Development of chimeric navigator to capture and remove beta2-microglobulin in the blood

Abstract

Event Abstract Back to Event Development of chimeric navigator to capture and remove beta2-microglobulin in the blood Yusuke Kambe1 and Tetsuji Yamaoka1 1 National Cerebral and Cardiovascular Center Research Institute, Department of Biomedical Engineering, Japan Introduction: Decreased renal functions lead to the increased blood level of β2-microglobulin (β2mg), resulting in serious complications. To reduce the β2mg level, special dialysis has been conducted clinically, but there remain issues such as unexpected removal of serum components. Thus, a new methodology is required to remove β2mg selectively. We reported a novel strategy “drug-navigated clearance system (DNCS)”[1],[2], whose concept is that an etiologic agent is navigated from its original metabolic pathway to another one by “navigator” molecules (Fig. 1). Based on DNCS, we here addressed the development of a “chimeric navigator” which captures and removes β2mg. The α3 chain of H2-Db, which is a mouse MHC class I, was selected as the β2mg-capturing molecule, whereas mouse apolipoprotein E N-terminal domain (mApoE-NTD) was used to navigate β2mg to the liver LDL receptor (LDLR) for being evaluated in a mouse model. These two proteins were fused as the chimeric navigator and its characteristics were evaluated in vitro. Materials and Methods: Gene sequences of mApoE-NTD and H2-Db α3 were amplified by PCR, whose products were cloned into a expression vector, with the origonucleotide coding (GGGGS)3 being between the products. Thus, the chimeric navigator is composed of mApoE-NTD–(GGGGS)3–H2-Db α3. BL21 (DE3) cells transformed with the vector were cultured to produce the navigator. To evaluate the β2mg-capturing ability of the navigator, β2mg and the navigator were mixed in PBS and an immunoprecipitation assay with anti-β2mg or anti-H2-Db α3 antibodies were conducted. The immunocomplexes were analyzed by SDS-PAGE and western blotting with anti-β2mg antibody. A fluorescent navigator containing Umikinoko Green (UKG) fluorescent protein (mApoE-NTD-(GGGGS)3-UKG) was generated to evaluate the specific binding of mApoE-NTD to LDLR. The fluorescent navigator was mixed with phospholipid (DMPC) to form a complex, which was then added to the culture medium of mouse liver (NMuLi) cells. Additionally, the complex of commercial mApoE (CmApoE) and DMPC was added to the medium at 0, 1, 5, and 10-fold mole ratios to the navigator–DMPC complex. Cells were observed by a fluorescent microscope. Results: Figure 2 shows the immunoprecipitation results. β2mg appeared to bind not only to anti-β2mg antibody (arrows) but also to anti-H2-Db α3 antibody (arrowheads) (A). Similarly, the navigator bound to both anti-H2-Db α3 antibody (arrow) and anti-β2mg antibody (arrowhead) (B). Fluorescent images of NMuLi cells incubated with the fluorescent navigator and various concentrations of CmApoE are shown in Fig.3. The fluorescent intensity of the navigator bound to the cells decreased with an increase in the CmApoE concentration. Discussion: As anti-β2mg and anti-H2-Db α3 antibodies had no affinity to the navigator and β2mg, respectively, the binding between β2mg and anti-H2-Db α3 antibody was mediated by the navigator. Similarly, β2mg mediated the navigator’s binding to anti-β2mg antibody, indicating that the navigator successfully captured the target β2mg. This ability is likely due to the affinity between the MHC class I and β2mg[3]. Binding of the fluorescent navigator to NMuLi cells was inhibited competitively by CmApoE, suggesting that the binding of the navigator to the cells is mediated by LDLR as well as CmApoE-NMuLi cell binding[4]. Conclusions: The chimeric navigator could capture β2mg and specifically bind to LDLR. JSPS KAKENHI Grant Number 15K16335