Structure Of Carbohydrate Chains Research Articles

Experimental and computational characterization of dynamic biomolecular interaction systems involving glycolipid glycans.

On cell surfaces, carbohydrate chains that modify proteins and lipids mediate various biological functions, which are exerted not only through carbohydrate-protein interactions but also through carbohydrate-carbohydrate interactions. These glycans exhibit considerable degrees of conformational variability and often form clusters providing multiple binding sites. The integration of nuclear magnetic resonance spectroscopy and molecular dynamics simulation has made it possible to delineate the dynamical structures of carbohydrate chains. This approach has facilitated the remodeling of oligosaccharide conformational space in the prebound state to improve protein-binding affinity and has been applied to visualize dynamic carbohydrate-carbohydrate interactions that control glycoprotein-glycoprotein complex formation. Functional glycoclusters have been characterized by experimental and computational approaches applied to various model membranes and artificial self-assembling systems. This line of investigation has provided dynamic views of molecular assembling on glycoclusters, giving mechanistic insights into physiological and pathological molecular events on cell surfaces as well as clues for the design and creation of molecular systems exerting improved glycofunctions. Further development and accumulation of such studies will allow detailed understanding and artificial control of the "glycosynapse" foreseen by Dr. Sen-itiroh Hakomori.

Various biological functions of carbohydrate chains learned from glycosyltransferase-deficient mice

Carbohydrate chains are attached to various proteins and lipids and modify their functions. The complex structures of carbohydrate chains, which have various biological functions, are involved not only in regulating protein conformation, transport, and stability but also in cell–cell and cell–matrix interactions. These functional carbohydrate structures are designated as “glyco-codes.” Carbohydrate chains are constructed through complex reactions of glycosyltransferases, glycosidases, nucleotide sugars, and protein and lipid substrates in a cell. To elucidate the functions of carbohydrate chains, I and my colleagues generated and characterized knockout (KO) mice of galactosyltransferase family genes. In this review, I introduce our studies about galactosyltransferase family genes together with related studies performed by other researchers, which I presented in my award lecture for the Ando-Tajima Prize of the Japanese Association for Laboratory Animal Science (JALAS) in 2019.

Differences of glycocalyx composition in the structural elements of placenta in preeclampsia

IntroductionGlycans expressed in the fetal-maternal interface were shown to exert immunomodulating effects and to mediate interactions between the cells. The aim of this study was to investigate alterations in the structure of carbohydrate chains of glycocalyx in placental tissue in pregnancies complicated with preeclampsia (PE). MethodsA histochemical analysis of placental tissues was performed with a panel of biotinylated lectins. We analyzed placental tissues in women who had severe or moderate PE and compared them to placentas from women with normal pregnancies. ResultsThere was decreased content of terminal residues of α(2,6)-linked sialic acid (as stained by SNA lectin) in the carbohydrate chains of glycocalyx of the endothelium of placental terminal villi in patients with moderate preeclampsia. The composition of the glycocalyx of syncytiotrophoblast in patients of this group did not differ from the control group. Amount of the glycans with terminal β-Gal- (ECL) and α-mannosyl residues (ConA) in the syncytiotrophoblast and capillary endothelium of the placenta was significantly higher in the group with severe PE compared to the control group. The increased content of sialoglycans with α(2,6)-linked sialic acids residues were discovered in the syncytium, and the decreased content of α(2,3)-linked sialic acids residues – in the endothelium of terminal villi in preeclampsia. DiscussionThe most prominent alteration of the glycocalyx composition was found in the placentas of women with severe preeclampsia. It is likely that the modified glycome of syncytiotrophoblast and capillary endothelium may play an important role in pathogenesis of preeclampsia.

