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1.
Glycosylation is a common post-translational modification of proteins. Although its significance in biological system is well recognized, approaches to analyze carbohydrate function are limited. This is because of difficulty in obtaining homogeneous glycoproteins from natural sources. Due to the progress of the carbohydrate and peptide chemistry, syntheses of various homogeneous glycopeptides and glycoproteins, which are suitable for biological studies, have been achieved by chemical means. In this review, we briefly summarize recent advances in the field of glycopeptide synthesis after 1999. 相似文献
2.
Sugar residues on proteins 总被引:16,自引:0,他引:16
Glycoproteins have become increasingly important in the structure and function of many different mammalian systems; for example, membrane glycoproteins and glycoprotein hormones. It is, therefore, important to understand their chemistry, which would include an understanding of both the carbohydrate and protein parts of the molecule. Since the chemical characterization of the protein moiety has been extensively examined and the techniques for its characterization are well worked out, only the carbohydrate portion of glycoproteins will be reviewed in this article. The chemical nature of the carbohydrate moiety of glycoproteins will be examined. First, the types of monosaccharides present in animal systems, especially those in the mammalian systems, will be described. Next, various types of simple and complex carbohydrate chains will be discussed to establish the diversity, size, and number of chains present in the carbohydrate units in different glycoproteins. Then, the type of linkages of the carbohydrate to the protein will be examined to determine if the primary sequence of protein is important in determining the size and type of carbohydrate chains present in glycoproteins. Finally, the current methods of structural elucidation such as monosaccharide sequence, intersugar bonds, and anomeric linkages in the carbohydrate moiety of glycoproteins will be reviewed. These methods include the techniques of periodate oxidation, methylation, partial acid hydrolysis, and specific glycosidase digestion of glycoproteins, as well as the latest techniques using micromethods of carbohydrate quantitation and characterization involving gas chromatography and mass spectrometry. The function of the carbohydrate in glycoproteins will also be considered. First, hormone glycoproteins will be discussed in their relationship to the immunological and biological function of the glycoprotein when the carbohydrate is sequentially removed. Next, the function of the carbohydrate in the turnover of glycoproteins will be discussed. These topics will be considered in order to develop an understanding of a specific function(s) of the carbohydrate in glycoproteins. 相似文献
3.
Uma D. Palanisamy Abid Hussain Sayeedha Iqbal Kenny Sproule Christopher R. Lowe 《Journal of molecular recognition : JMR》1999,12(1):57-66
The concepts of rational design and solid phase combinatorial chemistry were used to develop affinity adsorbents for glycoproteins. A detailed assessment of protein–carbohydrate interactions was used to identify key residues that determine monosaccharide specificity, which were subsequently exploited as the basis for the synthesis of a library of glycoprotein binding ligands. The ligands were synthesised using solid phase combinatorial chemistry and were assessed for their sugar‐binding ability with the glycoenzymes, glucose oxidase and RNase B. Partial and completely deglycosylated enzymes were used as controls. The triazine‐based ligand, histamine/tryptamine (8/10) was identified as a putative glycoprotein binding ligand, since it displayed particular affinity for glucose oxidase and other mannosylated glycoproteins. Experiments with deglycosylated control proteins, specific eluants and retardation in the presence of competing sugars strongly suggest that the ligand binds the carbohydrate moiety of glucose oxidase rather than the protein itself. Copyright © 1999 John Wiley & Sons, Ltd. 相似文献
4.
Mucin-carbohydrate directed monoclonal antibody 总被引:2,自引:0,他引:2
A Kurosaka S Fukui H Kitagawa H Nakada Y Numata I Funakoshi T Kawasaki I Yamashina 《FEBS letters》1987,215(1):137-139
To raise monoclonal antibodies recognizing cancer-associated alterations of the carbohydrate structure of glycoproteins, Balb/c mice were immunized with human colonic cancer cells (LS 180 from ATCC). One of the generated hybridomas produced a monoclonal antibody that bound to the carbohydrate moiety of mucin-type glycoproteins from LS 180. The antibody did not bind to glycoproteins from another colonic cancer cell line, SW 1116, or to glycolipids from any of the colonic cancer cell lines. The antibody bound to ovine and bovine submaxillary mucins (OSM and BSM). NeuAc alpha 2----6Ga1NAc seemed to be involved in the epitope. 相似文献
5.
