Major histocompatibility class one molecule associates with glucose regulated protein (GRP) 78 on the cell surface

doi:10.1016/S0198-8859(01)00269-5

Human Immunology

Volume 62, Issue 8, August 2001, Pages 764-770

https://doi.org/10.1016/S0198-8859(01)00269-5 Get rights and content

Abstract

Glucose regulated protein 78 (GRP78) is a member of the heat shock protein (hsp) 70 family. It is an endoplasmic reticulum (ER) chaperone, whose function is generally thought to be limited to the structural maturation of nascent glycoproteins. However, recent observations have shown that ER chaperones, such as GRP78, display peptide-binding activity. These peptide-binding activities along with the observation that heat shock proteins associated with peptides can elicit antigen-specific CTL responses suggest additional roles for these proteins. In this study we provide evidence that GRP78 is not only resident in the ER, but also exists on the cell surface. Furthermore, using biochemical and imaging studies we have found that GRP78 associates with MHC class I on the cell surface. Its presence on the cell surface is not dependent on MHC class I expression. In the absence of MHC class I its cell surface expression is upregulated.

Introduction

Activation of the adaptive immune system relies on the recognition of small ligands bound to major histocompatibility (MHC) molecules by T-cells. A large body of work has defined two distinct pathways by which protein antigens—peptides derived from “intracellular” or “extracellular” antigens—are processed by two intracellular pathways and bind to MHC class I and MHC class II molecules, respectively [1].

The assembly and peptide loading of MHC class I and class II molecules is overseen by molecular chaperones in various intracellular compartments 2, 3, 4. Heat shock proteins (hsps) are highly conserved molecules that act as molecular chaperones and control the folding of proteins, preventing their aggregation [4]. The composite function of the ER lumenal chaperones is generally thought to be limited to the structural maturation of nascent polypeptides. However, recent observations have shown that certain chaperones such as GRP94 (gp96), GRP78 (BiP), and protein disulfide isomerase (PDI) display peptide-binding activity that suggests additional roles for these proteins 5, 6, 7, 8, 9, 10. It is now being suggested that these ER chaperones bind peptides suitable for assembly onto MHC class I molecules 6, 7, 11, 12, 13. The functional significance of these peptide-binding ability is demonstrated by the observations that vaccinations of mice with GRP94 can elicit substantial immune response 6, 11, 12, 13, 14. This property suggests the existence of an uncharacterized mechanism by which hsps display antigen and activate MHC class I-restricted T-cells. Up until now, it has been suggested that the strong immune response is due to the ability of the antigen presenting cells (APCs) to internalize hsp-peptide complexes and that, following internalization, peptides bound to hsps are released and targeted for assembly onto class I molecules. The possibility that the hsp-peptide complexes might exist on the cell surface and are able to stimulate a T-cell response has never been considered.

In this study using fluorescent microscopy and biochemical methods, we have found for the first time that MHC class I molecules associate on the cell surface with GRP78, a member of the hsp70 family. In the absence of MHC class I, the expression of GRP78 on the cell surface is increased. Even though the functional significance of this novel interaction remains to be elucidated, we believe that this finding will shed some light on the reported activation of CD8 + T-cells by heat shock proteins.

Section snippets

Materials

All fine chemicals were purchased from Sigma Chemical Co. (St. Louis, MO). Hybridoma cells W6/32 secreting MHC class I specific mAb, were obtained from the American Type Culture Collection (ATCC, MD). Goat polyclonal serum against GRP78 was obtained from Autogen Bioclear (Wiltshire, UK). MCA485 mAb specific for MHC class I as well as MCA1115 mAb specific for β₂-m were obtained from Serotec (Oxford, UK). Rabbit anti-goat Ig conjugated to Fluorescein isothiocyanate (FITC) or horse-radish

Heterotypic associations of MHC class I molecules

We set out to investigate the homo and heterotypic cell surface associations of MHC class I molecules using biochemical studies. ECV-304 cells were surface-labelled with Biotin-NHS reagent, lysed in a variety of nonionic detergents and the MHC class I molecules were immunoprecipitated with W6/32 mAb at 4°C.

Two-dimensional gel electrophoresis of the immunoprecipitates revealed (as expected) the MHC class I heavy chain and β₂-microglobulin (β₂-m), as well as a 70 kDa protein with apparent pI of

Discussion

GRP78 is an ER lumen chaperone that is involved in the folding and assembly of MHC class I antigens. It has been shown to transiently associate with MHC class I in the ER 17, 18, 19.

This study provides strong evidence that MHC class I and GRP78 are associated on the cell surface. This association survives the solubilization step of biochemical methods when nonionic zwitteritonic detergents such as Brij-96 and CHAPS are utilized.

