The Internet Journal of Genomics and Proteomics ISPUB.COM Volume 4 Number 2

Genotyping S328F SNP of ACAT2 among CVD patients and normal population from North Western India and its associated lipid profile P Balgir, G Kaur, Divya

Citation P Balgir, G Kaur, Divya. Genotyping S328F SNP of human ACAT2 among CVD patients and normal population from North Western India and its associated lipid profile. The Internet Journal of Genomics and Proteomics. 2008 Volume 4 Number 2.

Abstract Acyl-coenzyme A: cholesterol acyltransferase 2 (ACAT2, EC2.3.1.26) is an integral membrane protein located in the rough endoplasmic reticulum (ER). It maintains a balance between the availability of free and esterified cholesterol and is critically important for cell function. Purpose: The present study discusses the standardization of protocol to delineate the role of SNP S328F in ACAT2 . Methods: SNP S328F stability prediction was done using CUPSAT and various parameters like Primer concentration, amount of taq and glycerol etc were studied to set a protocol using Polymerase Chain reaction to achieve a product of 127 . Restriction digestion was done overnight using BsmA1 enzyme to study the genotypes. The present study was used to study the SNP in 200 healthy subjects and 166 CAD patient group.Results: Higher frequency of C allele was observed in CAD patients and higher levels of TC were observed to be related to the presence of allele T which is associated with the presence of hydrophobic amino acid F at position 328. Discussion and Conclusion: Presence of T allele leads to more accumulation of cholesterol esters as it increases the protein stability in the cytoplasmic region and consequently making the individual more susceptible to atherosclerosis.

INTRODUCTION Keeping in mind the importance of nonsynonymous SNPs, Acyl-coenzyme A: cholesterol acyltransferase 2 (ACAT2, one such SNP S328F present in Gene ACAT2 (geneID: 39) EC2.3.1.26) is an enzyme found both in the liver and in the linked via Contig Annotation; was selected from NCBI SNP intestine. It is responsible for the synthesis of cholesteryl database (http://www.ncbi.nlm.nih.gov/Entrez/). The esters from (LDLs and HDLs) cholesterol and (long chain) variation at this site is a result of change from C to T at fatty acids either from our diets or internally, from the body nucleotide position 19354. This nsSNP in exon 8 leads to change of hydrophilic neutral serine to hydrophobic [1]. The enzyme appears to modulate the potentially toxic effects of cholesterol in cell membranes, by attaching a fatty Phenylalanine at position 328 (ref SNP ID: rs12213253). The present study aimed at in-silico prediction of the effect acid to the free hydroxyl group of cholesterol [2]. Unlike free (unesterified) cholesterol that partitions in the lipid bilayer, of above mentioned amino acid change on the stability of the cholesteryl esters exist as oily lipid droplets in the cytosol, protein in that specific region. Based on change in stability and its predicted functionality it was of interest to study the acting as a means for cholesterol storage [3]. A balance between the availability of free and esterified cholesterol is prevalence of this SNP in normal population as well as pathology samples, for which a novel PCR- RFLP critically important for cell function [4]. The gene for the enzyme is localized on no. 6. Seven Single methodology has been developed. nucleotide polymorphisms (SNPs) have been listed at the MATERIAL AND METHODS locus out of which four are synonymous and three are non- SNP STABILITY PREDICTION synonymous. Nonsynonymous SNPs (nsSNPs) might directly or indirectly affect functionality of the protein, alone Change in amino acid at this position may change the or by altering its interactions with other proteins in a multi- structural stability of the protein which was analyzed using a protein complex, thereby increasing/decreasing the activity web server CUPSAT (Cologne University Protein Stability Analysis Tool). This program uses structural environment [5]. This in turn may contribute to the etiology of genetic specific atom potentials and torsion angle potentials to diseases or susceptibility to diseases [6].