Two alpha-macroglobulins were isolated from the plasma of the frog. Murine clearance studies were performed with these proteins after they were reacted with proteinase. These studies indicated that clearance behavior was more complex than observed with avian or human homologues, since it involved not only specific receptors for the complex, but also carbohydrate recognition. Recognition of one of these macroglobulins by both the alpha-macroglobulin receptor and carbohydrate receptors required conformational change in the inhibitor. Clearance studies were then performed in frogs to probe the nature of carbohydrate receptor recognition. Model glycoproteins were employed to avoid the problem of heterogeneous carbohydrate end groups in the alpha-macroglobulins. These studies demonstrated that the N-acetylglucosamine/mannose receptor is the major carbohydrate recognition system in frogs. 相似文献
6.
A strategy is developed in this study for identifying sialylated glycoprotein markers in human cancer serum. This method consists of three steps: lectin affinity selection, a liquid separation and characterization of the glycoprotein markers using mass spectrometry. In this work, we use three different lectins (Wheat Germ Agglutinin, (WGA) Elderberry lectin,(SNA), Maackia amurensis lectin, (MAL)) to extract sialylated glycoproteins from normal and cancer serum. Twelve highly abundant proteins are depleted from the serum using an IgY-12 antibody column. The use of the different lectin columns allows one to monitor the distribution of alpha(2,3) and alpha(2,6) linkage type sialylation in cancer serum vs that in normal samples. Extracted glycoproteins are fractionated using NPS-RP-HPLC followed by SDS-PAGE. Target glycoproteins are characterized further using mass spectrometry to elucidate the carbohydrate structure and glycosylation site. We applied this approach to the analysis of sialylated glycoproteins in pancreatic cancer serum. Approximately 130 sialylated glycoproteins are identified using microLC-MS/MS. Sialylated plasma protease C1 inhibitor is identified to be down-regulated in cancer serum. Changes in glycosylation sites in cancer serum are also observed by glycopeptide mapping using microLC-ESI-TOF-MS where the N83 glycosylation of alpha1-antitrypsin is down regulated. In addition, the glycan structures of the altered proteins are assigned using MALDI-QIT-MS. This strategy offers the ability to quantitatively analyze changes in glycoprotein abundance and detect the extent of glycosylation alteration as well as the carbohydrate structure that correlate with cancer. 相似文献
7.
Recent investigations have implicated aberrant glycosylations in various malignancies, including epithelial ovarian cancer (EOC). The protocol here identifies O-linked carbohydrate patterns in EOC plasma glycoproteins through chemical cleavage and purification of these glycans. Dialyzed plasma is subjected to reductive beta-elimination with alkaline borohydride to release O-linked oligosaccharides from glycoproteins. Enrichment of released glycans, as well as removal of peptide and other contaminants, is followed by carbohydrate pattern analysis with MALDI-FT-ICR-MS. 相似文献
8.
J P Feugeas D Néel A A Pavia A Laham Y Goussault C Derappe 《Biochimica et biophysica acta》1990,1030(1):60-64
The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity. 相似文献
9.
