scholarly journals Translocation of peptides through microsomal membranes is a rapid process and promotes assembly of HLA-B27 heavy chain and beta 2-microglobulin translated in vitro.

1991 ◽  
Vol 115 (4) ◽  
pp. 959-970 ◽  
Author(s):  
F Lévy ◽  
R Larsson ◽  
S Kvist
Keyword(s):  
Mhc Class I ◽  
Heavy Chain ◽  
Peptide Binding ◽  
Class I ◽  
Hla B27 ◽  
Heavy Chains ◽  
Microsomal Membranes ◽  
Beta 2 ◽  
Beta 2 Microglobulin

We have translated major histocompatibility complex (MHC) class I heavy chains and human beta 2-microglobulin in vitro in the presence of microsomal membranes and a peptide from the nucleoprotein of influenza A. This peptide stimulates assembly of HLA-B27 heavy chain and beta 2-microglobulin about fivefold. By modifying this peptide to contain biotin at its amino terminus, we could precipitate HLA-B27 heavy chains with immobilized streptavidin, thereby directly demonstrating class I heavy chain-peptide association under close to physiological conditions. The biotin-modified peptide stimulates assembly to the same extent as the unmodified peptide. Both peptides bind to the same site on the HLA-B27 molecule. Immediately after synthesis of the HLA-B27 heavy chain has been completed, it assembles with beta 2-microglobulin and peptide. These interactions occur in the lumen of the microsomes (endoplasmic reticulum), demonstrating that the peptide must cross the microsomal membrane in order to promote assembly. The transfer of peptide across the microsomal membrane is a rapid process, as peptide binding to heavy chain-beta 2-microglobulin complexes is observed in less than 1 min after addition of peptide. By using microsomes deficient of beta 2-microglobulin (from Daudi cells), we find a strict requirement of beta 2-microglobulin for detection of peptide interaction with the MHC class I heavy chain. Furthermore, we show that heavy chain interaction with beta 2-microglobulin is likely to precede peptide binding. Biotin-modified peptides are likely to become a valuable tool in studying MHC antigen interaction and assembly.

scholarly journals Overproduction and secretion of beta 2-microglobulin by a rat thymic epithelial cell line that expresses MHC class I heavy chain

1991 ◽  
Vol 98 (4) ◽  
pp. 559-565
Author(s):  
C. Dargemont ◽  
D. Dunon ◽  
J. Salamero ◽  
M.A. Deugnier ◽  
J. Davoust ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Cell Line ◽  
Mhc Class I ◽  
Heavy Chain ◽  
Class I ◽  
Thymic Epithelial Cell ◽  
Epithelial Cell Line ◽  
Beta 2 ◽  
Beta 2 Microglobulin ◽  
Thymic Epithelial Cell Line

Major histocompatibility complex (MHC) class I antigens are constituted of dimers consisting of a peripheral light chain, beta 2-microglobulin (beta 2m) and a transmembrane heavy chain whose cell surface expression depends on its assembly with beta 2m. In contrast, soluble beta 2m can be secreted in the absence of heavy chain expression. The presence of beta 2m in medium conditioned by a rat thymic epithelial cell line, IT45-R1 (IT45) prompted us to investigate whether beta 2m could be secreted by cells that express MHC class I antigens. IT45 cells produce three to five times more beta 2m in the culture supernatant than another rat thymic epithelial cell line, IT26-R21 (IT26). The IT45 cell line exported beta 2m through a constitutive pathway of secretion, as indicated by the kinetics of production and localization of intracellular beta 2m. Although cells from the IT45 cell line expressed a much higher amount of beta 2m as compared to IT26 and NBT II cells (a rat bladder epithelial cell line), all three of these cell lines expressed the same amount of membrane and intracellular MHC class I heavy chain. These data are thus consistent with a constitutive secretion of beta 2m dependent upon an overexpression of MHC class I light chain as compared to the heavy chain. The amount of beta 2m mRNA and the ratio of beta 2m versus MHC class I heavy chain transcripts were higher in IT45 than in IT26 cells, indicating that overexpression of beta 2m in IT45 cells could be due to an enhanced level of beta 2m mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Participation of a novel 88-kD protein in the biogenesis of murine class I histocompatibility molecules.

