Optimized High-Yield Purification of Obesity-Associated Melanocortin 4 Receptor

Author(s): Minseon Kim, Soyeon Jo, Ji-Ho Jeong, Yongae Kim*

Journal Name: Protein & Peptide Letters

Volume 28 , Issue 1 , 2021


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Abstract:

Background: Obesity has emerged as a global public health challenge associated with increased risk of hyperlipidemia and hypertension. It contributes to high sympathetic activity and increased catecholamine levels. The hypothalamic melanocortin system is known to regulate the energy homeostasis. The role of melanocortin 4 receptor (MC4R) has been demonstrated pharmacologically and in animal studies, which showed that severe obesity in MC4R knockout mice was caused by increased food intake and decreased energy consumption. Over 70 multiple different mis- -sense and nonsense mutations in hMC4R have been found at a high frequency of 2-8% in severe early onset or hereditary obesity. The single amino acid variation (D90N) located in the second transmembrane domain (TM2) of MC4R results in accelerated growth and childhood onset obesity. Interestingly, the functional characterization of D90N hMC4R mutant TM2 (m-hMC4R-TM2) revealed normal cell surface expression and binding with agonist similar to the hMC4R wild-type TM2 (wt-hMC4R-TM2) but loss of signal transduction mediated via Gs/adenylyl cyclase activation. It is essential to delineate the three-dimensional structure of MC4Rs in order to elucidate their functional aspects.

Objective: In this study, we demonstrate the optimized expression and isolation of wt/m-hMC4R-TM2 proteins under different chemical cleavage reaction times and purification procedures via SDS precipitation. The solid-state NMR spectroscopy was carried out to study the structure of wt/m-hMC4R- TM2 protein in the anisotropic phospholipid bicelles.

Methods: The KSI-wt/m-hMC4R-TM2 fusion proteins developed in cell culture with LB medium. In order to isolate the expressed fusion protein from the cell, ultrasonication, Ni-NTA affinity chromatography, dialysis, and lyophilization techniques were used. Then, to obtain a protein with higher purity and higher yield, the CNBr chemical cleavage time was subdivided into 30 minutes, 1 h, 2 h, 3 h, and 4 h. Purification process was performed using FPLC, and 100 mM KCl and dialysis were used to remove the SDS. CD spectrometer, MALDI-TOF, solution-state NMR, and solid-state NMR were used to confirmed purity and structure of the wt/m-hMC4R-TM2.

Results: The precipitation method was used to remove the SDS bound to proteins as KCl-SDS. We optimized the 2 h cleavage reaction times for both wt-hMC4R-TM2 and m-hMC4R-TM2 depending on the purity based on mass spectra and 1H-15N HSQC spectra and the yield after final purification. The 1D 1H-15N CP (Cross polarization) solid-state NMR spectra suggest that the wt/m-hMC4R- TM2 undergo rotational diffusion around a perpendicular axis along the bilayer normal.

Conclusion: We expressed wt/m-hMC4R-TM2 in E.coli and optimized the isolation and purification process, especially CNBr chemical cleavage time. The efficiency of KCl-SDS precipitation was confirmed via MALDI-TOF MS and the pure proteins obtained using this method were characterized by CD spectroscopy and solution-state NMR. The results of 1H-15N HSQC spectra in solution- state NMR also show the probability for structural studies. The 1D 1H-15N CP solid-state NMR spectra indicate that most of the residues in both the wt/m-hMC4R-TM2 peptides are integrated into the membrane.

Keywords: Fast Protein Liquid Chromatography, obesity, melanocortin 4 receptor, trans-membrane, chemical cleavage, phospholipid bicelle.