R. Fischer-Colbrie M. Schachinger R. Zangerle H. Winkler 《Journal of neurochemistry》1982,38(3):725-732
Abstract: Chromogranin A and two other proteins (A1 and A2 ) of the soluble proteins of bovine chromaffin granules were isolated by extraction from polyacrylamide gels after electrophoresis. The carbohydrate content of these proteins was 5%, with galactose, N -acetylgalactosamine, and sialic acid as the main sugars. Membranes of chromaffin granules were solubilized with sodium dodecyl sulphate (SDS) and three glycoproteins were isolated by sequential affinity chromatography on Concanavalin A (Con A) and wheat germ lectin (WGL) Sepharose columns. Two glycoproteins, designated GP II and III, were found to have a high carbohydrate content of about 30%. Mannose, galactose, N -acetylgalactosamine, and sialic acid were the main sugars. In addition membrane-bound dopamine β-hydroxylase was isolated by this procedure. No significant differences between the carbohydrate composition of the membrane-bound and the soluble enzyme were revealed. It was shown that all four subunits of dopamine β-hydroxylase possess carbohydrate chains with an affinity for Con A. The isolation methods established in this study will be useful for immunological studies on these glycoproteins. 相似文献
10.
Govindaswami Ragupathi 《Cancer immunology, immunotherapy : CII》1996,43(3):152-157
Carbohydrate antigens such as GM2, GD2 and GD3 (gangliosides), Lewisy and globo-H (neutral glycolipids and glycoproteins), and Tn, TF and sTn (glycoproteins) are overexpressed in a variety of
cancers. Antibodies against several of these carbohydrate antigens have been detected in sera from patients treated with cancer
vaccines, and have been associated with a more favorable prognosis. Clinical responses have been reported after treatment
with monoclonal antibodies against some of these antigens. Hence cell-surface carbohydrate antigens have been identified as
suitable targets for immune attack by both active and passive immunotherapies. Different approaches have been adopted to induce
immune responses against these carbohydrate antigens. These includes vaccination with whole or lysed tumor cells, purified
or synthetic carbohydrates, immunogenic carbohydrate derivatives, or carbohydrates conjugated with immunogenic carriers and
administered with immunological adjuvants. In the case of gangliosides, immunization with either whole tumor cells or cell
lysates has only occasionally induced responses against carbohydrate antigens, and the antibodies were generally IgM antibodies
of low titer. Compared with other methods of vaccination, conjugate vaccines have consistently induced the highest titer of
IgM and IgG antibodies against gangliosides and other carbohydrate antigens. Preclinical and clinical studies with conjugate
carbohydrate vaccines have induced IgM and IgG antibody responses capable of inducing complement-mediated cytotoxicity of
tumor cells in vitro and associated with prolonged disease-free and overall survival in patients.
Received: 6 August 1996 / Accepted: 20 September 1996 相似文献
11.
D Baeckstr?m G C Hansson O Nilsson C Johansson S J Gendler L Lindholm 《The Journal of biological chemistry》1991,266(32):21537-21547
Two mucin-type glycoproteins detected by the monoclonal antibody C50, which reacts with the carcinoma-associated sialyl-Lewis a and sialyl-lactotetraose epitopes, were found in secreted and solubilized materials from the colon carcinoma cell line COLO 205. The larger glycoprotein (H-CanAg; heavy cancer antigen) was predominantly found in extracts of cells grown in vitro or as nude mice xenografts whereas the smaller species (L-CanAg; light cancer antigen) was the major component in spent culture medium and serum from grafted mice. Using detergent in the extraction buffer doubled the yield of H-CanAg, suggesting that this glycoprotein is membrane bound whereas the yield of L-CanAg was relatively unaffected. The two glycoproteins were purified from xenograft extracts and spent culture medium using perchloric acid precipitation, monoclonal antibody affinity purification, ion exchange chromatography, and gel filtration. Both glycoproteins were unaffected by reduction and alkylation in guanidine HCl. