Comparative Study on Human A-Glucosidase

Comparative Study on Human Α-Glucosidase Q. Ong1 and L. Le 1,2,* 2

1 School of Biotechnology of Ho Chi Minh International University, Vietnam Life Science Laboratory of the Institute for Computational Science and Technology at Ho Chi Minh City, Vietnam [email protected]

*

Abstract— α-glucosidase is an enzyme in human encoded by GAA gene [1]. Normally, this enzyme is essential for the degradation of glycogen to glucose in lysosomes. However, with patients of diabetes mellitus type 2, α-glucosidase causes the unwanted elevation of blood glucose level. Therefore, αglucosidase is now the target of anti-diabetic drugs. In this study, structure of human α-glucosidase was investigated using the approaches of bioinformatics tools. The results from phylogenetic tree also suggest the Mus musculus for animal testing. Finally, some approved α-glucosidase inhibitor drugs from Drugbank database are tested for binding affinity energy and pharmacophore features.

parameters (BLOSUM 62 matrix [4]). These sequences were the materials for progressive multiple sequence alignment (MSA) using Clustal X [5] with input ordered and Phylip output format. The MSA step was conducted several times to optimize by removing the most different sequences with very low identity degrees in order to gain the reliable alignment of homologies. The results from MSA were then used for refining the construction of phylogenetic tree which served to analyze the evolutionary relationship of α- glucosidase and its homologies in other species.

Keywords— α-glucosidase, diabetes mellitus type 2, multiple sequence alignment, phylogenetic tree.

Obtain 3D Structure of Human α-Glucosidase

I. INTRODUCTION α-Glucosidase inhibitors are oral anti-diabetic drugs used for diabetes mellitus type 2 that work by preventing the digestion of carbohydrates (such as starch and table sugar). Carbohydrates are normally converted into simple sugars (monosaccharides), which can be absorbed through the intestine. Hence, α-glucosidase inhibitors reduce the impact of carbohydrates on blood sugar. In order to support in vivo testing of α-glucosidase inhibitor, this research of αglucosidase will be defined on animal modeling and then observing the result before aplplying for human treatment. In addition, using computational tools including Clustal X, Treeview, VMD, Autodock Vina and other online softwares can test the drug compouds for α-glucosidase inhibitor. II.

MATERIALS AND METHODS

Multiple Sequence Alignment of Homologous α- Glucosidase Sequences Human α-glucosidase sequence (NCBI ID: ABI53718.1) as the template protein was collected from the NCBI database [2]. It is the full-length sequence of human α-glucosidase with 952 amino acids. The homologous αglucosidase amino acid sequences were taken from nonredundant protein database of Blastp [3] search with default *

Corresponding author.

Because the crystal structure of human α- glucosidase is still a mist, 3D structure of it was obtained using homology modeling provided by Swiss-Model workspaces with default parameters [6]. Phylogenetic Tree Construction The results of multiple sequence alignments were used as input data for constructing phylogenetic trees that would outline the interrelationships of the Homo sapiens enzyme and the other vertebrate species. To approach this, the PHYML [7] which implements the maximum likelihood method was used to estimate the phylogenetic tree using the options for amino acid data type, Jones, Taylor, and Thornton (JTT) substitution model and tree topology best search of NNI (Nearest Neighbor Interchange) and SPR (Subtree Prune and Regraft) search. Analysis of phylogenetic tree and suggest the suitable species for testing drug on animal Based on the results of phylogenetic study and the Three Rs principle which were first described by W.M.S. Russell and R.L. Burch in 1959 [8], one species was selected to be used as in vivo testing for α-glucosidase drug. Prepare for Docking Drug molecules were selected from Drugbank database (http://www.drugbank.ca).Several modifications from the original data were made using different software. VMD Visual molecular dynamics was used to visualize and separate the receptor for docking [9]. The PRODRG Server

© Springer International Publishing Switzerland 2015 V. Van Toi and T.H. Lien Phuong (eds.), 5th International Conference on Biomedical Engineering in Vietnam, IFMBE Proceedings 46, DOI: 10.1007/978-3-319-11776-8_86

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[10] was used to convert SDF and MOL2 file formats to the pdb format. All drug molecules were modified by using this server for obtaining drug molecules in the pdb file format. ADT / Auto-dock tools — Auto-dock was used to convert pdb files to pdbqt files for the docking process. All ligands and proteins in the pdb format were converted to the pdbqt format with the correction of charges for docking [11]. Python [12] is an integrative program that facilitates ADT and AutodockVina to run on the Linux platform.

residues W376, W402, D404, I441, D443, W481, W516, D518, M519, F525, R600, W613, D616, D645, F649, and H674 (Fig. 1B,1C). Asp-518 is predicted to be the essential carboxylate in the active site of human α-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized α-glucosidase cDNA in transiently transfected COS cells [16]. The residues Trp-516 and Asp-518 are also demonstrated to be critical for catalytic function (Fig. 2A)

Docking

Phylogenetic Tree Construction

The docking procedure requires the identification of the binding box position, which is the active site of the protein. The binding energy of these bound molecules on the protein was used for control docking. Furthermore, a grid box was prepared for docking drug molecules with a box size of 98x102x97Å spacing of 0.1 Å and a level 12 of exhaustiveness. Analyze of Docking Results The binding conformations of drug molecules with the protein were analyzed to find the basic interactions and characterize possible residues responsible for binding with the aid of VMD and ligand-scout [13] III.

