Enhanced Capacity of Antigen Presentation of HBc-VLP-Pulsed RAW264.7 Cells Revealed by Proteomics Analysis Fu Yang, Fang Wang,* Yingjun Guo, Qi Zhou, Yue Wang, Yixuan Yin, and Shuhan Sun* Department of Medical Genetics, Second Military Medical University, 800 Xiang Yin Road, Shanghai 200433, P. R. China Received July 19, 2008
Many recent studies have indicated that virus-like particles (VLPs) have many potential applications in the fields of vaccine development and gene therapy. However, we still know little about the subtle mechanisms involved in the presentation of VLPs by antigen presenting cells (APCs). To illustrate the mechanisms, we utilized two-dimensional electrophoresis and tandem MS to compare and identify differentially expressed proteins between hepatitis B virus core antigen VLP (HBc-VLP)-pulsed and control RAW264.7 cells. Of the 25 spots identified as differentially expressed (p < 0.05) between the two cell lines, 11 (corresponding to 11 unique proteins) were positively identified. Further analysis of two proteins, prohibitin and heat shock protein 70, confirmed that these proteins are expressed at higher levels in HBc-VLP-pulsed RAW264.7 cells compared with control cells. The proteins identified in this study will be useful in revealing the mechanisms that underlie VLP-APC interactions. Overall, this study also provides some useful suggestions for vaccine development and gene therapy. Keywords: virus-like particles • RAW264.7 • proteomics • antigen capture • antigen presentation
Introduction Virus-like particles (VLPs) are highly organized spheres that self-assemble from virus-derived structural antigens. They offer a promising approach to the production of vaccines against many diseases, because their repetitive, high-density display of epitopes is often effective in eliciting strong immune responses.1,2 Recent studies have indicated that human papilloma virus (HPV) VLPs bind to dendritic cells (DCs) and stimulate their maturation, including the up-regulation of major histocompatibility complex class I and II, CD86, CD80 and cytokine production.3,4 It is noteworthy that VLPs assembled from HPV major capsid protein L1 in yeast were capable of inducing protective immune responses against HPV subtypes 16 and 18, which cause cervical cancer in humans, thus, resulting in a safe, well-tolerated and highly immunogenic vaccine that received approval for marketing in 2006.5 However, the mechanisms of uptake, processing and presentation of the exogenous particles remain unclear. Indeed, DCs and macrophages have been shown to be involved in the processing of VLPs,6,7 but no direct evidence has been obtained to identify the subtle mechanisms involved in presentation of VLPs by APCs. The RAW264.7 cell line was initially derived from Balb/c mice injected with Abelson murine leukemia virus.8 This cell line secretes lysozymes, phagocytoses zymosan- and antibody* To whom correspondence should be addressed. Professor Shuhan Sun, Department of Medical Genetics, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, P.R.China; phone, +86-21-25070331; Fax, +86-21-25070331; e-mail,
[email protected]. Professor Fang Wang, Department of Medical Genetics, Second Military Medical University, 800 Xiangyin Road, Shanghai 200433, P.R.China; phone, +86-21-25070333; fax, +86-21-25070331; e-mail,
[email protected].
4898 Journal of Proteome Research 2008, 7, 4898–4903 Published on Web 10/09/2008
coated erythrocytes, expresses immunoglobulin Fc receptors, and is designated as a macrophage cell line.8 Subsequently, this cell line has extensively been used to study APC functions. Development of proteomics technology has provided a powerful tool to study the complicated biological processes in cells, which results in rapid profiling of global alterations in protein expression.9,10 In the present study, we developed an antigen delivery system based on hepatitis B virus core antigen (HBc)-VLP. We found that HBc-VLP was well-captured by RAW264.7 cells in vitro. Furthermore, two-dimensional electrophoresis (2-DE) and tandem MS (MS/MS) analysis were utilized to analyze differential protein expression between HBcVLP-pulsed and control RAW264.7 cells. We detected significantly modulated expression of 25 protein spots, identified 11 differentially expressed proteins from 11 of these spots, and validated the differential expression patterns of certain identified proteins by Western blotting. Proteins identified included those that function in diverse biological processes such as RAW264.7 cell capture and presentation of VLPs. These results illustrate the utility of differential proteomic analyses for obtaining new information and illuminating the mechanism of RAW264.7 cell capture and presentation of VLPs.
Materials and Methods The Construction, Characterization, and Purification of Recombinant HBc Particles. HBc-VLPs were generated as follows. The truncated HBc gene (aa 1-144) was cloned by PCR from the complete viral genome of Hepatitis B virus (subtype adr) and was introduced into the NdeI/XhoI restriction sites of expression vector pET28a (Merck) under a T7/lac promoter. Escherichia coli BL21 (DE3) were transfected with the plasmids 10.1021/pr800547v CCC: $40.75
2008 American Chemical Society
research articles
Enhanced Capacity of Antigen-Presentating Cells and the cells were induced by isopropyl b-D-thiogalactoside (Sigma). The protein was obtained from inclusion body and purified by avidity chromatography on a Ni2+-agarose column (GenScript). The particles were obtained after routine renaturation. Endotoxin levels in VLPs preparation were measured using the Limulus amebocyte lysate test (BioWhittaker, Inc.) and were in each case