[1]
Opella, S.J. Structure determination of membrane proteins in their native phospholipid bilayer environment by rotationally aligned solid-state NMR spectroscopy. Acc. Chem. Res., 2013, 46(9), 2145-2153.
[http://dx.doi.org/10.1021/ar400067z] [PMID: 23829871]
[2]
Dawson, P.E.; Kent, S.B.H. Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem., 2000, 69, 923-960.
[http://dx.doi.org/10.1146/annurev.biochem.69.1.923] [PMID: 10966479]
[3]
Nilsson, B.L.; Soellner, M.B.; Raines, R.T. Chemical synthesis of proteins. Annu. Rev. Biophys. Biomol. Struct., 2005, 34, 91-118.
[http://dx.doi.org/10.1146/annurev.biophys.34.040204.144700] [PMID: 15869385]
[4]
Schlegel, S.; Klepsch, M.; Gialama, D.; Wickström, D.; Slotboom, D.J.; de Gier, J.W. Revolutionizing membrane protein overexpression in bacteria. Microb. Biotechnol., 2010, 3(4), 403-411.
[http://dx.doi.org/10.1111/j.1751-7915.2009.00148.x] [PMID: 21255339]
[5]
Wagner, S.; Bader, M.L.; Drew, D.; de Gier, J.W. Rationalizing membrane protein overexpression. Trends Biotechnol., 2006, 24(8), 364-371.
[http://dx.doi.org/10.1016/j.tibtech.2006.06.008] [PMID: 16820235]
[6]
Peti, W.; Page, R. Strategies to maximize heterologous protein expression in Escherichia coli with minimal cost. Protein Expr. Purif., 2007, 51(1), 1-10.
[http://dx.doi.org/10.1016/j.pep.2006.06.024] [PMID: 16904906]
[7]
Baker, L.A.; Baldus, M. Characterization of membrane protein function by solid-state NMR spectroscopy. Curr. Opin. Struct. Biol., 2014, 27, 48-55.
[http://dx.doi.org/10.1016/j.sbi.2014.03.009] [PMID: 24865155]
[8]
Garman, E.F. Development in X-ray crystallographic structure determination of biological macromolecules. Science, 2014, 34, 1102-1108.
[http://dx.doi.org/10.1126/science.1247829]
[9]
Carlson, E.D.; Gan, R.; Hodgman, C.E.; Jewett, M.C. Cell-free protein synthesis: applications come of age. Biotechnol. Adv., 2012, 30(5), 1185-1194.
[http://dx.doi.org/10.1016/j.biotechadv.2011.09.016] [PMID: 22008973]
[10]
Shoichet, B.K.; Kobilka, B.K. Structure-based drug screening for G-protein-coupled receptors. Trends Pharmacol. Sci., 2012, 33(5), 268-272.
[http://dx.doi.org/10.1016/j.tips.2012.03.007] [PMID: 22503476]
[11]
Hofmann, K.P.; Scheerer, P.; Hildebrand, P.W.; Choe, H.W.; Park, J.H.; Heck, M.; Ernst, O.P.A. A G protein-coupled receptor at work: the rhodopsin model. Trends Biochem. Sci., 2009, 34(11), 540-552.
[http://dx.doi.org/10.1016/j.tibs.2009.07.005] [PMID: 19836958]
[12]
Teller, D.C.; Okada, T.; Behnke, C.A.; Palczewski, K.; Stenkamp, R.E. Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs). Biochemistry, 2001, 40(26), 7761-7772.
[http://dx.doi.org/10.1021/bi0155091] [PMID: 11425302]
[13]
Martinelli, C.E.; Keogh, J.M.; Greenfield, J.R.; Henning, E.; van der Klaauw, A.A.; Blackwood, A.; O’Rahilly, S.; Roelfsema, F.; Camacho-Hübner, C.; Pijl, H.; Farooqi, I.S. Obesity due to melanocortin 4 receptor (MC4R) deficiency is associated with increased linear growth and final height, fasting hyperinsulinemia, and incompletely suppressed growth hormone secretion. J. Clin. Endocrinol. Metab., 2011, 96(1), E181-E188.
[http://dx.doi.org/10.1210/jc.2010-1369] [PMID: 21047921]
[14]
Xi, B.; Chandak, G.R.; Shen, Y.; Wang, Q.; Zhou, D. Association between common polymorphism near the MC4R gene and obesity risk: a systematic review and meta-analysis. PLoS One, 2012, 7(9), e45731.