RESULTS

3D Structure Generation of Human α-Glucosidase The 3D structure of human α-glucosidase was generated by SWISS-MODEL web server using crystal structure of the N-terminal domain of sucrase-isomaltase [14]. According to this alignment, the human α-glucosidase shares around 44.4% sequence identity and 89.0% sequence similarity with the template (Fig. 1A) Obtain 3D Structure of Human α-Glucosidase Because the crystal structure of human α-glucosidase is still a mist, 3D structure of it was obtained using homology modeling provided by Swiss-Model workspaces with default parameters [15]. Analysis of 3D Structure and Propose the Active Site of Human α-Glucosidase The structure of this model has 3 main part: N-termimal domain (residues 1-346), C-terminal domain (residues 724952) and the sub-domain (residues 347-723). The proposed active-site pocket here was composed to residues of

The phylogenetic tree was constructed using the result of ClustalX software. As the result, the proteins in each species share high degree of sequence similarity and common tertiary structure motif as well as the enzymatic specificity (Fig. 2B). Though all of these organisms are closely related to human beings (gorillas, gibbob, chimpanzee and monkeys) but they are limited for testing in terms of resources and animal research ethics. Therefore the Mus musculus (House mouse) is the best candidate for animal testing because they are satisfied the Three Rs principle. Testing the Best α-Glucosidase Inhibitors Structure Using AutoDock Vina After screening top ten approved drugs as α-glucosidase inhibitors from Drug Bank database, 3 best drugs were selected for studying pharmacophore features. They are Acarbose, Methyldopa and Voglibose. Based on docking result, Acarbose is the drug molecule had the lowest binding affinity energy among those 3 (Fig. 3). IV.

DISCUSSION

This research using the approaches of modern bioinformatics tools to give the most accurate results of Human α-glucosidase 3D structure. However, due to the rapidly increase of computational software developers, the results from updated tools may be quite different from what using in this study. Therefore, further studies should give the comparison on the results among similar softwares to give the most significant data. In addition, study on pharmacophore features of experimental drug-liked compounds as α- glucosidase inhibitors will be the next level of this research in future, which gives correlated data between computational and practical studies.

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Comparative Study on Human Α-Glucosidase

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Fig. 1 A. 3D structure of human α-glucosidase generated by SWISS-MODEL. B. The proposed active-site pocket with residdues W376, W402, D404, I441, D443, W481, W516, D518, M519, F525, R600, W613, D616, D645, F649, and H674. C. Binding pockket (red).

Fig. 2 A. Multiple sequences alignment using ClustalX, Trp-516 and Asp-518 (in Red column) are also demonstrated to be criitical for catalytic function. T (version 1.6.6) in which suggests Mus Musculus for in vivo testing. B. Phylogenetic tree constructed by TreeView


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Fig. 3 A, B, C are the 3D structures of Acarbose, Metthyldopa and Voglibose respectively with their binding affinity energy and a 2D structures with pharmacophore features. V.

CONCLUSION

α-Glucosidase inhibitors have high potentiial to be used as the early treatment for diabetes mellitu us type 2 by preventing the digestion of carbohydrates in our body. This research suggest Mus musculus as the animal model for αglucosidase drug testing. In addition, Acarbose A was suggested as the best drug for blocking α-gluccosidase as its lowest binding affinity energy and also shown many important pharmacophore features that can be used for further study on α-glucosidase as a target treatment for diabetes mellius type 2.

Altschul SF, Gish W, et al. Basic loocal alignment search tool. Journal of Molecular Biology, 1990. 2155(3): p. 403-410. Henikoff S and Henikoff JG. Amino aciid substitution matrices from protein blocks. Proceedings of the Nattional Academy of Sciences, 1992. 89(22): p. 10915-10919. Higgins,D.G., et al. (1992) CLUSTAL L V: improved software for multiple sequence alignment. CABIOS 88,189-191. Arnold K., Bordoli L., et al. (2006). The SWISS-MODEL Workspace: A web-based environm ment for protein structure homology modeling. Bioinformatics, 222,195-201. Guindon S and Gascuel O. A Simple, F Fast, and Accurate Algorithm to Estimate Large Phylogenies by Maxim mum Likelihood. Systematic Biology, 2003. 52(5): p. 696-704. Russell, W.M.S. and Burch, R.L., (19599). The Principles of Humane Experimental Technique, Methuen, Londdon. Visual Molecular Dynamics: www.ks.uiiuc.edu/Research/vmd The PRODRG Server : davapc1.bioch.duundee.ac.uk/prodrg/ ADT/AutoDockTools-AutoDock:autodoock.scripps.edu/resources/adt Sanner M (1999) Python: a programminng language for software integration and development. J Mol Grapphics Mod 17:57–61. Wolber G. Langer T (2005) LigandScouut: 3-D Pharmacophores Derived from Protein-Bound Ligands annd Their Use as Virtual Screening Filters. J. Chem. Inf. Model 445(1); 160-169. Sim, L. et al., Structural basis for subbstrate selectivity in human maltase-glucoamylase and sucrase-isom maltase N-terminal domains. J.Biol.Chem (2010). Arnold K., Bordoli L., Kopp J., andd Schwede T. (2006). The SWISS-MODEL Workspace: A web-baased environment for protein structure homology modelling. Bioinform matics, 22,195-201. Hong, Yeongjin, et al. "The Lec23 Chinnese hamster ovary mutant is a sensitive host for detecting mutationss in α-glucosidase I that give rise to congenital disorder of glycosylattion IIb (CDG IIb)." Journal of Biological Chemistry 279.48 (2004): 49894-49901. First author:Quang D. Ong Institute: International University Street: Quarter 6, Linh Trung W Ward, Thu Duc District City: Ho Chi Minh Country: Vietnam Emai: [email protected] Corresponding author: Ly Le Institute: Institute for Computationnal Science and Technology Street: Quang Trung Software C City, Tan Chanh Hiep Ward, District 12 City: Ho Chi Minh Country: Vietnam Emai: [email protected]

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