Characterization of Variable Region Structures and Antigen-Binding Properties of OxLDL-Specific Human Monoclonal Antibody Isolated from PBMC and Atherotic Plaques of Atherosclerosis Patients

PURPOSE: Atherosclerosis is an inflammatory response to the accumulated cholesterol in the artery wall. Oxidative modification of low-density lipoprotein (LDL) plays an important role in the pathogenesis of atherosclerosis and the oxidized LDL (oxLDL) may represent a target for autoantibodies involved in the pathogenesis of the disease. The aim of this study was; first, analysis of the Ag-binding characteristics of anti-oxLDL autoantibodies isolated from atherosclerosis patients' PBMC; second, determination of the VH and VL sequences of the Fab produced from the plaques and comparison them with those from PBMC. This study will improve the knowledge of the mechanism which are related to the pathogenesis of atherosclerosis and provide a basis for developing of new therapeutics which can regulate the pathogenesis. MATERIALS AND METHODS: Soluble forms of the anti-oxLDL Fabs isolated from PBMC were purified using Ni-NTA agarose, and their affinities and specificities to native LDL and oxLDL were measured by surface plasmon resonance. Using phage display, we have also cloned anti-oxLDL monoclonal antibodies from atherosclertic plaques. The VH and VL sequences of the Fab produced from the plaques were compared with those from PBMC. The total RNA were isolated from atherotic plaques. PCR was then used to amplify cDNA synthesized from RNA and overlapping extention PCR was performed to produce a Fab library. Fab genes were cloned into the phagemid vector, pComb3X-ss which contains gene Ⅲ coding minor coat protein pⅢ of phage. Recombinant pComb3X-ss containing the Fab genes was transformed into E. coli XL1-Blue to produce recombinant phage Fab. phage Fabs which showed high affinities to oxLDL have been selected, and their phagemids have been purified for sequencing the variable regions of the Fab. To analyze activation-induced cytidine deaminase (AID) mRNA transcripts by RT-PCR, cDNA which synthesized from RNA isolated from atherosclerotic plaques was used as template. RESULTS: The affinities of the anti-oxLDL Fabs isolated from PBMC were measured. It is shown that the purified eight anti-oxLDL Fabs originated from PBMC are all specific to oxLDL; the affinities to oxLDL (K_(D) : 1.77 × 10^(-6) ∼ 9.27 × 10^(-8)) are 10~1000 folds higher than to native LDL (K_(D) : 2.59 × 10^(-4) ∼ 1.07 × 10^(-7)) in each Fab. Fabs which include more positively charged amino acids than negatively charged amino acids tend to have higher affinities to oxLDL, although there are some exceptions. The VH and VL sequences of the anti-oxLDL autoantibodies isolated as recombinant phage Fabs from the atherosclerotic plaques were determined and compared with those from PBMC. The Fabs isolated from the plaques tend to be similar to those from PBMC; The VH3 gene family, JH4 segment, Vκ1 gene family and Jκ1 gene segment are frequently used; The length of CDR3 of the heavy chain are heterogeneous; The ratios of replacement-to-silent mutation (R/S) in the CDRs was higher than those in the FRs of most of VH and VL; Many positively charged amino acids occurs in the CDRs of VH and VL of the antibodies. Comparison of the genes of Fabs with their germline counterparts revealed that overall similarity of VH to germline genes was 84~98%, those of VL was 86~98%. Evidence of somatic hypermutation and class switch recombination was also obtained by analyzing the local expression of mRNA transcripts encoding AID in the atherosclerotic plaque. CONCLUSION: The oxLDL-specific human monoclonal antibody Fabs which have 10~1000 folds higher affinities to oxLDL than LDL were isolated from atherocsclerosis patients' PBMC. The Fabs which include more positively charged amino acids than negatively charged amino acids tend to have higher affinities to oxLDL. The human monoclonal antibodies containing binding properties with affinity to oxLDL higher than to LDL may be used as potential agents for detection or evaluation of oxLDL in human serum or exploring the characteristics of atherosclerotic artery. Sequences of anti-oxLDL Fabs isolated from atherosclerotic plaques tend to have similar characteristics to those from PBMC of the patients. Many positively charged amino acids occurs in the CDRs of VH and VL, suggesting it might be one of the properties that allow binding to negatively charged oxLDL. The overall results might suggest that in the both PBMC and atherotic plaques of atherosclerosis patients, anti-oxLDL antibodies are produced from B cell clones which have been selected through antigen-driven process and undergone affinity maturation, and are originated from similar B cell clones. It is additionally suggested that somatic hypermutation and class switch recombination might be occurred in the B cells found in the patients' plaques. In this study, we analyzed the molecular structures of human anti-oxLDL monoclonal autoantibodies to investigate the origin and mechanism of the antibody production which are related to the pathogenesis of atherosclerosis. We also produced human monoclonal antibodies as forms of Fab which are specific to oxLDL and may be useful in specific detection of oxLDL in human body.

Author dc:creator

• 서, 창원

Contributors dc:contributor

• 장, 영주
• 대학원 의학과
• 200324268

• autoantibody
• oxLDL
• phage diplay
• Fab
• atherosclerosis

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000000418
000000000418

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/2324