1 of 7 Genotyping S328F SNP of human ACAT2 among CVD patients and normal population from North Western India and its associated lipid profile predict ΔΔG, the difference in free energy of unfolding Figure 2 between wild-type and mutant proteins [7]. Input was fed as available protein structure in Protein Data Bank ID 1WL4 (http.//www.ebi.ac.uk) and the location of the residue which is mutated. The output consisted of information about mutation site, its structural features (solvent accessibility, secondary structure and torsion angles), and comprehensive information about changes in protein stability.

SAMPLE COLLECTION Blood samples were collected from normal healthy After amplification PCR product was checked on 10% population and patient group having CVD from Punjab with Polyacrylamide Gel and visualized using silver staining. informed consent of people. 3 ml of blood sample was used Spurious bands of high molecular weight were obtained to isolate Genomic DNA using the technique as followed by during the course of standardization procedure for ACAT2

Sambrook et al. [8]. The project was cleared from amplification. Results obtained using different protocols institutional Ethics Committee on Biomedical Human mentioned above are shown in fig2. research, Punjabi University, Patiala. RFLP ANALYSIS STANDARDIZATION OF PCR Restriction Enzyme was searched from New England Biolab The key to the PCR lies in the designing of the two website, http.//www.neb.com/ following enzymes was oligonucleotide primers. Sequence of ACAT2 SNP F328S found: was retrieved from NCBI and primers were designed using Figure 3 GENE RUNNER version 3.05. Six pairs of primers were predicted (Table 2). Different combinations of primer pair concentration along with other components like taq, annealing temperature and DNTPs were used for standardization of reaction mixture used for amplification (Table 1).

Figure 1 Table 1: Reaction mixture used for PCR: Various combinations of primer pair with Taq and DNTPs concentrations in the following three protocols were investigated. BsmA1 (cutting site GTCTC) was selected based upon the SNP recognition site and availability of the enzyme. Enzyme was procured from New England Biolabs. Protocol shown in table 3 was used for restriction digestion of 127bp product.

Table 3: Reaction mixture used for digestion of PCR product:

This reaction mixture was kept at 37° C for overnight digestion.

The data for prevalence of SNP S328F in different PCR programs were also standardized using different populations was retrieved from NCBI site annealing temperatures 63°C to 60°C. Finally 60°C resulted (http://www.ncbi.nlm.nih.gov/SNP/snp). This data was in a single band of product without any spurious products compared with Punjabi population for its significance in this and was used to further investigate samples. population.

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RESULTS AND DISCUSSION Reverse primer- SNP stability and structure: CUPSAT predicts the stability 5’GCTCTCAGGGTTACTTGTGCATC3’Tm-63.9 of protein mutants with accuracy in the range of 80–87% Selected pair of primer was also checked for the gene with a standard error of 0.78–1.15 kcal/mol. The amino acid specificity using NCBI BLAST tool. change from SER to PHE at position 328 leads to the overall (http://www.ncbi.nlm.nih.gov/BLAST). The same primer increased stability of the protein with favorable torsion angle pair was then checked for Insilco PCR using UCSC human and ΔΔG value of 0.55. genome browser (http://genome.ucsc.edu/cgi). A product of Figure 4 127bp was also obtained with this. Following figures Figure 1: Comprehensive Prediction Results showed results obtained with different protocols: Figure 6 Figure 2: 10% gels showing product obtained with different protocols.

RFLP ANALYSIS Band pattern obtained after restriction digestion of 127 bp product is shown below.

Figure 7 Figure 5 Table 2: Six pairs of primers scrutinized were as below

Homozygous CC – 67bp, 60bp Heterozygous CT – 127bp, 67bp, 60bp Homozygous TT- 127 bp

The overnight digested product was checked on 10% PAGE and visualized by silver staining. Gel was run at 150V for 1hr 30min (fig. 3).

