| Heat shock proteins (HSPs) are the most abundant and ubiquitous soluble intracellular proteins. HSPs perform essential biological functions under both physiological and stressful conditions, such as folding and unfolding of proteins, degradation of proteins, assembly of multisubunit complexes, thermotolerance and buffering the expression of mutations. They are grouped in ten families; members in a family exhibit a high degree of sequence identity, even between prokaryote and eukaryote members, whereas there is no homology between two members in differrent families. HSPs normally constitute up to 5% of the total intracellular proteins; however, under stresses their levels can rise to 15% or more. Typically, antigen presenting cells (APCs) present exogenous antigen on major histocompatibility complex class II (MHC) molecules, and endogenously synthesized antigen on MHC I molecules. In exceptional circumstances exogenous antigens can be presented on MHC I molecules, prime naive CD8+ T lymphocytes, and this phenomenon is called cross priming. HSPs can chaperone exogenous peptides and cross present them to MHC I molecules and lead to antigen specific CD8+ cytotoxic T lymphocyte (CTL) responses. The ability of HSPs, and the peptides chaperoned by them, to be introduced into the endogenous pathway makes them powerful adjuvants for the generation of CD8 + responses. They are the first adjuvants of mammalian origin. Immunization of mice with HSP-peptide complexes also elicits antigen specific MHC class II restricted CD4+ responses. The interaction of HSP-peptide complexes with APCs such as macrophages or dendritic cells leads, on the one hand, to presentation of antigenic peptides to CD8+ and CD4+ T lymphocytes and, on the other hand, to a cascade of non-antigen-specific events that activate APCs and promote immune responses. HSPs have peptide dependent capacity to elicit potent CTL responses and peptide independent ability to modulate innate immunological cells; therefore they serve as a bridge of the innate and adaptive immunities. Mycobacterial heat shock protien 65 (HSP65) belongs to HSP60 family. HSP65 has the ability to help the chaperoned peptide be internalized by antigen presenting cells (APCs), cross-presented to MHC-I molecules and prime peptide specific CD8+ T cells. It has been reported that the receptor for heat shock protein 60 on macrophage is saturable, specific and distinct from receptors for other heat shock proteins; and the receptor for HSP65 is different from human HSP60, that is, different heat shock protein 60 species share pro-inflammatory activity but not binding site on macrophages; Toll-like receptor (TLR-4) can be the signal receptor of HSP60 and Toll/IL-1 receptor signaling pathway is involved in transducing signals in innate immune cells, while some investigators report that the signaling pathway of HSP60 is TLR-4 independent. To date, seven putative HSP receptors have been identified, including CD91, CD40, LOX-1, CD36, TLR-2, TLR-4 and SR-A, but the receptor for HSP65 has not been identified yet. This project aims to explore the existance of receptor for mycobacterial HSP65 on immune cells and its signal transduction related. For doing this, we need to express HSP65-GFP (green fluorescent protein) fusion protein (HSP65-GFP) in which GFP is used as a reporter molecule. The experimental results are as follows:1. Construction, expression and purification of HSP65-GFP and HSP65 To obtain HSP65-GFP fusion protein, we constructed a reconbinant plasmid designated as pET28-HSP65-GFP. Procedures are performed as follows: The gene of GFP was obtained by using PCR method from the plasmid pET28-GFP and ligated to the downstream of HSP65 gene in plasmid pET28-HSP65. The accuracy of HSP65-GFP gene was verified by DNA sequencing. And then the plasmid pET28-HSP65-GFP was transformed into E. Coli. for expressing HSP65-GFP fusion protein. The protein was purified by Ni2+ Sepharose affinity chromatography and identified under fluorescent microscopy and by Western Blot Assay. LPS was eliminated by Q Sepharose to the degree less than 50EU/1mg. In order to study the specificity of the binding of HSP65-GFP fusion protein to cells, the plasmid pET28-HSP65 was transformed into E. Coli for expressing HSP65 protein. The protein was also purified by Ni2+ Sepharose affinity chromatography and identified by Western Blot Assay. LPS was eliminated by Q Sepharose to less than 50EU/1mg. 2. Binding assay of HSP65-GFP fusion protein to murine peritoneal macrophage To explore the receptor for HSP65 on the cell surface, we observed the binding of HSP65-GFP to murine peritoneal macrophage. 2.1. Influence of temperature and time on the binding assay of HSP65-GFP to murine peritoneal macrophage We performed the binding assay of HSP65-GFP to murine peritoneal macrophage at 0 ℃,25 ℃,37 ℃respectively in serum free medium for 20min, 30 min, 45 min. The binding was observed under confocal microscopy. It was found that under the conditions of 0 ℃45 min and 25 ℃30 min, HSP65-GFP protein (1 μmol/l) can bind the cell membrane of murine peritoneal macrophage, indicating that there maybe receptors for HSP65 on murine peritoneal macrophage. We also found that at 0 ℃cells are easy to shrink, therefore 25 ℃30 min is selected to be the best condition of the binding assay and used in all the following experiments. 2.2. Influence of fetal serum on the binding assay of HSP65-GFP to murine peritoneal macrophage We found that 10% fetal bovine serum (FBS) can dramatically inhibit the interaction of HSP65-GFP and murine peritoneal macrophage. This inhibitory effect was subsequently indetified to be non-specific. Therefore we perform the following binding assays in serum free medium. 2.3. Competition assay of HSP65 on the binding of HSP65-GFP to murine peritoneal macrophage To study the specificity of the binding of HSP65-GFP to murine peritoneal macrophage, we added 10 folds excess HSP65 as well as HSP65-GFP and found that HSP65 failed to compete the binding of HSP65-GFP to murine peritoneal macrophage, that is, our current results do not support the existance of receptors for HSP65 on murine peritoneal macrophage. But it is too early to draw a conclusion until the following questions are clarified: the difference of the conformation between recombinant and native proteins; hinderance of GFP on the binding; weakness of biological fluorescent signals of GFP; the difference of the conformation between HSP fusion protein and HSP alone; the sensitivity of experiments;interference of LPS; the cell types used etc. 3. Binding assay of HSP65-GFP to human peripheral blood mononuclear cells (PBMCs) The attached and suspending cells were obtained from human PBMCs, half of which were treated at 42 ℃for 1 hour and the other half in 37℃. We observed the binding of HSP65-GFP to both attached cells and suspending cells, meanwhile included murine peritoneal macrophage as a control. Under confocal microscopy, we found that HSP65-GFP could bind the attached cells of human PBMCs and this binding effect was enhanced after heat treatment. This result indicates that receptors for HSP65 may exist on the attached cells of human PBMCs. 4. Binding assay of HSP65-GFP fusion protein to U937 cell line The fact that HSP65-GFP can bind the attached cells of human PBMCs prompted us to do some experiments on U937 cell line (human monocyte). We performed the binding assay of HSP65-GFP to U937 cells and found that HSP65-GFP was unable to bind U937 cells. 5. Binding assay of HSP65-GFP fusion protein to human platelet Platelets was reported to be a factor to regulate the level of HSP65 in mouse, hence we tested the binding of HSP65-GFP to human platelet. Under confocal microscopy, we found that HSP65-GFP failed to bind isolated human platelets. All together, we successfully produced recombinant HSP65-GFP fusion protein and HSP65. Using recombinant HSP65-GFP fusion proteins as molecular probes, we provided the evidence that the receptors for HSP65 might exist on murine peritoneal macrophage and the attached cells of human... |
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