S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Biomedical Sciences (대학원 의과학과) Theses (Ph.D. / Sc.D._의과학과)
Identification and characterization of antibodies to CTD of RPB1 enriched in the sera of centenarians
백세인 혈청에 높은 농도로 존재하는 RPB1 CTD에 대한 항체의 동정과 특성 규명
- 의과대학 의과학과
- Issue Date
- 서울대학교 대학원
- Aging; centenarian; phage display; RNA polymerase II; RPB1; carboxy terminal domain (CTD); phosphorylation
- 학위논문 (박사)-- 서울대학교 대학원 : 의과학과, 2016. 8. 정준호.
- The humoral immunity has been evolved to protect the host. For an individual it is one of the greatest biological advantages to harbor effective repertoire of humoral immunity toward the various hostile agents like bacteria, virus or cancers. In this context, it has been a long-waiting unanswered question whether the centenarians do have a special repertoire of humoral immunity giving them the ability to survive longer than general population. Recently the phage display technology made it possible to define the circulating repertoire of humoral immunity. This study was designed to define the circulating repertoire of antibodies specific to centenarians.
To achieve this goal, the sera of three populations
centenarian, old (healthy volunteers between age 60 and 79), and young (healthy volunteers younger than 43), were collected. The IgG fractions were purified from the centenarians sera, pooled and used to enrich specific phages from the phage display of combinatorial peptide library through biopanning. The phages reactive to the pooled IgG fractions of centenarians, but not to those of the other groups were selected by a phage enzyme immunoassay and their sequences were determined by nucleotide sequencing. Interestingly, phage clones encoding two homologous peptides, YSATLRY and YSPTLFY, were repeatedly found. In enzyme immunoassay, these two peptides, whether displayed on phage or chemically synthesized and conjugated to bovine serum albumin, reacted with centenarians IgG fractions with much higher frequency than other groups IgG fractions. As the antibody titers to these two peptides were highly correlated with each other, it was expected that these two peptides actually represent the same epitope of an antigen.
To characterize and define the antigen represented by these two homologous peptides, polyclonal antibodies reactive to these peptides was purified from human sera. From the human cancer cell line LoVo, proteins were immunoprecipated by the purified polyclonal antibodies and subjected to SDS-polyacrylamide gel electrophoresis. The identity of the antigen was determined by mass spectrometry analysis and turned out to be the DNA-directed RNA polymerase II subunit RPB1 (RPB1)
Identity of the antigen was once more confirmed in immunoblot analysis using several antibodies specific to the antigen. For establishing the stable source of the antibody, a phage display of combinatorial antibody library was constructed using mRNA prepared from peripheral mononuclear cell fractions of volunteer possessing the antibody to the epitope. Through biopanning on YSATLRY, several clones reactive to both of the peptides, and the antibodies were successfully selected. Especially, one of these monoclonal antibodies had high affinity to the phosphorylated C-termial domain (CTD) of RPB1.
To extensively define the phosphorylated form of RPB1 CTD, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 antibody (trastuzumab) with artificial sequences of 7–18 amino acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides representing typical phosphorylation patterns on the functional unit (YSPTSPS)2 of the RPB1 CTD. Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity to peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5), or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RPB1 CTD in immunoblot analysis. Interestingly, in genome-wide chromatin immunoprecipitation sequencing analysis, pCTD-2ndS2 precipitated predominately RNA polymerase II on the exonic regions of genes, which suggests that the phosphoserine at the second residue of the second repeat on the functional unit (YSPTSPS)2 is a mediator of exon definition.
In conclusion, we confirmed that centenarians do have a specific antibody in much higher frequency than the general population, and the antigen was identified as RPB1. We also have developed antibodies against its multiple phosphorylated form.