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, relative molecular masses were estimated to be 600-800 kDa for H-CanAg and 150-300 kDa for L-CanAg. Carbohydrate analysis revealed that the CanAg glycoproteins were highly glycosylated (81-89% carbohydrate by weight), carrying carbohydrate chains with average lengths of 13-18 sugars which were rich in fucose and sialic acid (2-3 residues/chain for each sugar). L-CanAg isolated from spent medium was glycosylated to a higher degree than its counterpart from xenograft extract. Immunochemical studies of the intact glycoproteins showed that both H-CanAg and L-CanAg expressed the monoclonal antibody-defined, sialic acid-containing carbohydrate epitopes CA203 and CA242 as well as the Lewis a blood group antigen whereas only H-CanAg appeared to carry the sialyl-Lewis x epitope. The amino acid compositions were typical of mucins, containing high amounts of serine, threonine (more than 25% together), and proline (11-18%). Significant differences in amino acid composition between H-CanAg and L-CanAg were found. A rabbit antiserum against the cytoplasmic C-terminal part of the MUC1 gene product, core protein of the carcinoma-associated polymorphic epithelial mucin (PEM) and DU-PAN-2, reacted with H-CanAg. After deglycosylation with trifluoromethanesulfonic acid, H-CanAg but not L-CanAg was recognized by the monoclonal antibodies SM-3 and HMFG-2, directed to the tandem repeat of the PEM apoprotein. However, these antibodies which react with PEM from mammary carcinomas without prior deglycosylation were unable to recognize intact H-CanAg, probably as a consequence of a more extensive glycosylation of this glycoprotein.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
12.
R. W. STODDART 《Biological reviews of the Cambridge Philosophical Society》1979,54(3):199-234
1. The nucleus has a distinctive carbohydrate chemistry, the main features of which are the lack of glycosphingolipid, the high density of carbohydrate per unit area of nuclear membrane, the presence of glycosaminoglycan in the nuclear matrix and possibly the nuclear membranes, and the existence of glycosylated non-histone proteins. 2. The nucleus has considerable autonomy in its metabolism of glycosaminoglycan and has a capacity for glycosyl transfers involving glycosyl dolichyl phosphates and pyrophosphates. This latter activity probably resides in the nuclear membranes. 3. The soluble fraction of the nucleoplasm contains the total cellular CMP-sialic-acid synthetase and, hence, all sialic acid metabolism passes through the nucleus, which may have a regulatory role. Uncertainty remains as to the sialic acid content of the glycoproteins of the nucleus and it is likely to vary between cell types. 4. Malignancy is associated with several alterations in the glycosylation of nuclear membranes, including increased levels of sialic acids in the glycoproteins of the inner nuclear membrane: changes in glycosylation of the matrix and chromatin are not yet well defined. In malignancy, some nuclear glycoproteins may possibly appear in other cellular membranes. 相似文献
13.
Mucin glycoproteins in neoplasia 总被引:30,自引:0,他引:30
Mucins are high molecular weight glycoproteins that are heavily glycosylated with many oligosaccharide side chains linked O-glycosidically to the protein backbone. With the recent application of molecular biological methods, the structures of apomucins and regulation of mucin genes are beginning to be understood. At least nine human mucin genes have been identified to date. Although a complete protein sequence is known for only three human mucins (MUC1, MUC2, and MUC7), common motifs have been identified in many mucins. The pattern of tissue and cell-specific expression of these mucin genes are emerging, suggesting a distinct role for each member of this diverse mucin gene family. In epithelial cancers, many of the phenotypic markers for pre-malignant and malignant cells have been found on the carbohydrate and peptide moieties of mucin glycoproteins. The expression of carbohydrate antigens appears to be due to modification of peripheral carbohydrate structures and the exposure of inner core region carbohydrates. The expression of some of the sialylated carbohydrate antigens appears to correlate with poor prognosis and increased metastatic potential in some cancers. The exposure of peptide backbone structures of mucin glycoproteins in malignancies appears to be due to abnormal glycosylation during biosynthesis. Dysregulation of tissue and cell-specific expression of mucin genes also occurs in epithelial cancers. At present, the role of mucin glycoproteins in various stages of epithelial cell carcinogenesis (including the preneoplastic state and metastasis), in cancer diagnosis and immunotherapy is under investigation. 相似文献
14.