Structure of carbohydrate chains of fucolectin from the bark of golden rain shrub Laburnum anagyroides

A reductive LiBH4-ButOH cleavage of N-glycosylamide carbohydrate-peptide bond allowed splitting off of oligosaccharide chains of the fucolectin, the bark agglutinin from the shrub golden rain Laburnum anagyroides (LABA). Four N-glycans were isolated by HPLC, and their structures were elucidated by monosaccharide analysis and 1H NMR (500 MHz) spectroscopy: Man2Fuc1XyllGlcNAc2 (M2FX), Man3XyllGlcNAc2 (M3X), Man3FuclXyllGlcNAc2 (M3FX), and Man3XyllFucIGlcNAc3 (NM3FX). All the N-glycans contain D-xylose and three of them, L-fucose; they were found to be in a 1 : 8 : 1 : 3 ratio.

Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) fusion protein, a novel immunosuppressive agent, was expressed in transgenic rice cell suspension culture and its characteristics and in vitro activities were investigated. The expression vector pMYN409 was constructed to express hCTLA4Ig under the control of rice α-amylase 3D (RAmy3D) promoter. Transgenic calli were prepared by particle bombardment mediated transformation and were screened for hCTLA4Ig expression using ELISA. Under the induction condition by sugar starvation, suspension-cultured rice cells secreted hCTLA4Ig into the media up to 31.4 mg/L in flask culture. The rice-derived hCTLA4Ig (hCTLA4Ig P) was purified from the culture media with affinity chromatography using protein A and compared with CHO-derived hCTLA4Ig (hCTLA4Ig M). Recombinant hCTLA4Ig P has molecular weight of ∼50 kDa on SDS–PAGE under reducing condition, which is a little different from that of hCTLA4Ig M probably due to the difference of carbohydrate chain structures. Purified hCTLA4Ig P was biologically active and was confirmed to suppress T-cell proliferation.

Pituitary–ovarian axis during lactational amenorrhoea. II. Longitudinal assessment of serum FSH polymorphism before and after recovery of menstrual cycles

The association of normal serum levels of immunoassayable gonadotrophins with anovulation during lactational amenorrhoea (LA) has not been fully explained. Serum FSH polymorphism was analysed in 10 women during LA between days 60 and 70 post-partum and again, in the mid-follicular phase (MFP), after resuming menstrual cyclicity. FSH microheterogeneity was characterized according to charge, using preparative isoelectric focusing, and according to the inner structure of carbohydrate chains, using lectin chromatography. A significantly higher proportion of FSH charge isoforms isolated below pH 4.10 and a lower proportion of FSH isoforms bearing highly branched oligosaccharides were observed during LA when compared to MFP. Further analysis with higher resolution showed that FSH charge isoforms, isolated in the lower pH range in LA, corresponded to FSH molecules bearing highly branched and biantennary oligosaccharides. FSH isoforms bearing hybrid-type oligosaccharides were only present during LA. The circulating FSH isoform mix was significantly less bioactive in LA than in MFP. LA is characterized by a more acidic mix of FSH isoforms, containing hormone bearing less processed oligosaccharides, with decreased biopotency in comparison with the follicular phase. This FSH microheterogeneity may be one of the critical factors contributing to incomplete follicular development and anovulation during LA.

Preventive and Therapeutic Effects of α1-Acid Glycoprotein in Mice Infected with B. anthracis

We studied the effects of alpha1-acid glycoprotein preparations on the survival rate of BALB/c mice infected with the lethal dose of B. anthracis STI-1. Apart from native alpha1-acid glycoprotein from donor blood, we studied 3 glycoforms differing in the affinity for concanavalin A and structure of carbohydrate chains. The protective effect of alpha1-acid glycoprotein preparations did not depend on its dose and was observed 3 months after treatment (0.3 mg per mouse). The protective effect was revealed in mice receiving alpha1-acid glycoprotein preparations 2 h before infection and 24 h after inoculation of the bacterial culture. In the latter case the survival rate of animals was much higher compared to that observed in preventive administration of alpha1-acid glycoprotein. The protective effect practically did not depend on the time of treatment with glycoforms. Pretreatment with alpha1-acid glycoprotein preparations significantly decreased plasma interferon-gamma concentration. Administration of the test preparations 24 h after infection decreased the concentration of tumor necrosis factor-alpha.

