Characterization of the N-linked high-mannose oligosaccharides of the insulin pro-receptor and mature insulin receptor subunits

The insulin receptor is synthesized as a 190,000-Mr single-chain precursor that contains exclusively asparagine-N-linked high-mannose-type carbohydrate chains. In this study we have characterized the structure of the pro-receptor oligosaccharides. IM-9 lymphocytes were pulse-chase-labelled with [3H]mannose, and the insulin pro-receptor was isolated by immunoprecipitation and SDS/polyacrylamide-gel electrophoresis. The pro-receptor oligosaccharides were removed from the protein backbone with endoglycosidase H and analysed by h.p.l.c. Immediately after a [3H]mannose pulse the largest oligosaccharide found in the pro-receptor was Glc1Man9GlcNAc2; this structure represented only a small fraction (3%) of the total. The predominant oligosaccharides present in the pro-receptor were Man9GlcNAc2 (25%) and Man8GlcNAc2 (48%). Smaller oligosaccharides were also detected: Man7GlcNAc2 (18%), Man6GlcNAc2 (3%) and Man5GlcNAc2 (3%). The relative distribution of the different oligosaccharides did not change at 1, 2 or 3 h after the pulse with the exception of the rapid disappearance of the Glc1Man9GlcNAc2 component. The mature alpha- and beta-subunits of the insulin receptor are known to contain both high-mannose-type and complex-type oligosaccharides. We have also examined here the structure of the high-mannose chains of these subunits. The predominant species in the alpha-subunit was Man8GlcNAc2 whereas in the beta-subunit it was Man7GlcNAc2. These results demonstrate that most (approx. 75%) oligosaccharides of the insulin pro-receptor are chains of the type Man8GlcNAc2 or Man9GlcNAc2. Thus, assuming that a Glc3Man9GlcNAc2 species is transferred co-translationally, carbohydrate processing of the pro-receptor appears to be very rapid and limited to the removal of the three glucose residues and one mannose residue. Further mannose removal does not occur until the pro-receptor has been proteolytically cleaved. In addition, the degree of mannose trimming appears to be different in the alpha- and beta-subunits.

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The structure of the oligosaccharides of alpha3beta1 integrin from human ureter epithelium (HCV29) cell line.

Acta Biochimica Polonica ◽

10.18388/abp.2002_3808 ◽

2002 ◽

Vol 49 (2) ◽

pp. 491-500 ◽

Cited By ~ 18

Author(s):

Anna Lityńska ◽

Ewa Pocheć ◽

Dorota Hoja-Lukowicz ◽

Elzbieta Kremser ◽

Piotr Laidler ◽

...

Keyword(s):

Cell Line ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Beta Subunits ◽

Ionization Mass ◽

Complex Type ◽

Peptide Characterization ◽

Alpha3beta1 Integrin ◽

Human Ureter

There is a growing line of evidence that glycosylation of alpha and beta subunits is important for the function of integrins. Integrin alpha3beta1, from human ureter epithelium cell-line HCV29, was isolated by affinity chromatography on laminin GD6 peptide. Characterization of its carbohydrate moieties was carried out using sodium dodecyl sulfate/polyacrylamide gel electrophoresis followed by Western blotting on Immobilon P and on-blot deglycosylation with peptide N-glycosidase-F. Profiles of N-glycans for each subunit were obtained by matrix-assisted laser desorption/ionization mass spectrometry. Our findings demonstrated, in both subunits of integrin alpha3beta1, the presence of complex type oligosaccharides with a wide heterogeneity. Bi- tri- and tetraantennary structures were the most common, while high-mannose type structures were minor. Also the presence of short poly-N-acetyllactosamine entities was shown. These results show that while the predominant oligosaccharides of both subunits are identical, some slight differences between them do exist.

