to picomole sensitivity without pre- or post-column derivatisation. The DX-500 chromatography system is viewed as a major innovation in HPAEC-PED due to an improved sensitivity and resolution as a result of a new amperometric cell design and new digital detector signal processing. Our investigations have utilised the DX-500 in the analysis of various oligosaccharide mixtures in direct comparison with the DX-300 system that it has superceded.

Chemo-Enzymatic Synthesis of a Glycopolymer Carrying Clustered-N-ACETYL-β-lactosamine Moieties

Abstract A polyacrylamide derivative having a β-linked N-acetyllactosamine moiety, a major component of oligosaccharide chains of glycoproteins, on each repeating unit was synthesized via a chemo-enzymatic process. p-Nitrophenyl N-acetyl-β-lactosaminide was prepared by an one-step enzymatic synthesis, using a β-galactosidase as catalyst, from p-nitrophenyl N-acetyl-β-d-glucosaminide as the glycosyl acceptor and lactose as the glycosyl donor. The nitro group was reduced to an amino function, which was then allowed to react with either acryloyl chloride or acrylic acid. The resulting p-acryloylaminophenyl N-acetyl-β-lactosaminide was polymerized with azobisisobutyronitrile as initiator in dimethyl sulfoxide to give a homopolymer with a number-average molecular weight ( M n ) of 3.2 × 105. High molecular-weight polymers carrying α-lactose, N-acetyl-β-d-glucosamine, and α-d-glucopyranose were also synthesized as reference polymers in similar manners. Solution properties of these poly-p-acryloylaminophenyl der...

Artificial Glycoconjugate Polymers

Well-defined polymers having pendant oligosaccharide chains as the recognition signals, termed “artificial glycoconjugate polymers”, are described. The first part of this review treats the applications of oligosaccharide-substituted polystyrenes to cell-specific biomedical materials using the oligosaccharides as the key participants in cell-substratum recognitions. Lactose-carrying polystyrene abbreviated as PVLA is a useful substratum for culture of parenchymal liver cells (hepatocytes). Owing to the hydrophilic-hydrophobic structure, PVLA formed compactpacked (super molecular) conformations in water and were adsorbed on the surface of polystyrene culture dishes. As the result, highly-concentrated galactose signals protruded from the material surfaces to interact strongly with asialoglycoprotein receptors of the cell surfaces. Cell-culture systems using the strong interactions between pancreatic cells and maltose-carrying polystyrene and between erythrocytes and a reducing glucose-carrying polystyrene are also of interest.The second part is concerned with our synthetic approaches to four different types of artificial glycoconjugate polymers having highly-concentrated carbohydrate signals. (1) Chemoenzymatic synthesis of polyacrylamide derivatives, especially, having β-linked N-acetyllactosamine moiety, a major component of oligosaccharide chains of glycoproteins. (2) Multi-branched graft copolymers. (3) Branched (1→6) -α-mannan polysaccharide having pendant α-mannose units. (4) Oligosaccharide-substiuted poly (γ-glutamate).

Chemical structure and conformational features of cell-wall polysaccharides isolated from Aphanoascus mephitalus and related species

The structures of cell-wall mannans isolated from Aphamoascus mephitalus, A. Fulvescens, A. verrucosus, and A. reticulisporus have been investigated by chemical analyses and 1D and 2D 1H and 13C NMR techniques. It was found that all of them consists of a relatively simple comb-like structure of the disaccharide repeating block {→ 6)-[α-Man p-(1 → 2)]-α-Man p-(1 →}. The conformations around the α-(1 → 2) and α-(1 → 6) linkages in thes kinds of polymers were also studied by using molecular mechanics and dynamics calculations, together with NOE data. The results are similar to those found within the oligosaccharide chains of glycoproteins, with a well-defined conformation for the α-(1 → 2) linkage and a certain restriction around the α-(1 → 6) bonding imposed by the 2-substitution.