Even though GRP78 is mostly found intracellularly its existence on

Acknowledgements

We would like to thank Dick Winant (Stanford PAN Facility) for help with mass spectroscopy and Ada Nikolaou for help with confocal microscopy. M.T. and K.T. were employed by Grant Nos. C12823 and C10845 awarded by the BBSRC.

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Glucose-related protein 78 (GRP78) is a chaperone protein localized primarily in the endoplasmic reticulum (ER) lumen, where it helps in proper protein folding by targeting misfolded proteins and facilitating protein assembly. In stressed cells, GRP78 is translocated to the cell surface (csGRP78) where it binds to various ligands and triggers different intracellular pathways. Thus, csGRP78 expression is associated with cancer, involved in the maintenance and progression of the disease. Extracellular exposition of csGRP78 leads to the production of autoantibodies as observed in patients with prostate or ovarian cancer, in which the ability to target csGRP78 affects the tumor development. Present on the surface of cancer cells and not normal cells in vivo, csGRP78 represents an interesting target for therapeutic antibody strategies. Here we give an overview of the csGRP78 function in the cell and its role in oncogenesis, thereby providing insight into the clinical value of GRP78 monoclonal antibodies for cancer prognosis and treatment.
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Translocation of 78-kDa glucose-regulated protein (GRP78) from endoplasmic reticulum (ER) to plasma membrane represents a paradigm shift beyond its traditional function as an ER chaperone protein. Cell surface GRP78 (csGRP78) exerts novel signaling functions, and mechanisms underlying its cell surface expression are just emerging. Acquired tamoxifen resistance of breast cancer cells is accompanied with elevated level of csGRP78. Therefore, the tamoxifen-resistant MCF7 breast cancer cells (MCF7-LR) represents a clinically relevant model to study mechanisms of csGRP78 expression. We discovered that a proline-rich region (PRR) containing three consecutive prolines close to the COOH-terminus of GRP78 is important for its ability to form a complex with the partner protein, CD44v, as demonstrated by in vitro glutathione S-transferase pull-down assay. Proline to alanine mutations at the PRR compromised GRP78 expression level on the cell surface as evidenced by purification of biotinylated cell surface proteins. Reconstitution of MCF7-LR cells with the PRR mutant after knockdown of endogenous GRP78 diminished the capacity of GRP78 to stimulate STAT3 activation. The enforced expression of a short peptide bearing the PRR region of GRP78 led to reduction of CD44v and Cyclin D1 protein levels as well as cell viability, accompanied with increase in apoptotic signaling including cleaved Caspase-3 and PARP. These findings suggest that the COOH-terminal PRR of GRP78 is critical for its interaction with CD44v as well as its cell surface expression, and enforced expression of the short peptide bearing the PRR region may provide a new approach to lower the viability of tamoxifen-resistant breast cancer cells.
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Cell surface BiP is involved in transducing signals from ligands as disparate as activated α2-macroglobulin and antibodies, takes part in physiologic processes such as inhibition of tissue factor (TF) procoagulant activity [43,44], and angiogenesis on endothelial cells [45]. The association of BiP with the major histocompatibility complex class I has also been reported [46]. Nevertheless, one of the most interesting fact is that cell surface BiP was utilized as host cell attaching receptor by diverse viruses, including Hepatitis E virus (HEV) [47], Coxsackie virus A9 [48], dengue virus serotype 2 [49], Borna disease virus (BDV) [50], avian leucosis virus subgroup J [51] and Japanese encephalitis virus (JEV) [52].
White spot syndrome virus (WSSV) is a dangerous threat to shrimp farming that also attacks a wide range of crustaceans. Knowledge of the surface protein-protein interactions between the pathogen and host is very crucial to unraveling the molecular pathogenesis mechanisms of WSSV. In this study, LvBiP (Litopenaeus vannamei immunoglobulin heavy-chain-binding protein) was identified as a novel WSSV binding protein of L. vannamei by a biotinylation based affinity chromatography method. By using pull-down and ELISA assays, the binding of recombinant LvBiP to WSSV was proved to be specific and ATP- dependent. The interaction was also confirmed by the result of co-immunoprecipitation assay. Immunofluorescence studies revealed the co-localization of LvBiP with WSSV on the cell surface of shrimp haemocytes. Additionally, LvBiP is likely to play an important role in WSSV infection. Treatment of gill cellular membrane proteins (CMPs) with purified rLvBiP and antibody that specifically recognizes LvBiP, led to a significant reduction in the binding of WSSV to gill CMPs. In the in vivo neutralization assay, rLvBiP and anti-LvBiP polyclonal antibody partially blocked the infection of WSSV. Taken together, the results indicate that LvBiP, a molecular chaperon of the HSP70 family, is a novel host factor involved at the step of attachment of the WSSV to the host cells and a potential candidate of therapeutic target.
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The Glucose Regulated Protein 78,000 (GRP78) is a member of the heat shock protein family in the endoplasmic reticulum to regulate protein quality control and degradation. We and others demonstrated that GRP78 is preferentially translocated onto the cell surface (CS-GRP78) and plays a novel role in cellular signaling, proliferation, migration, invasion, apoptosis, inflammation, and immunity which have major implications in cancer progression. CS-GRP78 acts as a multifunctional receptor by interacting with various ligands and other cell surface proteins to control key oncogenic signaling pathways. Activated α₂-macroglobulin (α₂M*) signals predominately through CS-GRP78 to regulate transcriptional activation of target genes, and, modulates metabolic regulation which might be an adaptive mechanism in rapidly proliferating cancer cells. Therapeutic agents against CS-GRP78 have the potential to target tumor cells with minimal harmful effects on normal cells. This review will focus on our understanding of CS-GRP78 and their influence on fundamental biological processes as well as its targeting agents which hold great therapeutic promise.
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This data implies that targeting cytokines in ER with drugs could be therapeutically exploited to alleviate inflammation and autoimmune diseases. Heat shock protein 70 kDa protein 5 (HSPA5), also named glucose-regulated protein 78 kDa (GRP78), takes part in cell signaling, viral entry and antigen representation [23–25]. 30%–75% RA patients showed overexpressed autoantibodies towards GRP78 in synovial fluid or serum [26,27].
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Secondly, we found a chaperon protein, GRP-78 likely to be more expressed in young animals after LPS injection. This protein is associated at the cell surface with major histocompatibility class 1 molecule, suggesting a role in cell-mediated immune response [65]. In addition, GRP-78 may also fulfil a protective role against endoplasmic reticulum stress-induced cell death [66].
Individual response to an immune challenge results from the optimization of a trade-off between benefits and costs of immune cell activation. Age-related immune disorders may have several mechanistic bases, from immune cell defects to chronic pro-inflammatory status and oxidative imbalance, but we are still lacking experimental data showing the relative importance of each of these mechanisms. Using a proteomic approach and subsequent biochemical validations of proteomics-derived hypotheses, we found age-dependent regulations in the liver of 3-months and 1-year old-mice in response to an acute innate immune activation. Old mice presented a chronic up-regulation of several proteins involved in pathways related to oxidative stress control. Interestingly, these pathways were weakly affected by the innate immune activation in old compared to young individuals. In addition, old mice suffered from lower glutathione-S-transferase activity and from higher oxidative damage at the end of the experiment, thus suggesting that they paid a higher immune-related cost than young individuals. On the whole, our data showed that a substantial fraction of the liver costs elicited by an activation of the innate immune response is effectively related to oxidative stress, and that ageing impairs the capacity of old individuals to control it.
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¹: Present address: University of Portsmouth, School of Biological Sciences, King Henry Building, King Henry I Street, Portsmouth, U.K. (M.T., K.T.).