High-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) is an established technique for the carbohydrate analysis of glycoproteins. HPAE-PAD is routinely used for determinations of monosaccharide, sialic acid, mannose-6-phosphate (M-6-P), and oligosaccharide contents of a glycoprotein. This is true for both the initial investigation of a glycoprotein and routine assays of recombinant therapeutic glycoproteins. This contribution reviews the fundamentals of HPAE-PAD, recent technological improvements, and advances in the last ten years in its application to carbohydrate analysis of glycoproteins. The application areas reviewed include monosaccharide determinations, sialic acid determinations, M-6-P determinations, sugar alcohol determinations, analysis of polysialic acids, neutral and charged oligosaccharide analysis, following glycosidase and glycosyltransferase reactions, and coupling HPAE-PAD to mass spectrometry (MS). 相似文献
15.
Susan A. Brooks 《Molecular biotechnology》2009,43(1):76-88
More than half of human proteins are glycosylated by a bewildering array of complex and heterogeneous N- and O-linked glycans. They function in myriad biological processes, including cell adhesion and signalling and influence the physical
characteristics, stability, function, activity and immunogenicity of soluble glycoproteins. A single protein may be glycosylated
differently to yield heterogenous glycoforms. Glycosylation analysis is of increasing interest in biomedical and biological
research, the pharmaceutical and healthcare industry and biotechnology. This is because it is increasingly apparent that glycosylation
changes in diseases, such as cancer, making it a promising target for development of clinically useful biomarkers and therapeutics.
Furthermore, as the non-human cells employed in expression systems glycosylate their proteins very differently to human cells,
and as glycosylation changes unpredictably under changing environmental conditions, glycans analysis for quality control,
optimum efficacy and safety of recombinant glycoproteins destined for human therapeutic use is paramount. The complexities
of carbohydrate chemistry make analysis challenging and while there are a variety of robust methodologies available for glycan
analysis, there is currently a pressing need for the development of new, streamlined, high throughput approaches accessible
to non-specialist laboratories. 相似文献
16.
Robert B. Trimble Frank Maley 《Biochemical and biophysical research communications》1977,78(3):935-944
Endo-β-N-acetylglucosaminidase H from can be useful in determining both the molecular weight of the protein moiety of glycoproteins and their inherent number of oligosaccharide chains. In the case of carboxypeptidase Y the molecular mass of the carbohydrate free protein was confirmed as 51,000 daltons. The native enzyme was shown to contain 4 oligosaccharide chains each averaging about 14 mannose residues. On treatment of mung bean nuclease I with the endoglycosidase, the molecular mass decreased from 39,000 to 31,000 daltons. The peptides produced on reduction of this enzyme with thiol were 18,700 and 12,500 daltons, indicating that carbohydrate had been present on both. Penicillium nuclease P1 was decreased in size from 40,000 to 30,000 daltons by the endoglycosidase. Although most of the carbohydrate was removed from each of the native enzymes by the endoglycosidase, denaturation of the glycoproteins was necessary to effect complete removal. Enzyme activitywas not affected by carbohydrate depletion of these glycoproteins, a result consistent with similar studies on other oligosaccharide-containing enzymes. 相似文献
17.
The proton nuclear magnetic resonance (1H-NMR) spectra of glycophorin and its tryptic sialoglycopeptides were investigated. From the intensities of the assigned resonances it was concluded that all of the residues in the sialoglycopeptides are sufficiently mobile in conformation to give sharp resonances, while in glycophorin this is true for only approximately 80% of the peptide backbone. The resonances of the central sequence of some 20 of the hydrophobic residues are strongly broadened. This region is probably that of alpha-helical structure which is known to aggregate. The linewidths and intensities of the resonances are not, or only slightly, affected by changing the ionic strength, temperature or by carboxymethylation of the Met-81 residue in glycophorin. Glycophorin was found to bind about 100 mol sodium dodecylsulphate/mol protein as derived from studies on linebroadening of the latter's C-3 to C-11 methylene resonances. The bound dodecyl-sulphate probably increases the mobilities of the hydrophobic residues in the protein as these resonance intensities are increased by the binding. The carbohydrate chains in glycophorin were conformationally mobile; no evidence was found for tight carbohydrate-protein interactions. The relevance of flexible carbohydrate chains in membrane glycoproteins is discussed in relation to cell surface chemistry. 相似文献
18.