The amount of Taq polymerase was tested in the range 0.25 to 3 U per reaction. As little as 0.5 U of enzyme can be used without a decrease in the yield of PCR product (data not shown). PCR was performed at 25, 30, 35, and 40 cycles, Based upon tm and GC% and secondary structure and including the initial cycle. Although PCR products can be structural features like Dimer formation, hairpin loop, bulge detected after 25 cycles, maximal PCR product were loop and internal loop formation in the primers, only one obtained with 30 cycles of PCR amplification without the pair was selected as follows: production of nonspecific PCR products (data not shown). Forward primer- 5’GGTTGGTCACTGGAAGATGTTG3’ Same amount of PCR product was obtained with higher no. Tm-62.5 of cycles. The range of template genomic DNA necessary to produce detectable PCR products was then investigated,

3 of 7 Genotyping S328F SNP of human ACAT2 among CVD patients and normal population from North Western India and its associated lipid profile from 25 to 200 ng of DNA. 25 ng of genomic DNA can be Figure 9 amplified without a significant reduction in the intensity of Table 4: Allele frequency of S328F among populations of the signals (data not shown). 25pM - 60pM of primer the world (http://www.ncbi.nlm.nih.gov/SNP/snp) concentration was investigated, spurious amplification was observed with higher concentration of primers. 30pM of primer concentration was found to be sufficient to obtain good amount of PCR product (fig.2). Digestion of PCR product was carried out by directly addition of enzyme digestion buffer, without a reduction in the efficiency of BsmA1digestion (fig. 3).

Figure 8 Figure 3: 10% PAGE showing different genotypes. Figure 10 Table 5: Allele frequency of Punjabi population in comparison with other populations

As shown in table 2, higher frequency of C allele (0.65) than T (0.35) was observed in this population. Highly significant This methodology can be used for population screening or difference was observed in the allele frequency (p<0.0001) large number of patient samples within a short period. Given when compared with other populations of the world that protocol has been standardized as to maximize the analytical show monomorphism at this locus as reported at NCBI power by obtaining product with lowest amounts of input. (Table 5). The populations used for comparison with Punjabi Improved efficiency and lower cost should be useful in a population were of mixed origin from different regions of clinical diagnostic laboratory or a research laboratory the world. European population consisted of Utah residents performing population screening. As the allele studied has with ancestry from northern and western Europe. Asian relevance to prediction of disease susceptibility and drug population comprised of individuals from Beijing and China. response, it has been analyzed in Indian North Western The other Asian population was of Japanese origin from population as a test case. The developed protocol was used Tokyo in Japan. African population was from Ibadan and for SNP typing S328F in 200 normal healthy individuals Nigeria. All these samples were analyzed for SNP from Punjab. distribution by sequencing. All of them were found to be homozygous at this locus whereas Punjabi population displayed heterozygosity. ACAT2 is found predominantly in the liver and intestine, the major site of ApoB-containing

lipoprotein assemble and secretion [910]. As studied by Joyce

et al [11] cholesterol esters formed in the lumen may be readily used by apoB and microsomal triglyceride transfer protein for lipoprotein particle assembly, whereas proteins in the cytosol associated with lipid droplet formation may target cholesterol esters for storage. Since the T allele

4 of 7 Genotyping S328F SNP of human ACAT2 among CVD patients and normal population from North Western India and its associated lipid profile increases protein stability in the cytoplasmic region of the the region and consequently the activity of the enzyme. C protein which probably increases the binding of cholesterol allele leads to presence of ser328 and T allele by its for conversion to cholesterol esters and its consequent replacement with Phe328. The phe328 is found to stabilize accumulation into lipid droplets and increasing the tendency the structure in the region (as per CUPSAT) and is found to for lipid storage instead of lipid metabolism and transport. be present at a frequency of 0.35 as compared to ser328 at Its presence in the population may have implications for 0.65. Presence of T allele may have implications for disease susceptibility specially influencing accumulation of susceptibility to atherosclerosis in this population. Further cytosolic cholesterol droplets. study of patient group was undertaken to discern the effect of this prediction. In literature two ACAT2 models have been proposed. Joyce et al [11] determined that the Ser249 of ACAT2 resides in the As we found that the SNP could affect the disease active site by studying the mutations of the residue which susceptibility a study was carried out in a group of random leads to inactivation of the enzyme. They predicted presence unrelated patients having CVD. Total 166 samples were of five transmembrane domains. They further localized the analyzed for occurrence of different alleles. Highly Ser249 to putative ER lumen by observing glycosilation and significant difference (p= < 0.0001) was observed in the accessibility to proteases of microsomal membranes. The individuals having CAD from normal individuals (Table 6). SNP S328F apparently lies near to the active site and may be Figure 11 a part of the triad of amino acids Ser-His-Asp residues in the Table 6: Genotype distribution among Punjabi population active site as catalytic triad present in enzymes that bind Normal vs. Patient groups. cholesterol substrate like serine esterase, ACAT and LCAT