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Major histocompatibility class one molecule associates with glucose regulated protein (GRP) 78 on the cell surface

Abstract

Introduction

Section snippets

Materials

Heterotypic associations of MHC class I molecules

Discussion

Acknowledgements

Cell

Trends Biochem Sci

J Biol Chem

Curr Opin Immunol

Hum Immunol

Supervising the foldfunctional principles of molecular chaperones

FASEB J

Molecular chaperones in cellular protein folding

Nature

TAP translocated peptides specifically bind proteins in the endoplasmic reticulum including gp96, protein disulfide isomerase and calreticulin

Eur J Immunol

Heat shock protein peptide complexes reconstituted in vitro elicit peptide specific cytotoxic T lymphocyte response and tumour immunity

J Exp Med

Interaction of endoplasmic reticulum chaperone GRP94 with peptide substrates is adenine nucleotide independent

J Biol Chem

Peptide binding and release by proteins implicated as catalysts of protein assembly

Science

Protein disulfide isomerase is the dominant acceptor for peptides translocated into the endoplasmic reticulum

Eur J Immunol

A mechanism for the specific immunogenicity of heat shock protein chaperoned peptides

Science

Cross-priming of minor histocompatibility antigen-specific cytotoxic T cells upon immunisation with the heat shock protein gp96

J Exp Med

Immunotherapy of cancers with autologous cancer-derived heat-shock protein preparations

Science