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Changes in glycan branching and sialylation of the Thy-1 antigen during normal differentiation of mouse T-lymphocytes

Biochemical Journal ◽

10.1042/bj2260519 ◽

1985 ◽

Vol 226 (2) ◽

pp. 519-525 ◽

Cited By ~ 11

Author(s):

S R Carlsson

Keyword(s):

T Lymphocytes ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Cell Types ◽

Complex Type ◽

Antigen Present ◽

High Mannose ◽

Normal Differentiation ◽

Immature Cells

The glycans of the Thy-1 antigen present on thymocytes and lymph-node T-lymphocytes were investigated after external labelling of the cells. Neuraminidase, endoglycosidase H and endoglycosidase F were used in combination with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing in order to characterize the nature of the glycans on 125I-labelled and immunoprecipitated Thy-1. Glycopeptides were prepared from Thy-1 obtained from cells labelled by periodate/boro[3H]hydride treatment. The glycopeptides were separated by affinity chromatography on concanavalin A-Sepharose and analysed by gel filtration. The results show that both types of cells possess Thy-1 molecules with three N-linked carbohydrate chains, of which one is of ‘high-mannose’ type and the other two of triantennary and biantennary ‘complex’ type. The ratio of triantennary/biantennary chains was decreased on Thy-1 of mature cells compared with that of immature cells, but instead more sialic acid was present on these chains. Deglycosylated Thy-1 appeared to be of the same size regardless of origin, indicating that only the carbohydrate moiety differs between Thy-1 molecules of the two cell types.

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Expression and characterization of single-chain variable fragment antibody against staphylococcal enterotoxin A in Escherichia coli

Canadian Journal of Microbiology ◽

10.1139/cjm-2014-0468 ◽

2014 ◽

Vol 60 (11) ◽

pp. 737-743 ◽

Cited By ~ 3

Author(s):

Weifeng Chen ◽

Li Hu ◽

Aiping Liu ◽

Jinquan Li ◽

Fusheng Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Food Poisoning ◽

Staphylococcal Enterotoxin ◽

Enzyme Linked Immunosorbent Assay ◽

Staphylococcal Enterotoxin A ◽

Single Chain Variable Fragment ◽

Single Chain

The staphylococcal enterotoxins (SEs) are potent gastrointestinal exotoxins synthesized by Staphylococcus aureus, which is responsible for various diseases including septicemia, food poisoning, and toxic shock syndrome, as well as bovine mastitis. Among them, staphylococcal enterotoxin A (SEA) is one of the most commonly present serotypes in staphylococcal food poisoning cases. In this study, the stable hybridoma 3C12 producing anti-SEA monoclonal antibody was established with an equilibrium dissociation constant (KD) of 1.48 × 10−8 mol·L−1, its ScFv-coding genes were obtained and then the anti-SEA single chain variable fragment (ScFv) protein was expressed in Escherichia coli. Characterization of the expressed target ScFv protein was analyzed by sodium dodecyl sulfate – polyacrylamide gel electrophoresis, Western blot, and enzyme-linked immunosorbent assay. The results demonstrated that the recombinant anti-SEA ScFv protein retained a specific binding activity for SEA, and the KD value of the soluble ScFv was about 3.75 × 10−7 mol·L−1. The overall yield of bioactive anti-SEA ScFv in E. coli flask culture was more than 10 mg·L−1.

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Partial characterization of the oligosaccharides of mouse thymocyte Thy-1 glycoprotein

Biochemical Journal ◽

10.1042/bj2210379 ◽

1984 ◽

Vol 221 (2) ◽

pp. 379-392 ◽

Cited By ~ 10

Author(s):

S R Carlsson ◽

T Stigbrand

Keyword(s):

