Identification and Characterization of chCR2, a Protein That Binds Chicken Complement Component 3d

Abstract

Complement receptor type 2 (CR2) is an important membrane molecule expressed on B cells and follicular dendritic cells. Human CR2 has been shown to play a critical role in bridging the innate complement-mediated immune response with adaptive immunity by binding complement component 3d (C3d). However, the chicken CR2 (chCR2) gene has not been identified or characterized. In this study, unannotated genes that contain short consensus repeat (SCR) domains were analyzed based on RNA sequencing data for chicken bursa lymphocytes, and a gene with >80% homology to CR2 from other bird species was obtained. The gene consisted of 370 aa and was much smaller than the human CR2 gene because 10–11 SCRs were missing. The gene was then demonstrated as a chCR2 that exhibited high binding activity to chicken C3d. Further studies revealed that chCR2 interacts with chicken C3d through a binding site in its SCR1–4 region. An anti-chCR2 mAb that recognizes the epitope 258CKEISCVFPEVQ269 was prepared. Based on the anti-chCR2 mAb, the flow cytometry and confocal laser scanning microscopy experiments confirmed that chCR2 was expressed on the surface of bursal B lymphocytes and DT40 cells. Immunohistochemistry and quantitative PCR analyses further indicated that chCR2 is predominantly expressed in the spleen, bursa, and thymus, as well as in PBLs. Additionally, the expression of chCR2 varied according to the infectious bursal disease virus infection status. Collectively, this study identified and characterized chCR2 as a distinct immunological marker in chicken B cells.