C57BL/6 마우스에서 비만에 의해 유도되는 ApoB-100에 대한 자가항체 연구

Abstract

비만은 단순한 하나의 질병이 아니라 여러 가지 대사 성 질환을 동반 함으로써 보건의료뿐 만 아니라 사회적인 문제로 의료비용과 관심이 크게 증가하고 있다. 특히 이러한 질병은 저밀도 지단백질의 대사 이상과 밀접한 관련을 보이고 있다. 저밀도 지단백질은 혈중 중성지방과 콜레스테롤의 주요 운반 형태이며, 조직에 존재하는 LDL 수용체에 결합하여 지방을 축적하거나 스캐빈저(청소)수용체에 의해서 탐식, 분해된다. 이 역할을 매개하는 물질은 저밀도 지단백질(Low Density Lipoprotein, LDL)의 단백질 성분인 Apolipoprotein B-100(ApoB-100)이다. 저밀도 지단백질은 지방질 함량에 따른 비중차이에 의해 VLDL, IDL 및 LDL로 구분하며, 이들은 각각 하나의 ApoB-100을 포함하고 있다. 본 연구에서는 비만이 유도됨에 따라 혈중 ApoB-100에 대한 자가항체가 유도 생성되는 것을 확인하였고, 비만도(Body Mass Index)와 자가항체 유도의 상관성을 조사하여 그 유의성을 분석하였다. 비만의 정도는 BMI인 체중/(신장의 제곱)(g/㎠)으로 정량화하였고, ApoB-100에 대한 자가항체의 유도 생성은 ApoB-100을 항원으로 사용한 Indirect ELISA법으로 측정하였다. 정상식이(조 지방 5%)를 섭취한 C57BL/6 마우스군의 BMI는 22주 동안 0.270±0.012에서 0.338±0.024로 증가한 반면, 60% High fat diet를 급식하여 비만을 유도한 마우스는 동일 기간에 0.280±0.017에서 0.438±0.024로 증가하였다. 비만에 따른 ApoB-100에 대한 자가항체의 유도 생성은 정상 대조군의 경우 동일 22주간 동안 -0.001±0.003으로부터 0.020±0.028로 변화가 없었으나, 비만유도군의 경우 -0.003±0.002로부터 2.328±1.691(Z=2.785, p=0.003)로 증가하였다. 비만 유도에 따른 자가항체 유도의 상관성은 유의(Spearman rho=0.49, p-value=0.001)한 것으로 검증되었다. 유도된 자가항체가 ApoB-100을 인식하는 특성을 근거로 비만 마우스의 혈청을 이용하여 본 연구진의 선행연구에서 작성한 ApoB-100의 mimetic peptide인 B4그리고 human LDL에 대한 반응 성을 검토하였다. 그 결과 hLDL과 hApoB-100은 동일한 양상으로 항혈청과 반응하였다. 흥미롭게도, 유도된 자가항체는 인공 모조항원인 B4를 인식할 뿐만 아니라 B4의 기본단위 단량체인 PB1에 대한 단클론 항체 22B4와도 경쟁반응을 보여 이들 두 항체들이 동일한 에피토프를 인식함을 예측할 수 있었다. 따라서, 유도된 자가항체의 에피토프를 단클론 항체 22B4를 이용하여 규명하고자 하였다. 실험 방법은 ApoB-100과 22B4을 결합시켜 에피토프 부분을 보호시킨 다음, protease에 의한 partial digestion후 Western blotting과 탐지된 펩티드의 N-terminal 아미노산 배열 결정에 의해 peptide mapping을 시도하였으나 결과를 얻지 못하였다. 이에 대하여 ATCC사의 제품인 항ApoB-100 단클론 항체 B1B6를 구매하여 동일 방법으로 에피토프 결정을 시도한 결과 ApoB-100의 3333-3337번 아미노산 배열을 인식하는 것을 확인할 수 있었다. 본 연구의 결과로부터, 비만도인 BMI보다 자가항체의 역가가 병리적인 비만의 보다 정확한 진단 지표로 활용될 가능성을 시사하고 있으며, 결론적으로 비만이 유도되면 이에 대한 생체 방어작용의 일환으로 Apo B-100에 대한 자가 항체가 유도 생산되어 저밀도 지 단백질의 지방 축적 기능을 차단함으로 체중의 증가를 억제하는 기전이 존재함을 제시할 수 있게 되었다. |Obesity is considered a pandemic and is associated with numerous metabolic disease comorbidities. Also, it is a major public health problem in respect of direct and indirect medical costs and social stigma. Obesity is closely related with lipid metabolism including lipid-rich lipoproteins. The lipoproteins are the major carrier of triglyceride and cholesterol to the peripheral tissues by the interaction between apolipoprotein B-100(Apo B-100) on themselves, and specific lipoprotein receptors on the tissue cells. Lipoproteins are classified into very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL) and low-density lipoproteins (LDL) based on the particle density, and each of them harbors one molecule of Apo B-100, respectively. From the previous experiments, there was a strong implication for the induction of Apo B-100 recognizing auto-antibodies with the progression of obesity from the high-fat diet fed mice. In the present study, the phenomena were re-confirmed and the correlation between body-mass index (BMI) and autoantibody induction was analyzed statistically. The characterization of the obesity-induced auto-antibody from C57BL/6 mice was also performed. BMI for the normal diet fed control group C57BL/6 mice was changed from 0.27±0.012 g/cm2 (at the 11th week of age) to 0.33±0.024 g/cm2 (at the 33rd week of age), whereas, BMI for 60% high fat diet fed obese group mice was increased from 0.028±0.017 g/cm2 to 0.438±0.024 g/cm2 for the same period of 22 weeks. Induction of autoantibody to ApoB-100 from the control lean group mice was negative and the antibody titers determined by indirect ELISA were 0.001±0.003 OD at 11th week to 0.020±0.028 OD at 33rd week of age. On the other hand, from the obese group mice, the antibody titer was significantly increased from -0.003±0.002 OD at 11th week to 2.328±1.69 OD at 33rd week of age. The correlation between BMI and antibody titers were analyzed statistically using Spearman coefficients. The Spearman rho value between BMI and the antibody induction titer from lean control group was -0.065(p=0.689) whereas, that from obesity group was 0.409(p=0.001). Therefore, the significant and solid co-relationship between BMI and autoantibody induction was confirmed and proved. The obesity induced auto-antibody recognized LDL as a natural antigen which was found from the previous experiments. Extended screening with other antigenic proteins, it was confirmed that the auto-antibody can recognize ApoB-100 and also B4RB2 which is an Apo B-100 mimetic artificial peptide used for obesity vaccine. Furthermore, the auto-antibody and B4RB2 specific monoclonal antibody 22B4 (mAb 22B4), were competed with each other in recognition reactions of ApoB-100, B4RB2 and also of LDL. The result explains that the