Gel Filtration ◽

Glycosylation Site ◽

The Other ◽

Complex Type ◽

Mouse Thymocyte ◽

Lentil Lectin ◽

High Mannose ◽

Sepharose 4B ◽

Oligosaccharide Structures

Four glycopeptides (I, IIA, IIB, III) with different oligosaccharide structures were isolated from purified mouse thymocyte Thy-1 glycoprotein. The glycoprotein was digested with Pronase, and the glycopeptide fraction was isolated by gel filtration and acetylated with [3H]acetic anhydride. The different glycan structures were separated by affinity chromatography on concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. Size determinations of intact and exoglycosidase- and endoglycosidase-digested glycopeptides were performed by gel filtration on Bio-Gel P-6, calibrated with glycopeptides of known structure. On the basis of these experiments and on the behaviour of the glycopeptides on the lectin columns, the following structures of the oligosaccharide chains were proposed: I, triantennary ‘complex-type’ with terminal fucose; IIA, biantennary ‘complex-type’ without fucose; IIB, biantennary ‘complex-type’ with fucose; III, a mixture of ‘high-mannose’ chains containing either five or six mannose residues (approx. 50% of each). Amino acid analysis of the glycopeptides showed that the predominant oligosaccharide at glycosylation-site Asn-23 was of ‘high-mannose’ type, whereas the other two sites (Asn-75 and Asn-99) were glycosylated with ‘complex-type’ chains. Both these sites were shown to be variably glycosylated. The major glycans linked to Asn-75 were of structures I and IIB, whereas all three ‘complex-type’ chains were represented at Asn-99. The results presented explain the previously reported carbohydrate heterogeneity of thymocyte Thy-1 glycoprotein.

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Immunochemical characterization of proteins from mouse liver plasma membranes

Biochemical Journal ◽

10.1042/bj1350827 ◽

1973 ◽

Vol 135 (4) ◽

pp. 827-832 ◽

Cited By ~ 15

Author(s):

James W. Gurd ◽

W. Howard Evans ◽

Harold R. Perkins

Keyword(s):

Mouse Liver ◽

Plasma Membranes ◽

Soluble Fraction ◽

Polyacrylamide Gel Electrophoresis ◽

Extraction Procedure ◽

Relative Distribution ◽

Two Dimensional ◽

Nucleotidase Activity ◽

Triton X

1. Antiserum was prepared in rabbits against a purified mouse liver plasma-membrane fraction. 2. The antiserum was made to react with an125I-labelled alkaline-EDTA extract of the plasma membranes, and the immunoprecipitate analysed by polyacrylamide-gel electrophoresis. Seven proteins were immunoprecipitated and a single glycoprotein present in the alkaline-EDTA-soluble fraction was found to be a major component. 3. The alkaline-EDTA-soluble fraction was analysed by two-dimensional immunoelectrophoresis and this procedure indicated the presence of six antigenic components. 4. The plasma membranes were also extracted with 1% deoxycholate–1% Triton X-100; 50% of the protein, 80% of the alkaline phosphodiesterase activity and 30% of the 5′-nucleotidase activity were solubilized. 5. Two-dimensional immunoelectrophoresis of the deoxycholate–Triton X-100 extract indicated the presence of six antigens. 6. The relative distribution of the six antigens among the fractions obtained during the extraction procedure was examined immunoelectrophoretically to provide information on their disposition within the membrane.

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X-ray and EM structures of a natively glycosylated HIV-1 envelope trimer

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798317013353 ◽

2017 ◽

Vol 73 (10) ◽

pp. 822-828 ◽

Cited By ~ 8

Author(s):

Harry B. Gristick ◽

Haoqing Wang ◽

Pamela J. Bjorkman

Keyword(s):

Crystal Structures ◽

Biochemical Characterization ◽

Complex Type ◽

Cryo Electron Microscopy ◽

High Mannose ◽

Complex Glycans ◽

Anti Hiv ◽

Hiv 1 ◽

Trimeric Envelope

The structural and biochemical characterization of broadly neutralizing anti-HIV-1 antibodies (bNAbs) has been essential in guiding the design of potential vaccines to prevent infection by HIV-1. While these studies have revealed critical mechanisms by which bNAbs recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env), they have been limited to the visualization of high-mannose glycan forms only, since heterogeneity introduced from the presence of complex glycans makes it difficult to obtain high-resolution structures. 3.5 and 3.9 Å resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation were solved, revealing a glycan shield of high-mannose and complex-type N-glycans that were used to define the complete epitopes of two bNAbs. Here, the refinement of the N-glycans in the crystal structures is discussed and comparisons are made with glycan densities in glycosylated Env structures derived by single-particle cryo-electron microscopy.

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Biosynthesis and processing of platelet GPIIb-IIIa in human megakaryocytes.