Analysis of carbohydrates in glycoproteins by high-performance liquid chromatography and high-performance capillary electrophoresis

We describe two methods for the analysis of oligosaccharide chains in glycoproteins by high-performance liquid chromatography (HPLC) and high-performance capillary electrophoresis (HPCE). O- and N-glycosidically linked oligosaccharides released from glycoproteins can be identified as their borohydride-reduced forms by anion-exchange HPLC with pulsed amperometric detection. N-Glycosidically linked oligosaccharides can also be analyzed as 2-aminopyridine derivatives by HPCE in direct zone electrophoresis mode in an acidic phosphate buffer and zone electrophoresis mode as borate complexes in an alkaline buffer. We also present a convenient procedure for the analysis of the constituent monosaccharides of these oligosaccharides chains by HPLC based on reversed-phase partition mode as 1-phenyl-3-methyl-5-pyrazolone derivatives.

Structural and functional characterization of mutants of recombinant single-chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys158, Ile159 and Ile160.

Single-chain urokinase-type plasminogen activator (scu-PA) is converted to urokinase by hydrolysis of the Lys158-Ile159 peptide bond. Site-directed mutagenesis of Lys158 to Gly or Glu yields plasmin-resistant mutants with a 10-20-fold reduced catalytic efficiency for the activation of plasminogen [Nelles et al. (1987) J. Biol. Chem. 262, 5682-5689]. In the present study, we have further evaluated the enzymatic properties of derivatives of recombinant scu-PA (rscu-PA), produced by site-directed mutagenesis of Lys158, Ile159 or Ile160, in order to obtain additional information on the structure/function relations underlying the enzymatic properties of the single- and two-chain u-PA moieties. [Arg158]rscu-PA (rscu-PA with Lys158 substituted with Arg) appeared to be indistinguishable from wild-type rscu-PA with respect to plasminogen-activating potential (catalytic efficiency k2/Km = 0.21 mM-1 s-1 versus 0.64 mM-1 s-1), conversion to active two-chain urokinase by plasmin (k2/Km = 0.13 microM-1 s-1 versus 0.28 microM-1 s-1), as well as its specific activity (48,000 IU/mg as compared to 60,000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 micrograms/ml versus 2.1 micrograms/ml). [Pro159]rscu-PA (Ile159 substituted with Pro) and [Gly159]rscu-PA (Ile159 converted to Gly) are virtually inactive towards plasminogen (k2/Km less than 0.004 mM-1 s-1). They are however converted to inactive two-chain derivatives by plasmin following cleavage of the Arg156-Phe157 peptide bond in [Pro159]rscu-PA and of the Lys158-Gly159 peptide bond in [Gly159]rscu-PA. [Gly158,Lys160]rscu-PA (with Lys158 converted to Gly and Ile160 to Lys) has a low catalytic efficiency towards plasminogen both as a single-chain form (k2/Km = 0.012 mM-1 s-1) and as the two-chain derivative (k2/Km = 0.13 mM-1 s-1) generated by cleavage of both the Arg156-Phe157 and/or the Lys160-Gly161 peptide bonds by plasmin. These findings suggest that the enzymatic properties of rscu-PA are critically dependent on the amino acids in position 158 (requirement for Arg or Lys) and position 159 (requirement for Ile). Conversion of the basic amino acid in position 158 results in a 10-20-fold reduction of the catalytic efficiency of the single-chain molecule but yields a fully active two-chain derivative. The presence of Ile in position 159 is not only a primary determinant for the activity of the two-chain derivative, but also of the single-chain precursor. Cleavage of the Arg156-Phe157 or the Lys160-Gly161 peptide bonds by plasmin yields inactive two-chain derivatives.

Structures of the sialylated oligosaccharide chains in swine tracheal mucin glycoproteins.

The structures of the major sialylated oligosaccharide chains in swine tracheal mucin glycoprotein were established. The oligosaccharide chains were released by treatment with alkaline borohydride and isolated by gel filtration on Bio-Gel P6 columns and chromatography on DEAE-cellulose. The neutral oligosaccharide chains in this glycoprotein have been characterized in previous studies (Rana, S.S., Chandrasekaran, E.V., Kennedy, J., and Mendicino, J. (1984) J. Biol. Chem. 259, 12899-12907; Chandrasekaran, E.V., Rana, S.S., Davila, M., and Mendicino, J. (1984) J. Biol. Chem. 259, 12908-12914). The present study reports the isolation of four monosialylated chains ranging in length from 6 to 14 sugar units, two disialylated chains containing 6 and 12 sugar units, and one trisialylated chain containing 9 sugar units. The structure of the sialylated oligosaccharides was determined by permethylation analysis and sequential hydrolysis with specific exoglycosidases. The following structures (where GalNAcol is N-acetylgalactosaminitol) were assigned to these oligosaccharides.