Song et al [12] proposed another model for ACAT2 which contains only two detectable transmembrane structure near N terminal region. They inserted two different antigenic tags (hemagglutinin, monoclonal antibody Mab1) at various hydrophilic regions flanking each of its predicted TMDs, Figure 12 and expressed the recombinant proteins in mutant Chinese Table 7: Lipid profile of CVD patients in relation to different hamster ovary cells lacking endogenous ACAT. The alleles. existence of these two TMDs has been predicted by the PhD algorithm and the TMpred algorithm. They identified the histidine residues frequently involved in the enzymatic catalysis due to reactive immodazolium nitrogen, which function as a hydrogen bond donor/ acceptor. Conserved histidine and serine residues are essential for ACAT2 activity Histidine and serine residues were identified by studying sites specific mutagenesis, and then expressing the mutants individually in AC29 cells by transient transfection. HDL levels among different genotypes were not found to be They found that H434 plays a significant role in ACAT significantly (p- 0.08) different from each other (Table No. catalysis due to its presence in hydrophobic region. 7) LDL levels were also not found to be significantly related to occurrence of different alleles. Total Cholesterol levels Sojin et al [12] further based on their site directed mutagenesis studies proposed that H360 and H399 are were found to be associated with higher frequency of T critical residues involved in ACAT catalysis. H360 and allele. Higher levels TC were associated with the presence of H399 are located in the cytoplasm side of the ER and F328 in the population (Table No.7). The risk ratio was explained why catalysis takes place in the cytoplasm or near found to be 1.4 times higher in individuals with T allele. The the cytoplasm side of the membrane. Mutated H399A in presence of aromatic hydrophobic amino acid Phenylalanine ACAT2 causes complete loss of enzyme activity. The in place of hydrophilic neutral serine residue leads to more presence of SNP C328T i.e. S328F near the active sites S249 accumulation of cholesterol esters as it increases the protein and H360 has potential to influence the structural stability of stability in the cytoplasmic region and consequently making

