Mode of interaction of calcium oxalate crystal with human phosphate cytidylyltransferase 1: a novel inhibitor purified from human renal stone matrix

Priyadarshini Pathak, Pradeep Kumar Naik, Dipankar Sengupta, Shrawan Kumar Singh, Chanderdeep Tandon

.

DOI: 10.4236/jbise.2011.49075                

Abstract

Nephrolithiasis is a common clinical disorder, and calcium oxalate (CaOx) is the principal crystalline component in approximately 75% of all renal stones. It is widely believed that proteins act as inhibitors of crystal growth and aggregation. Acidic amino acids present in these proteins play a significant role in the inhibition process. In this study, interaction of cal-cium oxalate with human phosphate cytidylyltrans-ferase 1(CCT), a novel calcium oxalate crystal growth inhibitor purified from human renal stone matrix has been elucidated in silico and involvement of acidic amino acids in the same. As only sequence of CCT is available, henceforth its 3-D structure was modeled via Homology modeling using Prime module of Schrodinger package. Molecular dynamic simulation of modeled protein with solvation was done by mac-romodel (Schrodinger). The quality of modeled pro-tein was validated by JCSG protein structure valida-tion (PROCHECK & ERRAT) server. To analyze the interaction of modeled protein CCT with calcium oxalate along with role played by acidic amino acids, ‘Docking simulation’ was done using MOE–Dock. Interaction between calcium oxalate and CCT was also studied by substituting acidic amino acid in the active sites of the protein with neutral and positively charged amino acids. The in silico analysis showed the bond formation between the acidic amino acids and calcium atom, which was further substantiated when substitution of these acidic amino acids with alanine, glycine, lysine, arginine and histidine com-pletely diminished the interaction with calcium ox-alate.