[http://dx.doi.org/10.1371/journal.pone.0045731] [PMID: 23049848]
[15]
Rossi, J.; Balthasar, N.; Olson, D.; Scott, M.; Berglund, E.; Lee, C.E.; Choi, M.J.; Lauzon, D.; Lowell, B.B.; Elmquist, J.K. Melanocortin-4 receptors expressed by cholinergic neurons regulate energy balance and glucose homeostasis. Cell Metab., 2011, 13(2), 195-204.
[http://dx.doi.org/10.1016/j.cmet.2011.01.010] [PMID: 21284986]
[16]
Liu, H.; Kishi, T.; Roseberry, A.G.; Cai, X.; Lee, C.E.; Montez, J.M.; Friedman, J.M.; Elmquist, J.K. Transgenic mice expressing green fluorescent protein under the control of the melanocortin-4 receptor promoter. J. Neurosci., 2003, 23(18), 7143-7154.
[http://dx.doi.org/10.1523/JNEUROSCI.23-18-07143.2003] [PMID: 12904474]
[17]
Kishi, T.; Aschkenasi, C.J.; Lee, C.E.; Mountjoy, K.G.; Saper, C.B.; Elmquist, J.K. Expression of melanocortin 4 receptor mRNA in the central nervous system of the rat. J. Comp. Neurol., 2003, 457(3), 213-235.
[http://dx.doi.org/10.1002/cne.10454] [PMID: 12541307]
[18]
Huszar, D.; Lynch, C.A.; Fairchild-Huntress, V.; Dunmore, J.H.; Fang, Q.; Berkemeier, L.R.; Gu, W.; Kesterson, R.A.; Boston, B.A.; Cone, R.D.; Smith, F.J.; Campfield, L.A.; Burn, P.; Lee, F. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell, 1997, 88(1), 131-141.
[http://dx.doi.org/10.1016/S0092-8674(00)81865-6] [PMID: 9019399]
[19]
Biebermann, H.; Krude, H.; Elsner, A.; Chubanov, V.; Gudermann, T.; Grüters, A. Autosomal-dominant mode of inheritance of a melanocortin-4 receptor mutation in a patient with severe early-onset obesity is due to a dominant-negative effect caused by receptor dimerization. Diabetes, 2003, 52(12), 2984-2988.
[http://dx.doi.org/10.2337/diabetes.52.12.2984] [PMID: 14633860]
[20]
Park, T.J.; Choi, S.S.; Gang, G.A.; Kim, Y. High-level expression and purification of the second transmembrane domain of wild-type and mutant human melanocortin-4 receptor for solid-state NMR structural studies. Protein Expr. Purif., 2008, 62(2), 139-145.
[http://dx.doi.org/10.1016/j.pep.2008.08.008] [PMID: 18809499]
[21]
Park, Y.G.; Song, J.Y.; Kim, Y.A. Optimized purification and characterization of expressed hMC4R-TM2. J. Korean Mag. Res. Soc., 2012, 16, 147-161.
[http://dx.doi.org/10.6564/JKMRS.2012.16.2.147]
[22]
Kim, J.S.; Cho, S.J.; Jang, H.J.; Kim, Y.A. Efficient purification of human transmembrane protein, mutant hMC4R TM2. Bull. Korean Chem. Soc., 2018, 39, 618-624.
[http://dx.doi.org/10.1002/bkcs.11440]
[23]
Müller, S.D.; De Angelis, A.A.; Walther, T.H.; Grage, S.L.; Lange, C.; Opella, S.J.; Ulrich, A.S. Structural characterization of the pore forming protein TatAd of the twin-arginine translocase in membranes by solid-state 15N-NMR. Biochim. Biophys. Acta, 2007, 1768(12), 3071-3079.
[http://dx.doi.org/10.1016/j.bbamem.2007.09.008] [PMID: 17980349]
[24]
Park, S.H.; Mrse, A.A.; Nevzorov, A.A.; De Angelis, A.A.; Opella, S.J. Rotational diffusion of membrane proteins in aligned phospholipid bilayers by solid-state NMR spectroscopy. J. Magn. Reson., 2006, 178(1), 162-165.
[http://dx.doi.org/10.1016/j.jmr.2005.08.008] [PMID: 16213759]


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Article Details

VOLUME: 28
ISSUE: 1
Year: 2021
Published on: 29 January, 2021
Page: [63 - 73]
Pages: 11
DOI: 10.2174/0929866527666200525162928

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