Peptide binding to soluble HLA-DR4 molecules produced by insect cells.

The baculovirus/insect cell system (Autographa californica multiple nuclear polyhedrosis virus/Spodoptera frugiperda Sf9 cells) was used to express the GPI‐anchored human melanoma tumor antigen, melanotransferrin or p97. This system served to study the expression and productivity of recombinant GPI‐anchored p97 by insect cells. The Sf9 cells expressed a cell surface GPI‐anchored form of p97 as well as a soluble form of p97 that did not appear to be derived from the GPI‐anchored form of p97. Both recombinant forms, although Endo H resistant, migrated slightly faster (∼88 kDa) than the native p97 (∼95–97 kDa). The insect GPI‐anchored p97 was sensitive to PI‐PLC, which exposed a detectable cross‐reacting determinant. The Sf9 cell surface p97 expression was similar to that of human melanoma (SK‐MEL‐28) cells, whereas the Sf9 cell specific secretion rate was 10‐fold higher. Also Sf9 cells retained considerably higher levels of p97 within the cell. The Sf9 cell surface expression of p97 varied with time after infection, with the maximum expression, which appeared independent of multiplicities of infection greater than 1, occurring at 48 h. After 48 h, levels of cell surface and secreted p97 fell whereas p97 retained within the cell increased, which possibly reflected the lytic nature of the expression system. The successful expression of GPI‐anchored human p97 by the baculovirus/insect cell system not only provides a source of p97 for further research but also is the basis of an alternative method for the commercial production of GPI‐anchored proteins. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 41–53, 1997.

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Expression of cell surface GPI-anchored human p97 in baculovirus-infected insect cells

Kennard

Shimizu

Gabathuler

et al. 1997

Biotechnol. Bioeng.

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A fungal metabolite which inhibits the interaction of CD4 with major histocompatibility complex‐encoded class II molecules

Gammon¹,

Chandler²,

Depledge³

et al. 1994

Eur J Immunol

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CD4, a cell-surface glycoprotein expressed on a subpopulation of T cells, is the receptor for class II molecules of the major histocompatibility complex (MHC II) and a receptor for the envelope glycoprotein (gp 120) of human immunodeficiency virus-1 (HIV-1). Screening of microbial metabolites for CD4-binding activity using an enzyme-linked immunosorbent assay based on the binding of the CD4-specific monoclonal antibody (mAb), anti-Leu3a, identified a family of compounds comprising several novel polyketides. The parent compound (411F, Vinaxanthone) is a C28 molecule probably arising from a dimerization of two C14 polyketide units. It strongly inhibited the interaction of anti-Leu 3a and that of several other D1/D2 epitope-specific mAb with CD4, but only weakly inhibited the binding of HIV-1 gp120. Binding of a representative MHC class II molecule, HLA-DRB*0401, was also inhibited by 411F with a comparable inhibitory concentration (IC50 = 1 microM). In functional assays 411F inhibited antigen-induced CD4-dependent T cell proliferative responses of peripheral blood mononuclear cells. At the clonal level 411F exhibited selectivity in that the compound inhibited peptide-induced CD4+ T cell proliferative responses but not alloantigen-induced CD8+ T cell proliferation. It is hypothesized that 411F, a polyanionic compound in aqueous solution at neutral pH, inhibits CD4-dependent functions by binding over a broad area of the positively charged amino-terminal D1 and D2 domains implicated in the interaction with MHC II molecules. 411F has the potential for development as an immunosuppressive agent with a novel mechanism of action.

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Detergent‐enhanced dissociation of endogenous peptides from PI‐DRB1*0401

Buelow¹,

Kuo²,

Paborsky³

et al. 1994

Eur J Immunol

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A variety of detergents have been shown to catalyze the dissociation of bound peptides from a soluble from of DRB1*0401. By using a class II molecule lacking the hydrophobic transmembrane region, the need for solubilizing the transmembrane protein was removed and enabled the specific interaction between the class II protein and the amphiphile to be identified. The presence of detergent increased the rate of association of added peptide and the percent occupancy of the receptor, presumably because the dissociation of endogenous peptide was the rate-limiting step in binding. The data help explain the differences reported between peptide binding to class II proteins on the surface of cells and binding to class II proteins solubilized in detergent. The interaction did not correlate with the critical micellar concentration of the detergent nor were all amphiphilic structures equally effective, consistent with a specific interaction between the amphiphile and the MHC class II protein. Of the eight detergents examined, octyl glucoside was the most efficient. These experiments did not distinguish between an allosteric mechanism or direct competition with the peptide for binding.

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Peptide binding to soluble HLA-DR4 molecules produced by insect cells.

Cited by 21 publications

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Expression of cell surface GPI-anchored human p97 in baculovirus-infected insect cells

Expression of cell surface GPI-anchored human p97 in baculovirus-infected insect cells

A fungal metabolite which inhibits the interaction of CD4 with major histocompatibility complex‐encoded class II molecules

Detergent‐enhanced dissociation of endogenous peptides from PI‐DRB1*0401

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