5 of 7 Genotyping S328F SNP of human ACAT2 among CVD patients and normal population from North Western India and its associated lipid profile the individual more susceptible to atherosclerosis. This study Sahlin, Curt Einarsson, Mats Eriksson, Bo Angelin, Hiroshi Tomoda, Satoshi Omura, Mark C. Willingham, Lawrence L. can be of fundamental importance to study the Rudel: ACAT2 Is Localized to Hepatocytes and Is the pharmacokinetics in relation to SNPs and to design specific MajorCholesterol-Esterifying Enzyme in Human Liver. inhibitors for ACAT2 to prevention of cardiovascular Circulation 2004;110: 2017-2023 5. Alper Uzun, Chesley M. Leslin, Alexej Abyzov and disease. Valentin Ilyin: Structure SNP (StSNP): a web server for mapping and modeling nsSNPs on protein structures with WEB LINKS linkage to metabolic pathways. Nucleic Acids Research, 2007; 35: W384-W392. http://cupsat.uni-koeln.de. 6. Jinghui Zhang1, David A. Wheeler, Imtiaz Yakub, Sharon Wei, Raman Sood, William Rowe, Paul P. Liu, Richard A. http://www.ncbi.nlm.nih.gov/Entrez/ Gibbs, Kenneth H. Buetow: SNPdetector: A Software Tool for Sensitive and Accurate SNP Detection. PLoS http://glinka.bio.neu.edu/StSNP/ Computational Biology October 2005; 1:53. 7. Vijaya Parthiban, M. Michael Gromiha1 and Dietmar Schomburg CUPSAT: prediction of protein stability upon http://www.ncbi.nlm.nih.gov/BLAST point mutations Nucleic Acids Research; 2006: 34, W239–W242 http://genome.ucsc.edu/cgi 8. Sambrook J, Fritsch EF, Miniatis T Molecular cloning: A Laboratory Manual (2nd Ed.) Cold Spring Harber (N.Y.) http.//www.neb.com Laboratory Press; 1989: Vol. 3. 9. Anderson, R.A., Joyce, C., Davis, M., Reagan, J.W., Clark, M., Shelness, G.S., and Rudel, L.L: Identification of a http.//www.ebi.ac.uk form of acyl-CoA:cholesterol acyltransferase specific to liver and intestine in nonhuman primates. J. Biol. Chem. CORRESPONDENCE TO 1998; 273: 26747–26754. 10. Cases, S., Novak, S., Zheng, Y.W., Myers, H.M., Lear, Dr. Praveen P. Balgir Department of biotechnology Punjabi S.R., Sande, E., Welch, C.B., Lusis, A.J., Spencer, T.A., university, Patiala-147 002 Phone no. – 09872886277 Krause, B.R., Erickson, S.K. and Farese Jr., R.V: ACAT-2, a Email- [email protected] second mammalian acyl-CoA:cholesterol acyltransferase, Its cloning, expression, and characterization. J. Biol. Chem. 1998; 273: 26755– 26764. References 11. Joyce CW, Shelness GS, Davis MA, Lee RG, Skinner K, 1. Chang T.Y, Chang C.C.Y, and Cheng D: Acylcoenzyme Anderson RA, Rudel LL. ACAT1 and ACAT 2 membrane A:cholesterol acyltransferase. Annu. Rev. Biochem 1997; topology segregates a serine residue essential for activity to 66: 613–638. opposite sides of the endoplasmic reticulum membrane. Mol 2. John R. Burnett, Lisa J. Wilcox, Dawn E. Telford, Sandra Biol Cell. 2000;11:3675–3687 J. Kleinstiver, P. Hugh R. Barrett, Roger S. Newton, and 12. Song Lin, Xiaohui Lu, Catherine C.Y. Chang, and Ta- Murray W. Huff: Inhibition of ACAT by avasimibe Yuan Chang: Human Acyl-Coenzyme A:Cholesterol decreases both VLDL and LDL production Acyltransferase Expressed in Chinese Hamster Ovary Cells: in miniature pigs. The Journal of Lipid Research 1999; 40: Membrane Topology and Active Site Location Molecular 1317-1327. Biology of the Cell 2003; 14: 2447–2460. 3. Lawrence L. Rudel, Richard G. Lee, Paolo Parini: ACAT2 13. Sojin Ana,b, Kyung-Hyun Choc, Woo Song Leea, Jie- Is a Target for Treatment of Coronary Heart Disease Oh Leed, Young-Ki Paikb, Tae-Sook Jeonga: A critical role Associated With Hypercholesterolemia Arterioscler Thromb for the histidine residues in the catalytic function of acyl- Vasc Biol. 2005; 25; 1112-1118 CoA:cholesterol acyltransferase catalysis: Evidence for 4. Paolo Parini, MD, Matthew Davis, Aaron T. Lada, Sandra catalytic difference between ACAT1 and ACAT2 FEBS K. Erickson, Teresa L. Wright, Ulf Gustafsson, Staffan Letters. 2006; 580: 2741–2749

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Author Information Praveen P. Balgir, PDF Department of Biotechnology, Punjabi university

Gurlovleen Kaur, Ph.D. Department of Biotechnology, Punjabi university

Divya, Ph.D. Department of Biotechnology, Punjabi university

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