obesity-induced auto-antibody and mAb 22B4 shared the same antigenic determinant on Apo B-100. The epitope was determined with mAb22B4 in substitute for auto-antibody. However, it was impossible to address the epitope for mAb22B4 on ApoB-100. However, with a commercially available ApoB-100 specific mAb B1B6, in contrast to the mAb22B4, the epitope on Apo B-100 was successfully determined by the same methodology. The epitope of B1B6 was determined to be the peptide containing the amino acids sequence of Ser-Asp-lle-Val– Ala. This sequence was exactly matched with the peptide on Apo B-100 at amino acids 3333-3337. Therefore, as mAb 22B4 recognizes not a linear but a conformational epitope on Apo B-100, it was impossible to determine the antigenic determinant on the Apo B-100. In summary, the followings were confirmed from the present studies that 1) obese individuals induced autoantibody against ApoB-100, 2) BMI and the autoantibody induction has a statistically significant co-relationship and 3) the obesity induced autoantibody recognized conformational antigenic determinant on Apo B-100 which can affect the lipoprotein receptor binding. It is strongly suggested that the obesity induced auto-antibody against Apo B-100 might alleviate the progression of obesity and also it can be developed as a biochemical molecule for diagnosis or treatment of obesity.; Obesity is considered a pandemic and is associated with numerous metabolic disease comorbidities. Also, it is a major public health problem in respect of direct and indirect medical costs and social stigma. Obesity is closely related with lipid metabolism including lipid-rich lipoproteins. The lipoproteins are the major carrier of triglyceride and cholesterol to the peripheral tissues by the interaction between apolipoprotein B-100(Apo B-100) on themselves, and specific lipoprotein receptors on the tissue cells. Lipoproteins are classified into very-low-density lipoproteins (VLDL), intermediate-density lipoproteins (IDL) and low-density lipoproteins (LDL) based on the particle density, and each of them harbors one molecule of Apo B-100, respectively. From the previous experiments, there was a strong implication for the induction of Apo B-100 recognizing auto-antibodies with the progression of obesity from the high-fat diet fed mice. In the present study, the phenomena were re-confirmed and the correlation between body-mass index (BMI) and autoantibody induction was analyzed statistically. The characterization of the obesity-induced auto-antibody from C57BL/6 mice was also performed. BMI for the normal diet fed control group C57BL/6 mice was changed from 0.27±0.012 g/cm2 (at the 11th week of age) to 0.33±0.024 g/cm2 (at the 33rd week of age), whereas, BMI for 60% high fat diet fed obese group mice was increased from 0.028±0.017 g/cm2 to 0.438±0.024 g/cm2 for the same period of 22 weeks. Induction of autoantibody to ApoB-100 from the control lean group mice was negative and the antibody titers determined by indirect ELISA were 0.001±0.003 OD at 11th week to 0.020±0.028 OD at 33rd week of age. On the other hand, from the obese group mice, the antibody titer was significantly increased from -0.003±0.002 OD at 11th week to 2.328±1.69 OD at 33rd week of age. The correlation between BMI and antibody titers were analyzed statistically using Spearman coefficients. The Spearman rho value between BMI and the antibody induction titer from lean control group was -0.065(p=0.689) whereas, that from obesity group was 0.409(p=0.001). Therefore, the significant and solid co-relationship between BMI and autoantibody induction was confirmed and proved. The obesity induced auto-antibody recognized LDL as a natural antigen which was found from the previous experiments. Extended screening with other antigenic proteins, it was confirmed that the auto-antibody can recognize ApoB-100 and also B4RB2 which is an Apo B-100 mimetic artificial peptide used for obesity vaccine. Furthermore, the auto-antibody and B4RB2 specific monoclonal antibody 22B4 (mAb 22B4), were competed with each other in recognition reactions of ApoB-100, B4RB2 and also of LDL. The result explains that the obesity-induced auto-antibody and mAb 22B4 shared the same antigenic determinant on Apo B-100. The epitope was determined with mAb22B4 in substitute for auto-antibody. However, it was impossible to address the epitope for mAb22B4 on ApoB-100. However, with a commercially available ApoB-100 specific mAb B1B6, in contrast to the mAb22B4, the epitope on Apo B-100 was successfully determined by the same methodology. The epitope of B1B6 was determined to be the peptide containing the amino acids sequence of Ser-Asp-lle-Val&#8211