Automated structural assignment of derivatized complex N-linked oligosaccharides from tandem mass spectra.

Glycoprotein function is controlled by several biological factors, one of them being the structure of carbohydrate chains (glycans) attached to specific amino acids of the protein backbone. Changes in glycan structures have been shown to modify the secondary and tertiary conformation of glycoproteins, thus their function. Powerful analytical tools are available for the characterization of sugar structures, and recently mass spectrometry (MS) has been increasingly useful for this purpose. Manual interpretation of tandem mass spectrum is possible but tedious. Automated interpretation should speed the analysis and enhance the results obtained. A new computer program for automated interpretation of tandem MS spectra of complex N-linked glycans oligosaccharides from mammals will be described. N-Linked oligosaccharides standards were derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) and analyzed by matrix-assisted laser desorption/ionization (MALDI)-tandem MS. Simulated tandem mass spectra of other common glycans were also generated to test the algorithm. The MALDI-MS/MS spectra featured resolved isotopic distributions for the [M + H](+) and fragment ions of oligosaccharides. These isotopic distributions complicated the automated analysis of the spectra and were removed to leave only monoisotopic peaks. An algorithm was written for this purpose, yielding simplified tandem mass spectra. Another algorithm is then used to determine the structure of the oligosaccharide. A score is then given to each structure, depending on agreement with experimental results. The program successfully assigned the true structure in 24 out of the 28 cases (86%) and the true structure was among the three top scoring structures in all cases.

Extrahepatic biliary atresia: A disease or a phenotype?

Extrahepatic biliary atresia: A disease or a phenotype?

Endoglycosidase and Glycoamidase Release of N‐Linked Oligosaccharides

AbstractCarbohydrate chain modifications are often used to monitor glycoprotein movement through the secretory pathway. This is because stepwise sugar‐chain processing is unidirectional and generally corresponds to the forward or anterograde movement of proteins. This unit presents a group of techniques for analysis of the general structure of carbohydrate chains on a protein and, therefore, oligosaccharide processing mileposts. It has been revised from a previous version to include endoglycosidase H, D, F2, and F3 digestions, as well as peptide:N‐glycosidase F (PNGase F), neuraminidase, endo‐β‐galactosidase, and endo‐α‐N‐acetylgalactosaminidase digestions.

細胞膜表面に関わる構造と機能 細胞膜表面糖鎖による細胞間コミュニケーション

All mammalian cells have “glycocalix” on their surface, which comprises glycolipids, glycoproteins, and proteoglycans. Structures of carbohydrate chains in the glycocalix are cell type specific, and are known to change dramatically during the process of cellular activation or differentiation. Thus, these carbohydrates have been regarded to be potential signaling molecules to show what kind of cells are present in the cellular society and which differentiation stages they are. In this context, it has been hypothesized that there must be “glyco-receptors”, which receive the signals and mediate cellular communications. Only a few cases, however, have been identified so far, and a little is known about how they mediate cellular communications. In the present review, we would like to summarize the results of these “glyco-receptor” researches and discuss the future of this research field.

Endoglycosidase and Glycoamidase Release of N‐Linked Oligosaccharides

AbstractCarbohydrate chain modifications are often used to monitor glycoprotein movement through the secretory pathway. This is because stepwise sugar‐chain processing is unidirectional and generally corresponds to the forward or anterograde movement of proteins. This unit offers a group of techniques that will help analyze the general structure of carbohydrate chains on a protein and, therefore, oligosaccharide processing mileposts. The sugar chains themselves are not analyzed, but their presence and structure are inferred from gel mobility differences after one or more enzymatic digestions. This approach is most often used in combination with [35S]Met pulse‐chase metabolic labeling protocols, but they can be applied to any suitably labeled protein (e.g., biotinylated or 125I‐labeled).

