인유두종 바이러스 16형 E7 종양 단백의 인산화에 따른 항원성의 변화
Change of the Antigenecity of Human Papillomavirus Type 16 E7 Oncoprotein according to Phosphorylation
대한암학회지 1998년 30권 2호 p.313 ~ 320
소속 상세정보
박노현/Noh Hyun Park
기선호/노주원/김재원/송용상/강순범/이효표/Sun Ho Kee/Joo Won Noh/Jae Weon Kim/Yong Sang Song/Soon Beom Kang/Hyo Pyo Lee
KMID : 0360319980300020313
Abstract
결론
본 연구에서는 E7의 인산화에 따른 영향 중 항원성의 변화 여부를 조사하고자 우선 인산
화가 이루어지지 않은 대장균 발현 E7에 대해 단주 항체를 제작하였고 결손 변체 제작을
통해 잠재적인 항원성 부위를 결정하였으며 CK Ⅱ를 사용한 in vitro 인산화 분석과 CK Ⅱ
저해제인 DRB를 이용한 실험을 통해 두 가지 단주 항체중 인산화가 예상되는 부위와 반응
하는 항체(IB10)가 E7 단백이 인산화됨으로써 항원성이 감소된다는 것을 증명하였다. 이는
E7의 인산화에 따라 항원성과 같은 생물학적 특성에 영향을 줄 수 있다는 사실을 보인 것
으로 이러한 기능의 변화는 Rb와의 결합 또는 별개의 기전을 거쳐서 자궁경부의 암화과정
에 일정한 영향을 줄 가능성을 시사하는 소견으로 생각된다.
Purpose : It was suggested that immunogenic region of E7 proteins of human
papillo-mavirus (HPV) type 16 encompass casein kinase (CK) Ⅱ phosphorylation site
and the resulting negative charge may affect the various biologic function of E7 protein.
This study was undertaken to analyze the change of antigenic characteristics of HPV
type 16, E7 oncoprotein according to phosphorylation.
Materials and Methods : We produced two monoclonal antibodies (VD6 and IB10)
which showed different reactivities to E7 proteins expressed from bacteria or extracted
from CaSki cell. These reaction were analyzed by Western blotting. Also the antigenic
sites estimation of these antibodies using nested deletion sets was done. On the basis of
above experiments, we performed in vitro phosphorylation assay using CK Ⅱ and its
specific inhibitor, DRB (5, 6-dichloro-1-β-D-ribofuranosylbenzimidazole), to analyze the
IB10 reactivity to E7 oncoproteins according to phosphorylation.
Results : In Western blot analysis, VD6 and IB10 antibodies reacted strongly to
bacterially expressed 27 protein. But using E7 extracted from CaSki cell, VD6 reacted to
2.0 kDa E7 protein whereas IB10 showed weak reactivity. The antigenic sites estimation
of these antibodies showed that antigenic site of VD6 was located in amino terminal
region and that of IB10 in the middle portion in the range of approximate amino acid 25
∼45. The antigenic site of IB10 might contain the possible phosphorylation sites
(Ser-31, 32) in E7. Considering this, the different reactivities of IB10 to E7 proteins
expressed in bacteria and extracted from CaSki cell might be due to phosphorylation. In
in vitro phosphorylation assay using CK Ⅱ the phosphorylation of E7 increased
according to reaction time. And this phosphorylation reduced the reactivity of IB10 to E7
protein whereas the reactivity of VD6 did not change. Also the reactivity of IB10 to E7
protein increased in a dose dependent manner with CK Ⅱ specific inhibitor, DRB treated
CaSki cell extracts.
Conclusion : These result showed the antigenecity is affected by the degree of
phosphorylation of E7 protein.
키워드
HPV 16; E7; Phosphorylation; Antigenecity;
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