1991 ◽  
Vol 112 (6) ◽  
pp. 1099-1115 ◽  
Author(s):  
E Degen ◽  
D B Williams
Keyword(s):  
Heavy Chain ◽  
Gel Permeation Chromatography ◽  
Class I ◽  
Specific Class ◽  
Antigenic Peptides ◽  
Heavy Chains ◽  
Golgi Transport ◽  
Beta 2 ◽  
Beta 2 Microglobulin ◽  
Rate Limiting

Chemical cross-linking and gel permeation chromatography were used to examine early events in the biogenesis of class I histocompatibility molecules. We show that newly synthesized class I heavy chains associate rapidly and quantitatively with an 88-kD protein in three murine tumor cell lines. This protein (p88) does not appear to possess Asn-linked glycans and it is not the abundant ER protein, GRP94. The class I-p88 complex exists transiently (t1/2 = 20-45 min depending on the specific class I heavy chain) and several lines of evidence suggest that p88 dissociates from the complex while still in the ER. Dissociation is not triggered upon binding of beta 2-microglobulin to the heavy chain (t1/2 = 2-5 min). However, the rate of dissociation does correlate with the characteristic rate of ER to Golgi transport for the particular class I molecule studied. Consequently, dissociation of p88 may be rate limiting for ER to Golgi transport. Class I molecules bind antigenic peptides, apparently in the ER, for subsequent presentation to cytotoxic T lymphocytes at the cell surface. p88 could promote peptide binding or it may retain class I molecules in the ER during formation of the ternary complex of heavy chain, beta 2-microglobulin, and peptide.

scholarly journals Efficient dissociation of the p88 chaperone from major histocompatibility complex class I molecules requires both beta 2-microglobulin and peptide.

1992 ◽  
Vol 175 (6) ◽  
pp. 1653-1661 ◽  
Author(s):  
E Degen ◽  
M F Cohen-Doyle ◽  
D B Williams
Keyword(s):  
Major Histocompatibility Complex ◽  
Major Histocompatibility Complex Class ◽  
Heavy Chain ◽  
Class I ◽  
Complex Class ◽  
Major Histocompatibility ◽  
Heavy Chains ◽  
Histocompatibility Complex ◽  
Beta 2 ◽  
Beta 2 Microglobulin

Previously, we showed that an 88-kD protein (p88) associates rapidly and quantitatively with newly synthesized murine major histocompatibility complex class I molecules within the endoplasmic reticulum (ER). This interaction is transient and dissociation of p88 appears to be rate limiting for transport of class I molecules from the ER to the Golgi apparatus. In this report, we examine the relationship between p88 interaction and assembly of the ternary complex of class I heavy chain beta 2-microglobulin (beta 2m), and peptide ligand. In both murine and human beta 2m-deficient cells, in which little or no transport of class I heavy chains is observed, p88 remained associated with intracellular heavy chains throughout their lifetime. In murine RMA-S cells, which are apparently defective in accumulating peptide ligands for class I within the ER, prolonged association of p88 with "empty" heavy chain-beta 2m heterodimers was also observed. However, p88 dissociated slowly in parallel with the slow rate of ER to Golgi transport of empty class I molecules in these cells. The close correlation between p88 association and impaired class I transport suggests that p88 functions to retain incompletely assembled class I molecules in the ER. We propose that conformational changes in class I heavy chains induced by the binding of both beta 2m and peptide are required for efficient p88 dissociation and subsequent class I transport.

scholarly journals Identification and Molecular Characterization of Z/ZE Lineage MHC Class I Heavy Chain Homologue and ^|^beta;2-microglobulin from Rock Bream Oplegnathus fasciatus

2014 ◽  
Vol 49 (3) ◽  
pp. 93-112 ◽  
Author(s):  
Chamilani Nikapitiya ◽  
Sung-Ju Jung ◽  
Myung-Hwa Jung ◽  
Jun-Young Song ◽  
Jehee Lee ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Mhc Class I ◽  
Heavy Chain ◽  
Class I ◽  
Oplegnathus Fasciatus ◽  
Rock Bream ◽  
Beta 2 ◽  
Beta 2 Microglobulin

scholarly journals An unstable beta 2-microglobulin: major histocompatibility complex class I heavy chain intermediate dissociates from calnexin and then is stabilized by binding peptide.

1994 ◽  
Vol 180 (6) ◽  
pp. 2163-2171 ◽  
Author(s):  
M Sugita ◽  
M B Brenner
Keyword(s):  
Heavy Chain ◽  
Class I ◽  
Peptide Transporter ◽  
Histocompatibility Complex ◽  
Beta 2 ◽  
Stable Association ◽  
Beta 2 Microglobulin ◽  
Relationship Of ◽  
Stepwise Assembly