On-target exoglycosidase digestions/MALDI-MS for determining the primary structures of carbohydrate chains.

One method used to determine the primary sequence of oligosaccharides is to digest them with exoglycosidases and analyze the resulting digestion products by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Previous research has demonstrated that these digestions can be performed on the MALDI target. However, the procedure requires the sample to be incubated at elevated temperatures, and complete digestion requires a few hours. We demonstrate new conditions that permit exoglycosidase digestions to be performed on the MALDI target at room temperature within 30 min. Oligosaccharide standards were digested with one or more exoglycosidases to show that the enzymes retain their activity and specificity under these new reaction conditions. Using this method, the primary sequences of carbohydrate chains can be determined in a relatively short amount of time.

Characterization and representative structures of N-oligosaccharides bound to apolipoprotein H

We studied the structure of N-linked carbohydrates bound to apolipoprotein H by a combination of two methods which make use of lectins. Digoxigenin-labelled lectins are used for the structural characterization of carbohydrate chains of glycoproteins. Concanavalin A lectin affinity chromatography was used to analyse apolipoprotein H according to the characteristics of its carbohydrate chain inner to sialic acid residues. Our results from digoxigenin-labelled lectins analysis showed that apolipoprotein H gave positive bands to SNA, DSA, GNA, PNA and AAA lectins. Apolipoprotein H gave a negative band when reacted with MAA lectin. When we applied apolipoprotein H onto the Concanavalin A lectin column no detectable amounts of protein were eluted with Concanavalin A buffer. After adding a buffer with low sugar concentration (10 mM glucoside) a large amount of apolipoprotein H was recovered. These molecules of apolipoprotein H weakly bound to the lectin. When a higher sugar concentration (500 mM mannoside) was added most of the sample applied was eluted. These molecules of apolipoprotein H firmly bound to the column having high affinity for the lectin. These results combined with those coming from the digoxigen-labeled lectins method enable us to understand the inner structure of carbohydrate chains with their outer branches. Molecules of apolipoprotein H which weakly bind to Concanavalin A could bear complex N-glycans organized in biantennary or truncated hybrid structures. Firmly bound apolipoprotein H referred to molecules rich in N-glycan hybrid structures. They have an outer branch belonging to the high mannose carbohydrate chains which explain the ability to bind to the column and an other main branch bearing the sequence galactose β-(1-4)- N-acetylglucosamine β-(1-2) mannose. Galactose could be the terminal sugar or, alternatively, be masked with sialic acid α-(2-6) terminally linked.

A challenge to the riddle of the carbohydrate chain

Abstract

Molecular biology and biochemistry of human recombinant follicle stimulating hormone (Puregon).

Follicle stimulating hormone (FSH) is a heterodimeric glycoprotein hormone produced in the anterior pituitary gland. The hormone is essential in the regulation of reproductive processes, such as follicular development and ovulation. It is clinically used for treatment of anovulation and in assisted reproduction technologies such as in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Until recently, the only source for human FSH has been the urine from post-menopausal women. Such a natural source implies limited availability and potential product variability. Thus, we have cloned the genes encoding the alpha- and beta-subunits of human FSH and transfected these into Chinese hamster ovary (CHO) cells. A CHO-clone was isolated capable of secreting intact glycosylated FSH with identical amino acid sequences to natural FSH. This cell line was grown in perfusion culture and enabled us to isolate highly pure FSH (> 99%). The complexity of the charge distribution of human recombinant FSH was demonstrated by Isoelectric focusing. The observed microheterogeneity is caused by the large number of carbohydrate chain structures which are added to the four potential glycosylation sites in the alpha beta-dimer. Furthermore, the carbohydrates show a variation in their degree of sialylation which reflects the different pl values of the individual isohormones. Despite the complexity of post-translational modification, the isoform distribution of recombinant FSH produced in a CHO-cell line and grown in perfusion culture is surprisingly similar to that observed with pituitary FSH and urinary FSH. In conclusion, we have shown that FSH-gene transfected CHO-cells are capable of stable serum-free production of recombinant FSH. A process has been developed which assures the consistent and reproducible production of highly-purified recombinant FSH.

