scholarly journals Furin Cleavage Potentiates the Membrane Fusion-Controlling Intersubunit Disulfide Bond Isomerization Activity of Leukemia Virus Env

2006 ◽  
Vol 80 (11) ◽  
pp. 5540-5551 ◽  
Author(s):  
Mathilda Sjöberg ◽  
Michael Wallin ◽  
Birgitta Lindqvist ◽  
Henrik Garoff
Keyword(s):  
Membrane Fusion ◽  
Disulfide Bond ◽  
Fusion Reaction ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Furin Cleavage ◽  
Membrane Fusion Protein ◽  
Peptide Complexes ◽  
Disulfide Bond Isomerization

ABSTRACT The membrane fusion protein of murine leukemia virus is a trimer of a disulfide-linked peripheral-transmembrane (SU-TM) subunit complex. The intersubunit disulfide bond is in SU linked to a disulfide bond isomerization motif, CXXC, with which the virus controls its fusion reaction (M. Wallin, M. Ekström, and H. Garoff, EMBO J. 23:54-65, 2004). Upon receptor binding the isomerase rearranges the intersubunit disulfide bond into a disulfide bond isomer within the motif. This facilitates SU dissociation and fusion activation in the TM subunit. In the present study we have asked whether furin cleavage of the Env precursor potentiates the isomerase to be triggered. To this end we accumulated the late form of the precursor, gp90, in the cell by incubation in the presence of a furin-inhibiting peptide. The isomerization was done by NP-40 incubation or by a heat pulse under alkylation-free conditions. The cells were lysed in the presence of alkylator, and the precursor was immunoprecipitated, gel isolated, deglycosylated, and subjected to complete trypsin digestion. Disulfide-linked peptide complexes were separated by sodium dodecyl sulfate-tricine-polyacrylamide gel electrophoresis under nonreducing conditions. This assay revealed the size of the characteristic major disulfide-linked peptide complex that differentiates the two isomers of the disulfide bond between Cys336 (or Cys339) and Cys563, i.e., the bond corresponding to the intersubunit disulfide bond. The analyses showed that the isomerase was five- to eightfold more resistant to triggering in the precursor than in the mature, cleaved form. This suggests that the isomerase becomes potentiated for triggering by a structural change in Env that is induced by furin cleavage in the cell.

scholarly journals Kinetic Analyses of the Surface-Transmembrane Disulfide Bond Isomerization-Controlled Fusion Activation Pathway in Moloney Murine Leukemia Virus

2005 ◽  
Vol 79 (22) ◽  
pp. 13856-13864 ◽  
Author(s):  
Michael Wallin ◽  
Robin Löving ◽  
Maria Ekström ◽  
Kejun Li ◽  
Henrik Garoff
Keyword(s):  
Membrane Fusion ◽  
Receptor Binding ◽  
Disulfide Bond ◽  
Murine Leukemia Virus ◽  
Leukemia Virus ◽  
Moloney Murine Leukemia Virus ◽  
Activation Pathway ◽  
Free Thiol ◽  
Disulfide Bond Isomerization ◽  
Murine Leukemia

ABSTRACT The surface (SU) and transmembrane (TM) subunits of Moloney murine leukemia virus (Mo-MLV) Env are disulfide linked. The linking cysteine in SU is part of a conserved CXXC motif in which the other cysteine carries a free thiol. Recently, we showed that receptor binding activates its free thiol to isomerize the intersubunit disulfide bond into a disulfide within the motif instead (M. Wallin, M. Ekström and H. Garoff, EMBO J. 23:54-65, 2004). This facilitated SU dissociation and activation of TM for membrane fusion. The evidence was mainly based on the finding that alkylation of the CXXC-thiol prevented isomerization. This arrested membrane fusion, but the activity could be rescued by cleaving the intersubunit disulfide bond with dithiothreitol (DTT). Here, we demonstrate directly that receptor binding causes SU-TM disulfide bond isomerization in a subfraction of the viral Envs. The kinetics of the isomerization followed that of virus-cell membrane fusion. Arresting the fusion with lysophosphatidylcholine did not arrest isomerization, suggesting that isomerization precedes the hemifusion stage of fusion. Our earlier finding that native Env was not possible to alkylate but required isomerization induction by receptor binding intimated that alkylation trapped an intermediate form of Env. To further clarify this possibility, we analyzed the kinetics by which the alkylation-sensitive Env was generated during fusion. We found that it followed the fusion kinetics. In contrast, the release of fusion from alkylated, isomerization-blocked virus by DTT reduction of the SU-TM disulfide bond was much faster. These results suggest that the alkylation-sensitive form of Env is a true intermediate in the fusion activation pathway of Env.