Proper assembly of the class I heavy chain (HC), beta 2-microglobulin (beta 2m), and peptide must occur in the endoplasmic reticulum (ER) in order for MHC class I molecules to be expressed on the cell surface. Newly synthesized class I HC bind calnexin, an ER resident chaperone. These calnexin-associated class I HC appeared to lack the stable association with beta 2m in peptide transporter-deficient T2 cells since beta 2m-unassociated class I HC-specific HC10 antibody, but not beta 2m-associated class I HC-specific W6/32 antibody, coimmunoprecipitated calnexin. To determine the precursor-product relationship of the pool of HC that bind peptide, class I-restricted peptides were added to lysates of T2 cells in vitro. These peptides stabilized preexisting beta 2m-associated HC complexes (beta 2m+:HC:pep-), but had no significant effect on the preexisting pool of calnexin-associated HC that lack beta 2m. Release of HC from calnexin appeared to be controlled by the binding of beta 2m, since beta 2m-deficient FO-1 cells showed a prolonged association of class I HC with calnexin, while beta 2m-transfected FO-1 cells displayed a more rapid dissociation of class I HC from calnexin. Consistent with this result, the dissociation of class I HC from calnexin did not appear to be dependent on peptide binding since the dissociation rates were similar in peptide transporter-deficient T2 cells and in wild-type T1 cells. From these observations, we speculate that in the stepwise assembly of class I molecules, calnexin may mediate dimerization of class I HC with beta 2m, and that the unstable beta 2m+:HC:pep- complexes, after dissociation from calnexin, subsequently bind peptide to complete the assembly.

scholarly journals Species-specific differences in chaperone interaction of human and mouse major histocompatibility complex class I molecules.

1995 ◽  
Vol 181 (1) ◽  
pp. 327-337 ◽  
Author(s):  
E Nössner ◽  
P Parham
Keyword(s):  
Heavy Chain ◽  
Hla Class I ◽  
Cell Receptor ◽  
Class I ◽  
Human Class ◽  
Heavy Chains ◽  
Mouse Major Histocompatibility Complex ◽  
Beta 2 Microglobulin ◽  
Mouse Class

Previous studies have shown that immature mouse class I molecules transiently associate with a resident endoplasmic reticulum protein of 88 kD that has been proposed to act as a chaperone for class I assembly. Subsequently, this protein was demonstrated to be identical to calnexin and to associate with immature forms of the T cell receptor complex, immunoglobulin, and human class I HLA heavy chains. In this paper we define further the interaction of human class I HLA heavy chains with chaperone proteins and find key differences with the complexes observed in the mouse system. First, calnexin and immunoglobulin binding protein (BiP) both associate with immature HLA class I heavy chains. The two chaperones are not found within the same molecular complex, suggesting that calnexin and BiP do not interact simultaneously with the same HLA class I heavy chain. Second, only free HLA class I heavy chains, and not beta 2-microglobulin (beta 2m)-associated heavy chains are found associated with the chaperones. Indeed, addition of free beta 2m in vitro induces dissociation of chaperone-class I HLA heavy chain complexes. The kinetics for dissociation of the class I HLA heavy chain-chaperone complexes and for formation of the class I HLA heavy chain-beta 2m complex display a reciprocity that suggests the interactions with chaperone and beta 2m are mutually exclusive. Mouse class I heavy chains expressed in human cells exhibit the mouse pattern of interaction with human chaperones and human beta 2m and not the human pattern, showing the difference in behavior is purely a function of the class I heavy chain sequence.

scholarly journals Activated human T lymphocytes express MHC class I heavy chains not associated with beta 2-microglobulin.

1990 ◽  
Vol 171 (5) ◽  
pp. 1431-1442 ◽  
Author(s):  
E Schnabl ◽  
H Stockinger ◽  
O Majdic ◽  
H Gaugitsch ◽  
I J Lindley ◽  
...  
Keyword(s):  
T Lymphocytes ◽  
Mhc Class I ◽  
Hla Class I ◽  
Class I ◽  
Heavy Chains ◽  
Human T Lymphocytes ◽  
Beta 2 ◽  
Beta 2 Microglobulin ◽  
Mhc Class I Molecules

We present here the molecular characterization of a new activation-induced surface structure on human T lymphocytes, termed LA45, with high homology (93% at protein level) to MHC class I molecules. Antigen modulation and sequential immunoprecipitation experiments revealed that LA45 and HLA class I proteins do not crossreact with the corresponding antibodies. Furthermore, LA45 is not associated with beta 2-m. On the other hand, we could show that the separation of HLA-A,B,C and beta 2m molecules, induced by SDS-denaturation, leads to a conformational change in the heavy chain in such a way that it becomes reactive with LA45. The 90/45 kD LA45 proteins thus appear to be non-beta 2m-associated MHC class I alpha chains that are selectively expressed by activated but not by resting human T lymphocytes.

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