The Effect of C680T GPR43 Gene Variations to Its Interaction with Short Chain Fatty Acid (SCFA) In Type 2 Diabetes Mellitus
Rizky Amalia1, Karyono Mintaroem2, Loeki Enggar Fitri3, Nashi Widodo4, Hidayat Sujuti5
1 Master Program in Biomedical Science, Faculty of Medicine, University of Brawijaya, Indonesia
2 Department of Pathology Anatomy, Faculty of Medicine, University of Brawijaya, Indonesia
3 Department of Parasitology, Faculty of Medicine, University of Brawijaya, Indonesia
4 Department of Biology, Faculty of Mathematics and Natural Science, University of Brawijaya, Indonesia
5 Department of Biochemistry, Faculty of Medicine, University of Brawijaya, Indonesia Email address: [email protected]
Abstract Diabetes is a disorder in the metabolism of carbohydrates, lipids and proteins with various causes. From the previous research, GPR43 is a potent clinical target for the treatment of type 2 diabetes. It can be activated by Short Chain Fatty Acid (SCFA) which is an organic fatty acid produced through fermentation of dietary fiber by bacteria in the distal intestine. The aim of this study was to analyze the effect of C680T GPR43 Gene Variations to Its Interaction with Short Chain Fatty Acid (SCFA) in silico. The study used 8 sequences of GPR43: ID AF024690, AC002511, EU432114, BC095535, BC096198, BC096199, BC096200, BC096201 and data of ligand (SCFA): acetic acid (CID 176), Butyric acid (CID 264) and Propionic acid (CID 1032). GPR43 was modelled using I-TASSER, Sequence and structural alignments were conducted using Bioedit and Superpose V.10, respectively. The Docking process was done using PyRx and molecular interaction was analyzed using Discovery Studio 2016. The result showed that three types of SCFA bind to GPR43 variants T and GPR43 variant M obtained a similar pattern. The binding affinity from the largest to the smallest is butyric acid > propionic acid > acetic acid. GPR43 variant T has greater affinity to the SCFA than the GPR43 variant M. There is no defferences of preference between GPR43 variant T and GPR43 variant M to bind SCFA.
Introductions
Diabetes is a disorder in the metabolism of carbohydrates, lipids and proteins with various causes. The global prevalence of diabetes worldwide continues to increase with the mortality rate is very high. It was estimated that 382 million peoples have diabetes and 5.1 million of them died in 2013 (IDF, 2013) [1].
Diabetes is a group of metabolic diseases characterized by hyperglycemia resulting from defects in pancreatic β-cells that unable to produce insulin adequately, insulin resistance in peripheral tissue, or both the chronic
hyperglycemia of diabetes is associated with long-term damage, dysfunction and failure of different organs [2].
Preclinical study on the treatment of diabetes mellitus showed that agonist G- protein coupled receptors 120 (GRP 120) and free fatty acid receptors 4 (FFAR 4) increased insulin sensitivity, induced weight loss and reduced [3]. Previous studies showed that the FFA receptor agonists among which GPR120, GPR43 and GPR41 are potential clinical targets for the treatment of type 2 diabetes mellitus [4]. Research on GPR41 and GPR43 showed KEYWORDS
GPR43 SCFA Acetic acid Propionic acid Butyric acid.
that both can be activated by a Short Chain Fatty Acid (SCFA) [5, 3]. SCFA bond in GPR43 lead to several physiological responses.
Activation of the GPR43 enter endocrine intestinal cells induces the production of peptide YY (PYY) and GLP-1. PYY inhibits intestinal transit and appetite, whereas GLP-1 is to be anorexigenic and stimulate insulin secretion [5].
SCFA is a short chain fatty acid consist of 2 to 6 carbon atoms. These organic fatty acids produced in the distal intestine via bacterial fermentation of microfiber material that escapes from the process in the upper gastrointestinal tract and enter the [6]. SCFA function other than as a source of energy, also serves as an essential nutrient that acts as a signaling molecule. Anti-inflammatory and metabolic effects of (SCFA) associated with the activation of GPR43 (FFAR2) and GPR41 (FFAR3) [3]. Most of SCFA in the colon consist of acetate, propionate and butyrate with a proportion of 60% acetic acid, propionic acid 25% and 15% butyric acid [6].
Based on those findings, the computational research was done to analyze the effect of SCFA on interactions with GPR43 in type 2 diabetes. Identify most potential SCFAs to activate the GPR43 receptor and prove that GPR43 gene variations influence the molecular interaction with the SCFA.
Materials and Methods Target Sequence Retrieval
The DNA sequences of GPR43: ID AF024690, AC002511, EU432114, BC095535, BC096198, BC096199, BC096200, and BC096201 was obtained from the sequence
database of NCBI
(http://www.ncbi.nlm.nih.gov), and data of ligand (SCFA): acetic acid (CID 176), butyric acid (CID 264), and propionic acid (CID 1032) from PubChem
(http://www.pubchem.ncbi.nlm.nih.gov).