The Journal of Cell Biology ◽

10.1083/jcb.104.6.1665 ◽

1987 ◽

Vol 104 (6) ◽

pp. 1665-1673 ◽

Cited By ~ 67

Author(s):

A Duperray ◽

R Berthier ◽

E Chagnon ◽

J J Ryckewaert ◽

M Ginsberg ◽

...

Keyword(s):

Myelogenous Leukemia ◽

Beta Subunits ◽

Metabolic Labeling ◽

Single Chain ◽

Calcium Dependent ◽

Fibrinogen Receptor ◽

Cell Extracts ◽

Large Numbers ◽

Molecular Masses ◽

High Mannose

Platelet membrane glycoprotein IIb-IIIa forms a calcium-dependent heterodimer and constitutes the fibrinogen receptor on stimulated platelets. GPIIb is a two-chain protein containing disulfide-linked alpha and beta subunits. GPIIIa is a single chain protein. These proteins are synthesized in the bone marrow by megakaryocytes, but the study of their synthesis has been hampered by the difficulty in obtaining enriched population of megakaryocytes in large numbers. To examine the biosynthesis and processing of GPIIb-IIIa, purified human megakaryocytes were isolated from liquid cultures of cryopreserved leukocytes stem cell concentrates from patients with chronic myelogenous leukemia. Immunoprecipitation of [35S]methionine pulse-chase-labeled cell extracts by antibodies specific for the alpha or beta subunits of GPIIb indicated that GPIIb was derived from a precursor of Mr 130,000 that contains the alpha and beta subunits. This precursor was converted to GPIIb with a half-life of 4-5 h. No precursor form of GPIIIa was detected. The glycosylation of GPIIb-IIIa was examined in megakaryocytes by metabolic labeling in the presence of tunicamycin, monensin, or treatment with endoglycosidase H. The polypeptide backbones of the GPIIb and the GPIIIa have molecular masses of 120 and 90 kD, respectively. High-mannose oligosaccharides are added to these polypeptide backbones co-translationally. The GPIIb precursor is then processed with conversion of high-mannose to complex type carbohydrates yielding the mature subunits GPIIb alpha (Mr 116,000) and GPIIb beta (Mr 25,000). No posttranslational processing of GPIIIa was detected.

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Isolation and characterization of the subunits of bovine follitropin

Biochemical Journal ◽

10.1042/bj1750029 ◽

1978 ◽

Vol 175 (1) ◽

pp. 29-34 ◽

Cited By ~ 2

Author(s):

K W Cheng

Keyword(s):

Glutamic Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Deae Cellulose ◽

Alpha Subunit ◽

Beta Subunits ◽

Methionine Residue ◽

Isolation And Characterization ◽

Receptor Assays

Highly purified bovine follitropin was dissociated into its alpha- and beta-subunits after treatment with 1 M-propionic acid. The dissociated subunits were fractionated by chromatography on DEAE-cellulose and further purified by gel filtration on Sephadex G-100. The isolated alpha- and beta-subunits were biologically inactive, but their recombinants regenerated 80% of the follitropin activity. The alpha-subunit of bovine follitropin recombined with the beta-subunits of bovine lutropin and thyrotropin to regenerate 70% of lutropin and 50% of thyrotropin activities respectively. The beta-subunit of bovine follitropin recombined with the alpha-subunit of either bovine lutropin or thyrotropin to regenerate about 75% of follitropin activity. Recombinations were monitored by specific radioligand-receptor assays and polyacrylamide-gel electrophoresis. The elution volumes of the alpha- and beta-subunits of bovine follitropin after gel filtration on Sephadex G-100 were almost identical. The amino acid composition of bovine follitropin-alpha was low in histidine, arginine, isoleucine and leucine, but relatively high in lysine, threonine and glutamic acid. The bovine follitropin-beta contained one methionine residue and low amounts of histidine and phenylalanine, but relatively high in aspartic acid, threonine and glutamic acid. The N-terminal residues of the alpha- and beta-subunits of bovine follitropin were identified to be phenylalanine and glycine respectively.