The carbohydrate-deficient glycoprotein syndromes are a recentlydelineated group of genetic, multisystemic diseases with majornervous system involvement. Three distinct variants have beenrecognized and there are probably many more. They are characterizedby a deficiency of the carbohydrate moiety of secretory glycoproteins,lysosomal enzymes and probably also membranous glycoproteins.The biochemical changes are most readily observed in serum transferrinand the diagnosis is usually made by isoelectric focusing ofthis glycoprotein. The deficiency of sialic acid, in particular,results in a cathodal shift and hence the presence of abnormalisoforms of transferrin with higher isoelectric points thannormal. The basis defects are probably in the processing andsynthesis of the carbohydrate moiety of glycoproteins; thereis indirect evidence for a deficiency of asparagine-N-linkedoligosaccharide transfer in type I (endoplasmic reticulum defect)and for a deficiency of N-acetylglucosaminyltransferase II intype II (Golgi defect). From the large number of patients detectedin only a few years, it is expected that these diseases willbecome as important as, for example, the lysosomal, peroxisomalor mitochondrial disorders. Their study will undoubtedly yielda wealth of new information on the function of glycoproteinsand of their carbohydrate moiety. endoplasmic reticulum glycoproteins glycosylation Golgi sialotransferrins 相似文献
19.
Colony-stimulating factors (CSFs) are a group of acidic glycoproteins which stimulate the proliferation and differentiation of hematopoietic progenitor cells in vitro and stimulate hemopoiesis in vivo. Human GM-CSF contains two N-linked carbohydrate side chains of the complex acidic type and several sites of O-linked carbohydrate clustered on serine and threonine residues near the N-terminus of the molecule. Previous studies have failed to detect a significant functional role for the carbohydrate modification characteristic of human GM-CSF. Using permanent cell lines and transient expression systems which produce moderate to high levels of native or carbohydrate-deficient forms of the growth factor, the role of carbohydrate modification in the biosynthesis and secretion of GM-CSF was studied. Unlike a number of other secreted glycoproteins, the transient time and secretory efficiency of several carbohydrate-deficient mutants of GM-CSF are indistinguishable from those of the native growth factor in BHK, 293, COS, and ldlD cells. Furthermore, normal human endothelial cells and fibroblasts, which normally produce the growth factor, can synthesize and secrete GM-CSF that lacks all forms of carbohydrate modification. These studies help to point out the range of roles played by carbohydrate modification in the biosynthesis, assembly, and secretion of glycoprotein hormones. 相似文献
20.
The covalent attachment of carbohydrate to proteins is a very common co- or post-translational event in the biosynthesis of glycoproteins. The type and heterogeneity of these oligosaccharides can affect a range of physico-chemical and biological properties of a glycoprotein. Thus the development of sensitive, reliable and robust analytical methods for carbohydrate analysis is important in the pharmaceutical industry, especially in the recombinant production of experimental and therapeutic glycoproteins. In this report we have reviewed methodology for the in-gel enzymatic release of N-linked oligosaccharides from glycoproteins separated by electrophoresis. These oligosaccharides are derivatised by reductive amination using 3-acetamido-6-aminoacridine (AA-Ac), a novel, highly fluorescent probe. A major advantage of this technique is that glycan derivatives are amenable to analysis by an array of chromatographic and mass spectrometric methods, allowing the resolution and characterisation of a wide variety of glycan structures. It is hoped that in due course the methodology described will be applied to proteomics studies, especially in identifying the role of carbohydrate in protein function and disease. 相似文献