Keywords

Calcium Oxalate; Matrix-Assisted Laser Desorption/ Ionization-Time of Flight

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Drach, G.W. (1992) Urinary lithiasis etiology, diagnosis, and medical management. Campbell’s Textbook of Urology, 2085.
[2]	Grover, P.K. and Ryall, R.L. (1998) Inhibition of calcium oxalate crystal growth and aggregation by prothrombin and its fragments in vitro. European Journal of Biology Chemistry, 263, 50.
[3]	Nakagawa, Y., Abram, V., Kezdy, F.J., Kaiser, E.T. and Coe, F.L. (1983) Purification and characterization of the principal inhibitor of calcium oxalate monohydrate crystal growth in human urine. The Journal of Biological Chemistry, 258, 12594-12600.
[4]	Atmani, F. and Khan, S.R. (1995) Characterization of uronic acid rich inhibitor of calcium oxalate crystallization isolated from rat urine. Urological Research, 23, 95.
[5]	Atmani, F., Mizon, J. and Khan, S. R. (1996) Identi- fication of uronic acid rich protein as urinary bikunin, the light chain of inter-a-inhibitor. European Journal of Biology Chemistry, 236, 984.
[6]	Wesson, J.A., Johnson, R.J. and Mazzali, M.A, et al. (2003) Osteopontin is a critical inhibitor of calcium oxalate crystal formation and retention in renal tubules. Journal of the American Society Nephrology, 14, 139-147.
[7]	Taller, A., Grohe, B., Rogers, K.A., Goldberg, H.A. and Hunter, G.K. (2007) Specific adsorption of osteopontin and synthetic polypeptides to calcium oxalate monohy- drate crystals. Biophysical Journal, 93, 1768-1777.
[8]	Sodek, J., Ganss, B. and McKee, M.D. (2000) Osteo- pontin. Critical Reviews in Oral Biology and Medicine, 11, 279-303.
[9]	Sorensen, E.S., Hojrup, P. and Petersen, T.E. (1995) Post-translational modifications of bovine osteopontin: identification of twenty-eight phosphorylation and three O-gly- cosylation sites. Protein Scienty, 4, 2040-2049.
[10]	Christensen, B., Nielsen, M.S., Haselmann, K.F., Petersen, T.E. and Sorensen, E.S. (2005) Posttranslationally modified residues of native human osteopontin are located in clusters: identification of 36 phosphorylation and five O-glycosylation sites and their biological implications. Biochemical Journal, 390, 285-292.
[11]	Addadi, L. and Weiner, S. (1985) Interactions between acidic proteins and crystals: stereochemical requirements in biomineralization. Proc Natl Acad Sci USA 82, 4110.
[12]	Lian, J.B., Prien, E.L., Glimcher, M.J. and Gallop, P.M. (1977) The presence of protein bound ccarboxyglutamic acid in calcium-containing renal calculi. Journal of Clinicine Investigation, 59, 1151.
[13]	Ryall, R.L., Fleming, D.E. and Grover, P.K. et al. (2000) The hole truth: intracrystalline proteins and calcium oxalate kidney stones. Molecular Urology, 4, 391.
[14]	Priyadarshini, Singh, S.K. and Tandon, C. (2009) Mass spectrometric identification of human phosphate cytidy- lyltransferase 1 as a novel calcium oxalate crystal growth inhibitor purified from human renal stone matrix. Clinica Chimica Acta, 408, 34-38.
[15]	Lee, J., Johnson, J., Ding, Z., Paetzel, M. and Cornell, R.B. (2009) Crystal structure of a mammalian CTP: phosphocholine cytidylyltransferase catalytic domain reveals novel active site residues within a highly conserved nucleotidyltransferase fold. The Journal of Biological Chemistry, 284, 33535-33548.
[16]	Jon, A.F., Heidi, A.C. and Claudia Kent. (1999) Enzymatic and cellular characterization of a catalytic fragment of CTP: Phosphocholine Cytidylyltransferase α. The Journal of Biological Chemistry, 274, 13384-13389.
[17]	Coe, F.L., Parks, J.H. and Asplin, J.R. (1992) The pathogenesis and treatment of kidney stones. New England Journal of Medicine, 327, 1141-1152.
[18]	Zerwekh, J.E., Holt, K. and Pak, C.Y. (1983) Natural urinary macromolecular inhibitors: attenuation of inhibitory activity by urate salts. Kidney International, 23, 838-841.
[19]	Coe, F.L., Parks, J.H. and Nakagawa, Y. (1991) Protein inhibitors of crystallization. Seminars in Nephrology, 11, 98-109.
[20]	Aggarwal, S., Tandon, C., Forouzandeh, M., Singla, S.K., Kiran, R. and Jethi, R.K. (2000) Role of biomolecules from human renal stone matrix on COM crystal growth. Molecular and Cellular Biochemistry, 210, 109-119.
[21]	Shirane, Y., Kurokawa, Y., Miyashita, S., Komatsu, H. and Kagawa, S. (1999) Study of inhibition mechanisms of glycosaminoglycans on calcium oxalate monohydrate crystals byatomic force microscopy. Urology Research, 27, 426-431.
[22]	Guo, S., Ward, M.D. and Wesson, J.A. (2002) Direct visualization of calcium oxalate monohydrate (COM) crystallization and dissolution with atomic force microscopy (AFM) and the role of polymeric additives. Langmuir, 18, 4284-4291.
[23]	Qiu, S.R., Wierzbicki, A. and Orme, C.A. et al. (2004) Molecular modulation of calcium oxalate crystallization by osteopontin and citrate. Proceedingof the Natlional Academy Science of the USA, 101, 1811-1815.
[24]	Jung, T., Sheng, X., Choi, C.K., Kim, W., Wesson, J.A. and Ward, M.D. (2004) Probing crystallization of calcium oxalate monohydrate and the role of macromolecule additives with in situ atomic force microscopy. Langmuir, 20, 8587-8596.
[25]	Weber, C.H., Park, Y.S., Sanker, S., Kent, C. and Ludwig, M.L. (1999) A prototypical cytidylyltransferase: CTP: glycerol-3 phosphate cytidylyltrnsferase from Bacillus subtilis. Structure 7, 9.
[26]	Bijarnia, R.K., Kaur, T., Naik, P.K., Singla, S.K. and Tandon C. (2008) In silico study on interaction of active binding sites of proteins with calcium oxalate monohydrate. Office of James Bumett, 2, 92-107.
[27]	Gul, A. and Rez, P. (2007) Models for protein binding to calcium oxalate surfaces. Urology Research, 35, 63-71.
[28]	Tandon, C., Aggarwal, S., Forouzandeh, M. and Jethi, R.K. (1998) Inhibitors of in vitro mineralization from rabbit aorta and their role in biomineralization. Journal of Cellular Biochemistry, 68, 287-297.
[29]	Nakagawa, Y., Ahmed, M., Hall, S.L., Deganello, S. and Coe F.L. (1987) Isolation from human calcium oxalate renal stones of nephrocalcin, a glycoprotein inhibitor of calcium oxalate crystal growth. Evidence that nephr- ocalcin from patients with calcium oxalate nephr- olithiasis is deficient in γ-carboxyglutamic acid. Journal of Clinical Investigation, 79, 1782-1787.
[30]	Nakagawa, Y., Abram, V., Parks, J.H., Lau, H.S.H., Kawooya, J.K. and Coe, F.L. (1985) Urine glycoprotein crystal growth inhibitors. Evidence for a molecular abnormality in calcium oxalate nephrolithiasis. Journal of Clinical Investigation, 76, 1455-1462.
[31]	Shiraga, H., Min, W., VanDusen, W.J. et al. (1992) Inhibition of calcium oxalate crystal growth in vitro by uropontin: Another member of the aspartic acid rich protein superfamily. Proceedingof the Natlional Academy Science of the USA, 89, 426-430.
[32]	Kalmar, G.B., Kay, R.J., Lachance, A., Aebersold, R. and Cornell, R.B. (1990) Cloning and expression of rat liver CTP: phosphocholine cytidylyltransferase: an amphi- pathic protein that controls phosphatidylcholine synthesis. Proceedingof the Natlional Academy Science of the USA, 87, 6029-6033.
[33]	Kaur, T., Bijarnia, R.K., Singla, S.K. and Tandon, C. (2009) Purification and characterization of an anticalcifying protein from the seeds of Trachyspermum ammi (L.). Protein and Peptides Letters, 16, 173-81.
[34]	Bijarnia, R.K., Kaur, T., Singla, S.K. and Tandon, C. (2009) A novel calcium oxalate crystal growth inhibitory protein from the seeds of Dolichos biflorus (L.). The Protein Journal, 28, 161-168.