Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins

Robbins, Phillips W.; Hubbard, S C; Turco, Salvatore J.; Wirth, Dyann F.

doi:10.1016/0092-8674(77)90153-2

Cited by 483 publications

(206 citation statements)

References 14 publications

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“…Unlike the non-secreted, high mannose-containing ectodomain (34), secreted, COOH-terminal truncated TSHR ectodomain variants are recognized by autoantibodies. Whether the complex carbohydrate comprises part of the epitope(s) for TSHR autoantibodies or whether lack of autoantibody recognition of the "high mannose" ectodomain is secondary to incorrect polypeptide folding (and, hence, retention in the endoplasmic reticulum) (42,43) is presently unknown. It is of interest (and paradoxical) that COOH-terminal truncation of the LH/CG receptor ectodomain at a position (amino acid residue 294) similar to the TSHR variants generates a non-secreted protein (44).…”

Section: Discussionmentioning

confidence: 99%

Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera

Chazenbalk

Jaume

McLachlan

et al. 1997

Journal of Biological Chemistry

110

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Previous attempts to generate autoantibody-reactive, secreted thyrotropin receptor (TSHR) ectodomain in mammalian cells have failed because of retention within the cell of material with immature carbohydrate. We have overcome this difficulty by performing progressive carboxyl-terminal truncations of the human TSHR ectodomain (418 amino acid residues including signal peptide). Three ectodomain variants (TSHR-261, TSHR-289, and TSHR-309) were truncated at residues 261, 289, and 309, respectively. Unlike the full ectodomain, ectodomain variants were secreted with an efficiency inversely proportional to their size. Secreted ectodomain variants contained ϳ20 kDa of complex carbohydrate. TSHR-261 was chosen for further study because it was secreted very efficiently and neutralized autoantibodies in Graves' patients' sera. This ectodomain variant was partially purified using sequential lectin and nickel-chelate chromatography, permitting the first direct visualization and quantitation of the mammalian TSHR. Most important, very small (nanogram) quantities of this material neutralized 70 -100% of TSHR autoantibody activity in all 18 Graves' sera studied.In summary, carboxyl-terminal truncation of the human

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Section: Discussionmentioning

confidence: 99%

Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera

Chazenbalk

Jaume

McLachlan

et al. 1997

Journal of Biological Chemistry

110

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“…Metabolic labeling was with either [~SS]methionine (>650 Ci/mmol, Amersham Corp.) in methionine-free medium or [3H]mannose (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) Ci/mmol, ICN Pharmaceuticals, Inc., Irvine, CA) in low glucose (50 uM) medium that contained 10% dialyzed fetal calf serum. Purified proteins were iodinated using Iodogen (Pierce Chemical Co., Rockford, IL) (30) as described previously (12).…”

Section: Purification Of Membrane Proteinsmentioning

confidence: 99%

“…Glycosidase Digestion: Digestion with endo-C~-N-acetylglucosaminidase F (Endo F) and endo-~-N-acetylgucosaminidase H (Endo H) was performed using slight modifications of published procedures (26,27). Briefly, immunoprecipitated antigens were eluted from the S. aureus pellet by its resuspension in Endo F buffer (100 mM NaP~ pH 6.1, 50 mM EDTA, 1% Triton X-100, 0.1% SDS, 1% 13-mercaptoethanol) or Endo H buffer ( 100 mM sodium citrate, pH 5.5, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride) and heating to 100*C for 5 min.…”

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confidence: 99%

Glycoproteins of the lysosomal membrane.

Lewis¹,

Green²,

Marsh³

et al. 1985

The Journal of Cell Biology

280

166

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Three glycoprotein antigens (120, 100, and 80 kD) were detected by mono-and/ or polyclonal antibodies generated by immunization with highly purified rat liver lysosomal membranes. All of the antigens were judged to be integral membrane proteins based on the binding of Triton X-114. By immunofluorescence on normal rat kidney cells, a mouse monoclonal antibody to the 120-kD antigen co-stained with a polyclonal rabbit antibody that detected the 100-and 80-kD antigens as well as with antibodies to acid phosphatase, indicating that these antigens are preferentially localized in lysosomes. Few 120-kD-positive structures were found to be negative for acid phosphatase, suggesting that the antigen was not concentrated in organelles such as endosomes, which lack acid phosphatase. Immunoperoxidase cytochemistry also showed little reactivity in Golgi cisternae, coated vesicles, or on the plasma membrane. Digestion with endo-/~-N-acetylglucosaminidase H (Endo H) and endo-/~-N-acetylglucosaminidase F (Endo F) demonstrated that each of the antigens contained multiple Nlinked oligosaccharide chains, most of which were of the complex (Endo H-resistant) type. The 120-kD protein was very heavily glycosylated, having at least 18 N-linked chains. It was also rich in sialic acid, since neuraminidase digestion increased the pl of the 120-kD protein from <4 to >8. Taken together, these results strongly suggest that the glycoprotein components of the lysosomal membrane are synthesized in the rough endoplasmic reticulum and terminally glycosylated in the Golgi before delivery to lysosomes. We have provisionally designated these antigens lysosomal membrane glycoproteins Igp120, Igpl00, lgp80.

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“…The newly synthesized Sindbis virus glycoproteins carry only large oligosaccharides similar in size to the lipid-linked oligosaccharide (Robbins et al, 1977). Subsequent processing to yield the high-mannose-and complex-type oligosaccharides is presumed to occur at sites destined to become mature high-mannose-and complex-type oligosaccharide, respectively, by enzymes of the host cell.…”

Section: Discussionmentioning

confidence: 99%

Heterogeneity of Sindbis Virus Glycoprotein E1 and its Modification by Host Cell Transformation

Miki

1984

Journal of General Virology

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SUMMARYThe electrophoretic properties of glycoprotein E1 of Sindbis virus grown in Rous sarcoma virus-transformed cells were compared with those of Sindbis virus grown in untransformed cells. Isoelectric focusing in a gel containing 9-5 M-urea and 2~ Nonidet P40 indicated that the glycoprotein E1 of Sindbis virus from the untransformed cells was electrochemically heterogeneous and consisted of four components, whose isoelectric points (pIs) ranged from 6-2 to 6.7; the E1 of Sindbis virus from the transformed cells contained an additional component with a pI of 6.1. After treatment with neuraminidase the observed charge heterogeneity disappeared regardless of whether the virus had come from the transformed or the untransformed cells. The five components were digested with Pronase and compared by gel filtration on Bio-Gel P-6; the four components with relatively higher pls released high-mannose-type and complex-type oligosaccharides which differed in their content of sialic acid. In contrast, the pI 6-1 component, which existed only in the transformed cell-derived SV glycoprotein El, released only complex-type oligosaccharide. These results indicate that the properties of glycoprotein E1 derived from both cell types are basically similar but that a small proportion of the E1 molecules from the transformed cells, with carbohydrate chains elongated by attachment of sialic acid residues, lacks simple-type oligosaccharides either because they are not attached or because they have been processed to the complex type. Glycoprotein E 2 appeared as at least three components in nonequilibrium pH gradient electrophoresis. However, no significant difference was observed between the cell types.

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Proposal for a common oligosaccharide intermediate in the synthesis of membrane glycoproteins

Cited by 483 publications

References 14 publications

Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera

Engineering the Human Thyrotropin Receptor Ectodomain from a Non-secreted Form to a Secreted, Highly Immunoreactive Glycoprotein That Neutralizes Autoantibodies in Graves' Patients' Sera

Glycoproteins of the lysosomal membrane.

Heterogeneity of Sindbis Virus Glycoprotein E1 and its Modification by Host Cell Transformation

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