Immunoblotting analysis of plasma protein processing in the secretory pathway of rat liver: identification of proteolytic conversion sites of complement pro-C3 and prohaptoglobin.

Using an immunoblotting technique, we have studied the processing of plasma proteins in subcellular fractions of rat liver including rough and smooth microsomes and the Golgi subfractions. Each subcellular fraction was directly subjected to SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting with antibodies against alpha 1-protease inhibitor, haptoglobin, and the third component of complement (C3) in combination with 125I-protein A or 125I-rabbit anti-(goat IgG)-IgG. The results demonstrated that proteolytic processing of precursors of complement C3 and haptoglobin occurs in different compartments along the secretory pathway; conversion of prohaptoglobin takes place in the endoplasmic reticulum, while that of pro-C3 occurs in the Golgi complex. The processing in oligosaccharide chains of glycoproteins was also analyzed. The Golgi fraction was characterized by the presence of the mature 56 kDa alpha 1-protease inhibitor, which was indistinguishable from the serum alpha 1-protease inhibitor in SDS-polyacrylamide gel electrophoresis. In contrast, the immature 51 kDa form was the only form of alpha 1-protease inhibitor found in the microsomal fraction. Similar results were obtained for the beta subunit of haptoglobin; the immature 33 kDa form was detected in the microsomal fraction, while the mature 36 kDa form was found in the Golgi fraction. Taken together, these results identified the intracellular sites where these plasma proteins are modified by selective proteolysis and/or glycosylation.

Advances in Selective Chemical Syntheses of Complex Oligosaccharides

AbstractA wide variety of complex oligosaccharides has now been made accessible as a result of methodological improvements in the sphere of chemical synthesis, which can be used for the study of conformations and interactions with protein‐receptor molecules. Such work is of particular importance because the oligosaccharide chains of glycoproteins and glycolipids anchored to the plasma membrane are important in cell‐cell interactions and are receptors for enzymes, hormones, proteins, and viruses; furthermore, they determine the antigen properties of cells. The methods of synthesis available for the production of selective linkages in oligosaccharides are dealt with, particular attention being paid to conversion of saccharides into oligosaccharides, which are important from the biological point of view.

Modification of cell membrane in variants of chinese hamster cells resistant to abrin

Stable and heritable variants of Chinese hamster ovary (CHO) cells which are resistant to different levels (0.1, 1.0 and 10 μg/ml) of the toxin abrin have been isolated and characterized. The frequency of resistant colonies to abrin was increased with the concentration of a chemical mutagen. There was no effect of cell density or cross-feeding on the recovery of variants. In experiments using fluorescein-labeled abrin and ricin which bind to terminal (non-sialylated) galactose residues of cell-surface oligosaccharides, parental cells exhibited strong binding toward both toxins, whereas no fluorescence was observed in the resistant clones. A fluorescein-conjugated lectin, BS II, which is specific for terminal N-acetyl- d-glucosaminyl residues, did not interact with the parental cells, but did with the resistant clones. This suggests that on the surface of resistant cells the number of terminal galactosyl residues of oligosaccharide chains in glycoproteins was reduced, exposing the penultimate N-acetyl- d-glucosaminyl residues. The number of available endogenous acceptor sites for galactosyl transferase in the abrin-resistant clones was directly proportional to the degree of resistance. In the presence of great excess of exogenous acceptor, the rates of galactosyl transfer were similar in all the abrin-resistant cell types tested, with levels ranging from 1.4 to 1.7 times parental cell values. Studies with tetraploid cell hybrids reveal that resistance was a recessive trait. Fluctuation analysis showed that abrin resistance occurred in CHO cell populations at a rate of 4−7 × 10 −8/cell/generation. The system may serve as a new marker for quantitative mutagenesis studies.