Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels
Abstract
:1. Introduction
2. Results
2.1. Expression, Purification and Disulfide Scrambling of Region-1 And -2 of the Catalytic Domain of PCSK9
2.1.1. Expression and Purification
2.1.2. Disulfide Scrambling
2.2. X-Pcsk9 Isomers Produced Antibody Against Native Pcsk9 in Mice
2.3. X-PCSK9 Immunogens Decreased Plasma Cholesterol Levels in C57BL/6J and Apoe−/− Mice
2.4. X-PCSK9 Immunogens Decreased Plasma Triglyceride Levels in C57BL/6J and Apoe−/− Mice
2.5. The Functional Effect of X-PCSK9 in LDL Receptor mRNA: X-PCSK9-B1 Treatment Increased Hepatic LDL Receptor mRNA Level in Both C57BL/6J and Apoe−/− Mice
2.6. The Functional Effect of X-PCSK9 in LDL Receptor Protein: X-PCSK9-B1 Treatment Increased the Levels of Hepatic LDL Receptor Proteins in Both C57BL/6J and Apoe−/− Mice
2.7. The Functional Effect of X-PCSK9 in Plasma PCSK9 Concentration: X-PCSK9-Isomers Treatment Increased the Levels of Plasma PCSK9 in Both C57BL/6J and Apoe−/− Mice
3. Discussion
4. Materials and Methods
4.1. Expression and Purification of the PCSK9 Catalytic Domain for Isomer Formation
4.2. Preparation of X-Isomers (X-PCSK9) from the Fully Reduced Proteins Using Oxidative Folding and Disulfide Scrambling
4.3. Q-TOF LC/MS
4.4. Calculations
4.5. Immunization of Male C57BL/6J and Apoe−/− Knockout Mice
4.6. Immunogenicity Determination Using ELISA
4.7. Analysis of Plasma Cholesterol and Triglyceride Levels
4.8. Analysis of Hepatic LDL Receptor mRNA and Protein Levels
4.8.1. Real-Time qPCR
4.8.2. Western Blot Analysis
4.8.3. Statistical Analysis
Acknowledgments
Author Contributions
Conflicts of Interest
References
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C57BL/6J Mice | |||||
Treatment | PBS | X-PCSK9-A1 | X-PCSK9-A2 | X-PCSK9-B1 | X-PCSK9-B2 |
Time effect (p-value) | 0.9025 | 0.0002 | <0.0001 | 0.0061 | 0.0006 |
Apoe−/− Mice | |||||
Treatment | PBS | X-PCSK9-A1 | X-PCSK9-A2 | X-PCSK9-B1 | X-PCSK9-B2 |
Time effect (p-value) | 0.0007 | 0.0002 | 0.001 | <0.0001 | 0.0007 |
C57BL/6J Cholesterol (mg/dL) | Day-0 | Day-28 | Day-42 | Day-56 | Day-90 | Day-120 |
PBS | 58 ± 13 | 63 ± 17 | 66 ± 12 | 56 ± 5.1 | 60 ± 6.1 | 63 ± 9.3 |
X-PCSK9-A1 | 60 ± 9.4 | 56 ± 7.6 | 50 ± 7.2 | 46 ± 4.5 | 50 ± 9.1 | 56 ± 12 |
X-PCSK9-A2 | 68 ± 5.1 | 59 ± 5.0 | 62 ± 6.0 | 58 ± 7.2 | 59 ± 7.6 | 61 ± 4.2 |
X-PCSK9-B1 | 66 ± 9.6 | 64 ± 6.3 | 71 ± 6.9 | 61 ± 3.8 | 59 ± 14 | 57 ± 12 |
X-PCSK9-B2 | 66 ± 3.8 | 61 ± 4.5 | 61 ± 1.8 | 62 ± 2.1 | 62 ± 3.4 | 65 ± 2.6 |
Apoe−/− Cholesterol (mg/dL) | Day-0 | Day-28 | Day-42 | Day-56 | Day-90 | Day-120 |
PBS | 270 ± 39 | 391 ± 46 | 403 ± 48 | 410 ± 98 | 467 ± 128 | 471 ± 128 |
X-PCSK9-A1 | 342 ± 50 | 384 ± 36 | 365 ± 63 | 348 ± 61 | 361 ± 69 | 342 ± 66 |
X-PCSK9-A2 | 358 ± 60 | 393 ± 58 | 362 ± 79 | 312 ± 50 | 304 ± 55 | 303 ± 62 |
X-PCSK9-B1 | 406 ± 64 | 357 ± 36 | ND | 335 ± 42 | 350 ± 58 | 340 ± 70 |