Sequence Alignment
Alignment was done to the sequence sample of GPR43 to know polymorphism area by aligning 8 sequences of GPR43 using ClustalW in Bioedit v.7.0.9 [7]. Gen sequent was translated to amino acids using Bioedit.
The differences of amino acid properties were analyzed using Peptide Property Calculator Webservices.
GPR43 Protein Modelling
Since the tertiary structure of GPR43 is not available in the protein structure databases, we used I-TASSER and validated using RAMPAGE to evaluate backbone conformation based on Ramachandran plot analysis and repaired using MODREFINER to generate 3D structures in good quality (Figure 1) [8]. Then analyzed the differences of protein structure between two models of GPR43 using Superpose v.10.
Figure 1. 3D Structure of GPR43 Human and its orientation in bilayer lipid
Molecular Docking and Interaction Analysis To predict site for interaction of GPR43 and SCFA, used the binding position of TAK-85 that already crystalographed with GPR-40 in
the PDB database as a reference. Molecular docking process was conducted using PyRx 0.8 software. Then visualised the interaction of both molecule, wildtype GPR43 and Polimorphism of GPR43 using Discovery Studio 2016.
Results and discussions
GPR43 gene variation analysis showed that the different nucleotide at position 680 is cytosine into thymine which in turn translated respectively into the amino acids threonine and methionine (Figure 2). Alignment results showed that there is a sequence of nucleotides that differ in sequence or classified polymorphic, which is contained in the sequence on the gene sequences with ID BC096201.
Figure 2. Alignment result of GPR43 gene and amino acid sequence
Alignment of amino acids showed that SNP in GPR43 caused a difference in one amino acid. In BC096200 (wild type), the amino acid is threonine (T) was turned into methionine (M) on BC096201 (polymorphic). Furthermore, BC096200 will be called GPR43 T variant and BC096201 will be called GPR43 M variant. It can be concluded from the alignment process earned their two different amino acid sequences. Both have a hydrophobic property, but methionine tends to be more hydrophobic than threonine which is a polar amino acid, while methionine is an aliphatic amino acid.
From the GPR43 variant M modeling resulted in a model with a C-score at 0.47 while the GPR43 variant T has C-score 12.52. Ramachandran plot value obtained is 82.3% for GPR43 variant M and 80.5% for variant T. Since Ramachandran plot the value of which is less than 90%, it can be said that the modeled protein has a slightly poor quality. Refined model obtained Ramachandran plot for the model GPR43 variant M as 92.7% whereas the T variant is 94%. Attaining Ramachandran plot values >90%, it can be said that they have a good quality. The results of three- dimensional visualization of the model variant models GPR43 variant T and M can be seen in Figure 3.
It can be seen that the model GPR43 T and M variants have a similar properties and structure, does not alter the conformation polymorphism protein. Only the side chains of amino acids are different. Superpose V.10 analysis results showed that both models have almost the same structure, and shown by the RMSD value as 1.19 Å. If the RMSD less than 2 Å, two protein structures can be said to be identical.
Figure 3. GPR43 3D model using I-TASSER. a) Analysis of differences in amino acid properties by using Peptide Property Calculator Webservices. b) Model GPR43 variant M and GPR43 variant T. c) Top-view of GPR43.
Docking result showed that energy on bonding GPR43 variant M with acetic acid at -2.8 Kcal/mol, the bond energy GPR43 variant M with butyric acid at -3.6 Kcal/mol, while the bond energy GPR43 variant M with propionic acid is -3.3 Kcal/mol. From these results, it can be concluded that the lowest energy is required for bonding with butyric acid that is known the most potential SCFA to activate GPR43 variant M is butyric acid, propionic acid and followed by acetic acid.
Molecule interaction involving the types of interactions occurred with hydrogen bonds and hydrophobic interactions between SCFA and GPR43. It is the contribution of various bond determine the binding energy score:
∆𝐺𝑏𝑖𝑛𝑑𝑖𝑛𝑔 = ∆𝐺𝐺𝑎𝑢𝑠𝑠+ ∆𝐺𝑟𝑒𝑝𝑢𝑙𝑠𝑖𝑜𝑛+ ∆𝐺𝐻𝑏𝑜𝑛𝑑+ ∆𝐺ℎ𝑦𝑑𝑟𝑜𝑝ℎ𝑜𝑏𝑖𝑐+ ∆𝐺𝑡𝑜𝑟𝑠𝑖𝑜𝑛
The free binding energies are Gaussian value, repulsion, hydrogen bonding, hydrophobic interactions and the effect of the amount of the bond rotates [9]. Based on these equations, the more the contribution of these effects, the binding energy will be more negative.
From the three types of docking process SCFA against GPR43 T and M variant obtained a similar pattern. The potential activation from the highest to the lowest are butyric acid > propionic acid > acetic acid (Figure 4).
Figure 4. Comparison of Binding energy between GPR43 (M) and GPR43 (T) interact with SFA
Generally, the required energy to bind SCFA on GPR43 M variant is smaller than on T variant.
So, the potential bond between SCFA with GPR43 M variant is greater than on T variant.
