Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle
Abstract
:1. Introduction
2. Results
2.1. Glycosylation and Phosphorylation of Casq1
2.1.1. Casq1 Glycosylation
2.1.2. Casq1 Phosphorylation
2.2. Glycosylation and Phosphorylation of Casq2
2.2.1. Casq2 Glycosylation
2.2.2. Casq2 Phosphorylation
2.3. Casq1 Crystal Structures
2.4. Multi-Angle Light Scattering
3. Discussion
3.1. Evolution
3.2. Glycosylation
3.3. Phosphorylation
3.4. Clinical Implications
4. Materials and Methods
4.1. Isolation of Cardiac and Skeletal Muscle Tissue
4.2. Recombinant Bovine Casq1 Gene Synthesis
4.3. Purification of Native Casq1 and Casq2
4.4. Purification of Recombinant Bovine Casq1
4.5. Mass Spectrometry
4.6. Multi-Angle Light Scattering
4.7. Crystallization
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Protein and Chain Identity | High-Ca2+ Native | High-Ca2+ Recombinant | |||||
---|---|---|---|---|---|---|---|
Chain A | Chain B | Chain C | Chain A | Chain B | Chain C | ||
High-Ca2+ recombinant | Chain C | 0.245 | 0.269 | 0.234 | 0.401 | 0.391 | 0 |
Chain B | 0.380 | 0.412 | 0.399 | 0.535 | 0 | – | |
Chain A | 0.438 | 0.464 | 0.435 | 0 | – | – | |
High-Ca2+ native | Chain C | 0.205 | 0.228 | 0 | – | – | – |
Chain B | 0.234 | 0 | – | – | – | – | |
Chain A | 0 | – | – | – | – | – |
Protein | btCasq2 | cfCasq2 | hsCasq2 | mmCasq2 | ocCasq2 | rnCasq2 | rnCasq1 | ocCasq1 | mmCasq1 | hsCasq1 | cfCasq1 |
---|---|---|---|---|---|---|---|---|---|---|---|
btCasq1 | 69.53 | 69.81 | 70.08 | 68.70 | 68.98 | 68.70 | 95.84 | 94.74 | 95.01 | 94.18 | 95.84 |
(93.35) | (92.80) | (93.35) | (92.80) | (91.97) | (92.24) | (99.45) | (98.89) | (99.45) | (99.17) | (99.17) | |
cfCasq1 | 69.21 | 70.30 | 69.75 | 68.39 | 68.94 | 68.39 | 96.19 | 96.73 | 95.37 | 96.41 | – |
(93.73) | (93.19) | (94.01) | (92.64) | (92.64) | (92.64) | (99.73) | (99.18) | (99.73) | (99.45) | – | |
hsCasq1 | 68.51 | 68.51 | 69.06 | 66.57 | 67.68 | 66.85 | 97.51 | 96.41 | 96.41 | – | – |
(93.37) | (93.09) | (93.65) | (92.54) | (92.27) | (92.54) | (100) | (99.17) | (99.72) | – | – | |
mmCasq1 | 69.00 | 68.73 | 68.46 | 67.39 | 68.19 | 66.85 | 99.19 | 95.37 | – | – | – |
(93.90) | (93.26) | (94.07) | (92.72) | (92.45) | (92.72) | (100) | (99.46) | – | – | – | |
ocCasq1 | 69.48 | 70.03 | 69.75 | 68.94 | 69.21 | 66.21 | 96.19 | – | – | – | – |
(94.01) | (93.46) | (94.28) | (92.92) | (92.92) | (90.19) | (99.46) | – | – | – | – | |
rnCasq1 | 69.35 | 69.35 | 69.09 | 68.01 | 68.82 | 67.47 | – | – | – | – | – |
(93.82) | (93.28) | (94.09) | (92.74) | (97.24) | (92.74) | – | – | – | – | – | |
rnCasq2 | 90.41 | 91.30 | 91.58 | 97.21 | 92.05 | – | – | – | – | – | – |
(98.96) | (97.70) | (98.42) | (99.49) | (97.69) | – | – | – | – | – | – | |
ocCasq2 | 94.04 | 93.85 | 93.95 | 91.28 | – | – | – | – | – | – | – |
(98.45) | (97.95) | (98.16) | (97.69) | – | – | – | – | – | – | – | |
mmCasq2 | 89.12 | 90.28 | 90.53 | – | – | – | – | – | – | – | – |
(98.70) | (97.70) | (98.42) | – | – | – | – | – | – | – | – | |