Carbohydrate structures of recombmant soluble lamp-1 and leukosialin containing sialyl Le x terminus

Recombinant soluble lamp-1 and soluble leukosialin can be produced from CHO cells which express sialyl Le x structures after stable transfection of fucosyltransferase-III. It was shown previously that those soluble lamp-1 and leukosialin are potent inhibitors for E-selectin-mediated adhesion of human colonic tumor cells (Sawada, R.; Tsuboi, S.; Fukuda, M. J. Biol. Chem., 1994, 269, 1425). In the present study, we have determined the amount of the sialyl Le x structure present in recombinant, soluble lamp-1 and soluble leukosialin. CHO cells were metabolically labeled with [ 3HJ-galactose and recombinant soluble lamp-1 and leukosialin were purified from the spent medium. Glycopeptides containing N-glycans derived from lamp-1 were fractionated by sequential lectin affinity chromatography. Similarly, O-glycans released from leukosialin were fractionated by Bio-Gel P-4 gel filtration. The terminal structures of carbohydrate chains were determined by sequential digestion with specific glycosidases. The results clearly indicate that soluble lamp-1 contains much more sialyl Le x structure than soluble leukosialin. Considering that soluble leukosialin and lamp-1 are almost equally effective as inhibitors for E-selectin-mediated adhesion, the results strongly suggest that densely clustered O-glycans are better presenters for E-selectin ligands than N-glycans.

Profiles of expression of carbohydrate chain structures during human intrahepatic bile duct development and maturation: A lectin-histochemical and immunohistochemical study

We investigated expression of carbohydrate chain structures during human intrahepatic bile duct development and maturation. Results of staining for ABH antigens were negative in the ductal plate and biliary cells migrating into the mesenchyma but were positive in newly formed bile ducts in the fetal liver and in maturing bile ducts in the postnatal liver. Staining for Lewis(a), sialyl Lewis(a) and Lewisb antigens was negative in the ductal plate but positive in migrating biliary cells, newly formed ducts and maturing ducts. Staining for sialyl Lewisx antigen was positive in the ductal plate, migrating biliary cells and newly formed ducts but negative in the postnatal liver. Lewis(y) antigen staining was negative in the ductal plate and migrating biliary cells but positive in newly formed ducts and maturing ducts. Staining for T and Tn antigens was negative in the ductal plate, migrating biliary cells and newly formed ducts but positive in maturing ducts. Staining for Lewisx, sialyl Tn and carcinoembryonic antigen was negative in all tissues. Receptors of Ulex europaeus agglutinin I and soybean agglutinin were absent in the ductal plate, migrating biliary cells and newly formed ducts but present in maturing large ducts. Staining for receptors of concanavalin A and Ricnus communis agglutinin I was negative in the ductal plate but positive in migrating biliary cells, newly formed ducts and maturing ducts. Staining for the receptor of succinylated wheat germ agglutinin was always positive. These findings suggest that carbohydrate chain structures change during intrahepatic bile duct development and maturation and that new carbohydrate chain structures gradually emerge as intrahepatic bile ducts develop and mature.

Profiles of expression of carbohydrate chain structures during human intrahepatic bile duct development and maturation: A lectin-histochemical and immunohistochemical study

Profiles of expression of carbohydrate chain structures during human intrahepatic bile duct development and maturation: A lectin-histochemical and immunohistochemical study

S9.27 The structure of carbohydrate chains of bean storage protein from phaseolus vulgaris

S9.27 The structure of carbohydrate chains of bean storage protein from phaseolus vulgaris