The kinetics of in vitro reoxidation and reduction of the inter heavy–light chain disulfide bond in an unusual murine immunoglobulin G myeloma protein lacking inter-heavy chain disulfide bonds

1979 ◽  
Vol 57 (3) ◽  
pp. 279-285 ◽  
Author(s):  
Maire E. Percy ◽  
Lebe Chang ◽  
Catherine Demoliou ◽  
Reuben Baumal
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Disulfide Bridge ◽  
Myeloma Protein ◽  
Kinetics Of

After 5 years of subcutaneous transfer in Balb/C mice, our MOPC 173 myeloma tumour line (originally an IgG2a,κ H2L2-producer) exclusively synthesized an unusual IgG2b,κ protein lacking inter-heavy (H) chain disulfide bonds. This protein was designated MOPC 173B. On sodium dodecyl sulfate – polyacrylamide gel electrophoresis, it migrated with an apparent molecular weight of 77 000; following complete reduction and alkylation, the mobilities of its constituent H and light (L) chains were found to differ slightly from those of MOPC 173 H2L2. MOPC 173B was serologically identical to another typical IgG2b,κ myeloma protein, MOPC 195, and peptide mapping studies showed that it possessed only the inter H–L disulfide bond characteristic of typical IgG2b,κ proteins. In a nondissociating solvent, the sedimentation coefficient of the protein was 6.3S even at concentrations as low as 0.2 mg/ml, indicating that noncovalent interactions existed between two half-molecule subunits. Since this unusual IgG myeloma protein contained only a single category of interchain disulfide bridge, the inter H–L bond, it was an ideal model system for characterization of the kinetics of formation and reduction of interchain disulfide bonds. The kinetics of the glutathione-catalyzed reoxidation of the inter H–L disulfide bridge in MOPC 173B followed an apparent second-order rate equation. In contrast, reduction of its inter H–L bridge under anaerobic conditions with dithioerythritol in excess, was strictly a first-order process and not a simple reversal of the reoxidation. These studies provide the basis for the more complex mathematical models that describe the reoxidation and reduction of typical immunoglobulin molecules.

scholarly journals Disulfide Bond Isomerization and the Assembly of MHC Class I-Peptide Complexes

Immunity ◽  
2002 ◽  
Vol 16 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Tobias P Dick ◽  
Naveen Bangia ◽  
David R Peaper ◽  
Peter Cresswell
Keyword(s):  
Mhc Class I ◽  
Disulfide Bond ◽  
Class I ◽  
Peptide Complexes ◽  
Disulfide Bond Isomerization

scholarly journals Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

1983 ◽  
Vol 3 (8) ◽  
pp. 1343-1352 ◽  
Author(s):  
A R Frackelton ◽  
A H Ross ◽  
H N Eisen
Keyword(s):  
Monoclonal Antibodies ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydroxyl Group ◽  
Atp Citrate Lyase ◽  
Ionic Detergents ◽  
Epidermal Growth

Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.

scholarly journals The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro

1981 ◽  
Vol 193 (3) ◽  
pp. 781-792 ◽  
Author(s):  
E A H Baydoun ◽  
D H Northcote
Keyword(s):  
Zea Mays ◽  
Atpase Activity ◽  
Membrane Fusion ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Root Cells ◽  
Dependent Atpase

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.

scholarly journals Expression of murine leukemia virus envelope glycoprotein gp69/71 on mouse thymocytes. Evidence for two structural variants distinguished by presence vs. absence of GIX antigen.

1975 ◽  
Vol 142 (2) ◽  
pp. 518-523 ◽  
Author(s):  
J S Tung ◽  
E Fleissner ◽  
E S Vitetta ◽  
E A Boyse
Keyword(s):  
Murine Leukemia Virus ◽  
Inbred Mice ◽  
Leukemia Virus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Antigen ◽  
Mouse Strains ◽  
Structural Variants ◽  
Virus Envelope ◽  
Murine Leukemia

Thymocytes of several mouse strains were tested for expression of the gp69/71 envelope component of murine leukemia virus by surface iodination, followed by immunoprecipitation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Theses strains included two congenic lines differing from their partner stocks with respect to expression of GIX antigen demonstrable in the cytoxicity assay. We conclude that:(a) two structural variants of gp69/71 can be expressed on mouse thymocytes, (b) these are distinguishable by a small difference in mobility in SDS gels, (c) one carries GIX antigen and the other not, (d) they are coded, or their expression is regulated, by different chromosomal loci that are not closely linked, and (e) both can be expressed together on the thymocytes of inbred mice. In the intact thymocyte plasma membrane, the sites of group-specific antigen shared by the two gp69/71 variants, unlike the GIX type specificity carried by only one of them, are probably inaccessible to antibody.

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