X-PCSK9-B2 | 357 ± 80 | 358 ± 43 | ND | 412 ± 38 | 333 ± 53 | 321 ± 57 |
C57BL/6J Mice (Cholesterol) | PBS | X-PCSK9-A1 | X-PCSK9-A2 | X-PCSK9-B1 | X-PCSK9-B2 |
Time effect (p-value) | 0.6978 | 0.0003 | 0.0044 | 0.0014 | 0.0114 |
A1 vs. PBS | A2 vs. PBS | B1 vs. PBS | B2 vs. PBS | ||
Treatment effect (p-value) | 0.0612 | 0.9665 | 0.0325 | 0.7035 | |
Apoe−/− Mice (Cholesterol) | PBS | X-PCSK9-A1 | X-PCSK9-A2 | X-PCSK9-B1 | X-PCSK9-B2 |
Time effect (p-value) | 0.0006 | 0.5566 | 0.1701 | 0.0069 | 0.7087 |
A1 vs. PBS | A2 vs. PBS | B1 vs. PBS | B2 vs. PBS | ||
Treatment effect (p-value) | <0.0001 | <0.0001 | <0.0001 | <0.0001 |
C57BL/6J Triglyceride (mg/dL) | Day-0 | Day-28 | Day-42 | Day-56 | Day-90 | Day-120 |
PBS | 12 ± 5.2 | 14 ± 5.5 | 13 ± 2.4 | 16 ± 2.7 | 13 ± 10 | 9.5 ± 3.2 |
X-PCSK9-A1 | 51 ± 33 | 25 ± 4.9 | 24 ± 9.5 | 28 ± 7.1 | 25 ± 11 | 19 ± 4.7 |
X-PCSK9-A2 | 30 ± 8.6 | 26 ± 2.5 | 53 ± 15 | 24 ± 5.6 | 24 ± 6.5 | 17 ± 2.0 |
X-PCSK9-B1 | 28 ± 11 | 43 ± 13 | 22 ± 7.0 | 39 ± 14 | 30 ± 10 | 24 ± 8.0 |
X-PCSK9-B2 | 26 ± 10 | 39 ± 11 | 17 ± 3.4 | 26 ± 2.3 | 29 ± 6.5 | 22 ± 5.9 |
Apoe−/− Triglyceride (mg/dL) | Day-0 | Day-28 | Day-42 | Day-56 | Day-90 | Day-120 |
PBS | 55 ± 13 | 59 ± 18 | 54 ± 19 | 40 ± 9.4 | 49 ± 3.4 | 37 ± 5.5 |
X-PCSK9-A1 | 64 ± 19 | 90 ± 33 | 75 ± 30 | 45 ± 17 | 50 ± 18 | 45 ± 19 |
X-PCSK9-A2 | 85 ± 30 | 68 ± 18 | 69 ± 28 | 40 ± 19 | 41 ± 27 | 43 ± 19 |
X-PCSK9-B1 | 82 ± 13 | 73 ± 21 | ND | 41 ± 5.5 | 48 ± 8.1 | 54 ± 9.9 |
X-PCSK9-B2 | 71 ± 25 | 55 ± 17 | ND | 67 ± 13 | 61 ± 9.2 | 40 ± 13 |
C57BL/6J Mice (Triglyceride) | PBS | X-PCSK9-A1 | X-PCSK9-A2 | X-PCSK9-B1 | X-PCSK9-B2 |
Time effect (p-value) | 0.3284 | 0.0045 | 0.1516 | 0.0434 | 0.4195 |
A1 vs. PBS | A2 vs. PBS | B1 vs. PBS | B2 vs. PBS | ||
Treatment effect (p-value) | <0.0001 | <0.0001 | <0.0001 | <0.0001 | |
Apoe−/− Mice (Triglyceride) | PBS | X-PCSK9-A1 | X-PCSK9-A2 | X-PCSK9-B1 | X-PCSK9-B2 |
Time effect (p-value) | 0.0756 | 0.0038 | 0.0029 | 0.0033 | 0.1784 |
A1 vs. PBS | A2 vs. PBS | B1 vs. PBS | B2 vs. PBS | ||
Treatment effect (p-value) | 0.5777 | 0.7993 | 0.5533 | 0.5051 |
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Jiang, C.; Nischal, H.; Sun, H.; Li, L.; Cao, Y.; Wei, P.; Chang, J.-Y.; Teng, B.-B. Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels. Int. J. Mol. Sci. 2018, 19, 640. https://doi.org/10.3390/ijms19020640
Jiang C, Nischal H, Sun H, Li L, Cao Y, Wei P, Chang J-Y, Teng B-B. Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels. International Journal of Molecular Sciences. 2018; 19(2):640. https://doi.org/10.3390/ijms19020640
Chicago/Turabian StyleJiang, Chuantao, Hersharan Nischal, Hua Sun, Li Li, Ying Cao, Peng Wei, Jui-Yoa Chang, and Ba-Bie Teng. 2018. "Non-Native Conformational Isomers of the Catalytic Domain of PCSK9 Induce an Immune Response, Reduce Lipids and Increase LDL Receptor Levels" International Journal of Molecular Sciences 19, no. 2: 640. https://doi.org/10.3390/ijms19020640