hsCasq2 | 95.26 | 93.68 | – | – | – | – | – | – | – | – | – |
(99.47) | (98.42) | – | – | – | – | – | – | – | – | – | |
cfCasq2 | 95.60 | – | – | – | – | – | – | – | – | – | – |
(98.96) | – | – | – | – | – | – | – | – | – | – |
Protein | Low-Ca2+ Native Bovine Casq1 | High-Ca2+ Native Bovine Casq1 | Low-Ca2+ Recombinant Bovine Casq1 | High-Ca2+ Recombinant Bovine Casq1 |
---|---|---|---|---|
PDB ID | 5KN0 | 5KN2 | 5KN3 | 5KN1 |
Data collection | – | – | – | – |
Space group | P1 | C2221 | C2221 | C2221 |
Cell dimensions | – | – | – | – |
a, b, c (Å) | 60.342, 92.994, 101.849 | 130.363, 169.194, 155.477 | 59.393, 146.06, 110.34 | 135.669, 165.604, 156.626 |
α, β, γ (°) | 71.122, 84.574, 73.485 | 90.0, 90.0, 90.0 | 90.0, 90.0, 90.0 | 90.0, 90.0, 90.0 |
Resolution (Å) | 49.66–2.73 (2.83–2.73) | 42.95–2.60 (2.70–2.60) | 49.20–1.85 (1.92–1.85) | 43.63–2.14 (2.21–2.14) |
Rmerge | 0.0318 (0.3289) | 0.0633 (1.416) | 0.0698 (0.8201) | 0.0922 (0.8878) |
<I>/<σI> | 14.84 (2.55) | 13.25 (1.34) | 13.40 (2.72) | 27.16 (5.0) |
Completeness (%) | 0.98 (0.94) | 0.99 (0.97) | 0.100 (0.97) | 0.99 (0.94) |
Multiplicity | 2.0 (1.9) | 7.3 (7.4) | 7.1 (6.6) | 7.1 (6.8) |
Refinement | – | – | – | – |
Resolution | 49.7–2.73 (2.83–2.73) | 42.95–2.60 (2.70–2.60) | 49.20–1.85 (1.92–1.85) | 43.63–2.14 (2.21–2.14) |
Unique reflections | 52,550 (5026) | 52,807 (5112) | 41,309 (3984) | 96,658 (9078) |
Rwork/Rfree | 0.1970/0.241 (0.3474/0.3834) | 0.2056/0.2411 (0.3302/0.3536) | 0.1833/0.2106 (0.2936/0.3201) | 0.1836/0.2011 (0.2429/0.2594) |
Number of atoms | – | – | – | – |
Macromolecules | 11,172 | 8456 | 2827 | 8466 |
Ion | 19 | 39 | 5 | 40 |
Ligand | 175 | 84 | 32 | 33 |
Water molecules | 33 | 5 | 336 | 461 |
R.m.s deviations | – | – | – | – |
Bond lengths (Å) | 0.005 | 0.005 | 0.003 | 0.005 |
Bond angles (°) | 0.71 | 0.84 | 0.57 | 0.69 |
Ramachandrans | – | – | – | – |
Favored (%) | 97.6 | 98.0 | 98.6 | 98.0 |
Outliers (%) | 0 | 0 | 0 | 0 |
Clashscore | 1.59 | 1.03 | 2.70 | 1.82 |
B-factors | – | – | – | – |
Protein | 76.71 | 108.25 | 43.83 | 59.42 |
Ligand/Ion | 100.15 | 139.00 | 69.90 | 74.69 |
Water | 59.89 | 67.90 | 44.92 | 51.10 |
TLS groups | 27 | 13 | 20 | 60 |
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Lewis, K.M.; Munske, G.R.; Byrd, S.S.; Kang, J.; Cho, H.-J.; Ríos, E.; Kang, C. Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle. Int. J. Mol. Sci. 2016, 17, 1539. https://doi.org/10.3390/ijms17091539
Lewis KM, Munske GR, Byrd SS, Kang J, Cho H-J, Ríos E, Kang C. Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle. International Journal of Molecular Sciences. 2016; 17(9):1539. https://doi.org/10.3390/ijms17091539
Chicago/Turabian StyleLewis, Kevin M., Gerhard R. Munske, Samuel S. Byrd, Jeehoon Kang, Hyun-Jai Cho, Eduardo Ríos, and ChulHee Kang. 2016. "Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle" International Journal of Molecular Sciences 17, no. 9: 1539. https://doi.org/10.3390/ijms17091539