The results of molecular interactions analysis, in the complex GPR43 M variant with acetic acid, there are two hydrogen bonds at SER86 and GLU166. In the complex GPR43 M variant with butyric acid obtained two hydrogen bonds between GLU164 and CYS166, whereas phi sigma bond to the amino acid TYR89 and PHE90 (Figure 5).
The interaction between GPR43 T variant with acetic acid, hydrogen bonding a hydrogen bond is exists on SER86. While the GPR43 T variant with butyric acid, there are two hydrogen bonds at SER86 and GLU166, as well as phi sigma bond on PHE89 and LYS65. While the molecular interactions that occur between the T variant GPR43 with propionate acid is the hydrogen bond and bond SER86 phi sigma on PHE89 (Figure 5).
Figure 5. Molecular interaction of SCFA on the complex of GPR43 M and T variant. a) GPR43 T variant complex with acetic acid; b) GPR43 T variant complex with butyric acid; c) GPR43 T variant complex with propionic acid; d) GPR43 M variant complex with acetic acid; e) GPR43 M variant complex with butyric acid; f) GPR43 M variant complex with propionic acid.
Discussions
GPR43 is one of the potential clinical targets for the treatment of type 2 diabetes mellitus [4] that both can be activated by a Short Chain Fatty Acid (SCFA) [5, 3]. This research was aimed to analyze the effect of GPR43 gene variations to its interaction with Short Chain Fatty Acid (SCFA) in silico using 8 DNA sequences obtained from the sequence database of NCBI. The strength of this study was done in 3D protein modeling of GPR43 T variant and M variant in silico which has never been done in previous studies.
According to the research of the alignment of amino acids showed that there is an SNP in
nucleotide number 680 of GPR43 that caused a difference in one amino acid. In BC096200 (wildtype), the amino acid is threonine (T) is turned into methionine (M) on BC096201 (polymorphic).
SNP is the most common genetic variations. Each SNP showed a difference of one nucleotide and occurred normally in a person's DNA. SNPs may role as a biomarker to help scientists find the genes associated with disease. When SNPs occur within a gene or gene regulators in the near area, it will play a direct role in the disease by affecting the function of the gene. Most SNPs have no effect on health or development. However, their
genetic differences have proven to be very important in the study of human health.
Researchers have found SNPs that can help predict an individual's response to a particular drug, susceptibility to environmental factors such as toxins, and the risk of developing certain diseases (Lister Hill National Center for Biomedical Communications, 2016).
It can be seen that three-dimensional visualization of the GPR43 model T variant and M variant was not found significant differences between the two dues to the amino acid Threonin and Methionin both have hydrophobic properties.
From the three types of docking process SCFA against GPR43 T and M variant obtained a similar pattern. The potential activation from the highest to the lowest are butyric acid >
propionic acid > acetic acid (Figure 4). It is caused by the influence of the length of active groups on each SCFA. The longer the active group, the stronger the bond, and vice versa.
Generally, the required energy to bind SCFA on GPR43 M variant is smaller than on T variant. So, the potential bond between SCFA with GPR43 M variant is greater than on T variant. This is likely due to slight differences in the properties of Methionin and Threonin amino acids. It is thought to make a difference in the treatment effect of SCFA for type 2 diabetes in individuals with different variant GPR43.
This study limitation was limited sample only downloaded from NCBI. For further study, we suggest researcher to do the same study with more sample in vivo so the result of the study can be used as the basics for therapy using SCFA in DM type 2. We also suggest to study further about the effect of GPR43 gene variations to its interaction with Short Chain Fatty Acid (SCFA) and the therapeutic effect in DM type 2 patient.
The weakness of this study is the limited number of samples which is only downloaded
from NCBI. For further research, it is advisable to do research with more sample, if possible, be done in vivo so that research can provide benefits as the basic for SCFA therapy in patients with Diabetes Mellitus. Also needs to be researched more about the interaction between GPR43 T variant and GPR43 M variant with SCFA and its therapeutic effect on patients with type 2 of diabetes.
Conclusions
The 3D model of GPR43 M and T variants had identical structure and polymorphism does not alter the protein conformation except the side chains of amino acids that are slightly different, so there is little difference in the surface protein and binding potential with SCFA GPR43 M variant is greater than that on T variant. This is because GPR43 polymorphism occurs in Methionine Threonine amino acids, while both are hydrophobic. The binding affinity of SCFA bind to GPR43 from the largest to the smallest is butyric acid > propionic acid >
acetic acid. GPR43 variant T has greater affinity to the SCFA than the GPR43 variant M. There are no differences of preference between GPR43 variant T and GPR43 variant M to bind SCFA.
What is known in this topic?
Model GPR43 T and M variants have a similar properties and structure, does not alter the conformation polymorphism protein;
The binding affinity of SCFA bind to GPR43 from the largest to the smallest is butyric acid> propionic acid > acetic acid
What this study adds
This study shows the effect of SCFA on interactions with GPR43 in type 2 diabetes.
Prove that GPR43 gene variations do not influence the molecular interaction with the SCFA.
Competing interests
The author declares no competing interest.
Authors’ contributions
Each author has read and agreed to the final version of this manuscript and has contributed to its content and to the management of the case.
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