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Characterization of the mutant-unc D-gene product in a strain of Escherichia coli K12. An altered β-subunit of the magnesium ion-stimulated adenosine triphosphatase

Biochemical Journal ◽

10.1042/bj1720523 ◽

1978 ◽

Vol 172 (3) ◽

pp. 523-531 ◽

Cited By ~ 26

Author(s):

D R H Fayle ◽

J A Downie ◽

G B Cox ◽

F Gibson ◽

J Radik

Keyword(s):

Escherichia Coli ◽

Adenosine Triphosphatase ◽

Polyacrylamide Gel Electrophoresis ◽

Beta Subunit ◽

Beta Subunits ◽

Normal Strain ◽

Diploid Strain ◽

Escherichia Coli K12 ◽

Low Ionic Strength

Membranes from a mutant strain of Escherichia coli K12 carrying the uncD409 allele were washed in low-ionic-strength buffers in the presence or absence of the proteinase inhibitor p-aminobenzamidine. Unlike membranes from a normal strain, those from strain AN463 (uncD409) did not become proton-permeable, as judged by NADH-induced atebrinfluorescence quenching, when the membranes were washed in the absence of p-aminobenzamide. Furthermore, ATP-dependent atebrin-fluorscence quenching in such washed membranes could not be reconstituted by the addition of solubilized Mg2+-stimulated adenosine triphosphatase preparations. The examination by two-dimensional polyacrylamide-gel electrophoresis of the polypeptide composition of the washed membranes from strain AN463 (uncD409) indicated the presence of a polypeptide of similar molecular weight to the normal beta-subunit of the Mg2+-stimulated adenosine triphosphatase, but with an altered isoelectric point. Both the normal and abnormal beta-subunits were identified in membranes prepared from a partial diploid strain carrying both the unc+ and uncD409 alleles. It is concluded that the uncD gene codes for the beta-subunit of the Mg2+-stimulated adenosine triphosphatase.

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Degradation pathway of plant complex-type N-glycans: identification and characterization of a key α1,3-fucosidase from glycoside hydrolase family 29

Biochemical Journal ◽

10.1042/bcj20170106 ◽

2018 ◽

Vol 475 (1) ◽

pp. 305-317 ◽

Cited By ~ 4

Author(s):

Shun Kato ◽

Megumi Hayashi ◽

Mai Kitagawa ◽

Hiroyuki Kajiura ◽

Megumi Maeda ◽

...

Keyword(s):

Glycoside Hydrolase ◽

Degradation Pathway ◽

Glycoside Hydrolases ◽

Glycoside Hydrolase Family ◽

Complex Type ◽

Gene Encoding ◽

Mannose Residue ◽

Growth Features ◽

Identification And Characterization

Plant complex-type N-glycans are characterized by the presence of α1,3-linked fucose towards the proximal N-acetylglucosamine residue and β1,2-linked xylose towards the β-mannose residue. These glycans are ultimately degraded by the activity of several glycoside hydrolases. However, the degradation pathway of plant complex-type N-glycans has not been entirely elucidated because the gene encoding α1,3-fucosidase, a glycoside hydrolase acting on plant complex-type N-glycans, has not yet been identified, and its substrate specificity remains to be determined. In the present study, we found that AtFUC1 (an Arabidopsis GH29 α-fucosidase) is an α1,3-fucosidase acting on plant complex-type N-glycans. This fucosidase has been known to act on α1,4-fucoside linkage in the Lewis A epitope of plant complex-type N-glycans. We found that this glycoside hydrolase specifically acted on GlcNAcβ1–4(Fucα1–3)GlcNAc, a degradation product of plant complex-type N-glycans, by sequential actions of vacuolar α-mannosidase, β1,2-xylosidase, and endo-β-mannosidase. The AtFUC1-deficient mutant showed no distinct phenotypic plant growth features; however, it accumulated GlcNAcβ1–4(Fucα1–3)GlcNAc, a substrate of AtFUC1. These results showed that AtFUC1 is an α1,3-fucosidase acting on plant complex-type N-glycans and elucidated the degradation pathway of plant complex-type N-glycans.

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