INTRODUCTION:

Lysosomes organelles which have acidified cytoplasm consist of degradative or hydrolytic enzymes specific for the degradation of cellular components¹. The group of enzymes which are involved in the degradation of terminal, non-reducing sugar units from oligosaccharides molecules are exo-glycosidases. Gene Hex-A on chromosome 15 in human provides instructions for making subunit of an enzyme called Beta-Hexosaminidase A (BHA).

The BHA are lysosomal isozymes that separate the glycosidic linkages of the non-reducing, terminal L-DN-acetylglucosamine (GlcNAc) or L-D-N-acetyl galactosamine (GalNAc) residues on glycolipids, glycoproteins, also glycosaminoglycans. The Activator is a substrate specific (GM2 ganglioside) co-factor for one of the hexosaminidase isozymes, hexosaminidase - A. The presence of an N-acetyl-L-D-glucosaminidase (NAG) was first reported in 1936².

Specifically the protein produced from the Hex-A gene forms the alpha subunit of the enzymes BHA³. One alpha subunit interacts with one beta subunit produced from Hex-B gene to form a functioning enzyme. This complex formed interacts with GM2 Activator to prevent the accumulation of GM2 gangliosides¹.Tay-Sachs is autosomal recessive genetic disorder which is caused by mutation in a vital enzyme BHA. Classical Tay-Sachs patients usually do not survive beyond 4 years of age⁴. There were many mutations listed out of which we chose the Ser279 to Pro279⁵. Tay-sach’s is neurodegenerative lysosomal storage diseases associated with excessive accumulation of GM2⁶. The history of TaySach’s diseases was reported are listed(Table 1). Due to Hex-A genedysfunctioning there is accumulation of a fatty substance called GM2 gangliosides especially in the nerve cells of the brain. This progressive accumulation of GM2 gangliosides leads to damage in the nerve cells¹.GM2A is an activator that initiates degradation of GM2 gangliosides³. It acts as substrate specific co-factor for BHA as it is a large binding pocket that can hold single chain phospholipids and fatty acids. It has TYR137 on the surface that plays a major role in lipid transfer from the membrane to the BHA⁷. It works by binding gangliosides and stimulates GM2 degradation by calcium independent phospholipase activity⁴. It only stimulates breakdown of GM2 and GA2 by the beta Hex-A. It extracts single GM2 units from membrane which is presented in soluble form to BHA for cleavage of N-acteyl D-galactosamine and conversion to GM3.Since the major interaction is between GM2A and BHA, the GSEP₂₈₃ loop(alpha loop) in Hex-A protein is reported as to be responsible in binding to GM2A³.

Table 1.TaySach’s history

Year	Event	Ref.
1881	Warren Tay: initial description of clinical characteristics, cherry red spot	²⁰
1896	Bernard Sachs: `Amaurotic family idiocy' prevalent in the Jews	²¹
1962	GM2 ganglioside identi¢ed as the compound stored in TSD	²²
1968	Demonstration of two major Hex isozymes in normal human tissue (Hex A and Hex B)	¹⁰
1969	Hex A, but not Hex B is de¢cient in Jewish TSD	¹²
1969	Isozyme patterns in non-Jewish TSD patients: (a) deficiency of both Hex A and B (SD); (b) no Hex deficiency AB-variant and B1-variant	¹³
1973	Immunological basis for the subunit compositions of Hex A (KL) and B (LL): deficiency of the K results in TSD, deficiency of L results in SD	²³
1979	Purification and characterization of the GM2 Activator protein: its deficiency results in the AB-variant form	²⁴
1984-1985	Isolation of cDNA clones encoding the K- and L-subunits	^15,25
1986	Identification of partial HEXA and HEXB gene deletions resulting in infantile TSD and SD	^14,17
1988	Identification of two major HEXA mutations responsible for TSD in Ashkenazi Jews	^26-28
1989-1992	Isolation of a cDNA clone encoding the GM2 activator	¹⁶
1991-1992	Association of a GM2A mutation with the AB-variant form	^18,19

GM2 is an intermediate in both the amalgamation (ER) also, degradation (lysosome) of the higher brain gangliosides, e.g. GM1. So as to hydrolyze GM2, Hex A needs the little, monomeric, heat stable GM2 activator protein. The Activator connects with both the carbohydrate and lipid part of the ganglioside and it takes out steric hindrance from the membrane by solubilizing a ganglioside. Also, Activator binding may interfere with a specific hydrogen bond between the acetamido-NH of the GalNAc residue and the carboxylic group of the NeuAc residue of GM2, making the GalNAc accessible for hydrolysis by Hex A⁸. The relative significance of these two factions is right now a matter of level headed discussion in the writing⁹.

Our main focus is to provide evidence that GSEP₂₈₃alpha loop of BHA interacts with GM2 Activator for the degradation of GM2 gangliosides, the amino acids involved in the interaction and comparative study of binding energy before and after mutation. We also provide our own hypothesis related to functioning of GM2 Activator with BHA.

MATERIALS AND METHODS:

The software and server used are Arguslab 4.0.1, PyMOL, and SPDBV.HADDOCK2.2: High Ambiguity Driven protein-protein DOCKing- is information operated flexible docking method for the modelling of biomolecular complexes. This docking online application differs itself from ab-initio docking methods encodes information from identified or predicted protein interfaces in ambiguous interaction restraints to drive the docking process. HADDOCK can deal with many classes of modelling problems such as protein-protein, protein-nucleic acids and protein-ligand complexes¹¹.

The protein3Dstructures were obtained from Protein Databank (PDB). BHA protein (PDB ID:2GK1_A) and GM2A (PDB ID:2AF9) were taken as receptor and ligand respectively. We preceded our studies in two ways, firstly the unmutated A chain of 2GK1 was docked with 2AF9 by using HADDOCK2.2 server. Secondly, the 2GK1_A was mutated (S279P) using SPDBV and PyMol. This mutated Hex-A protein (2GK1_A) was then docked with GM2A (2AF9) and the amino acid interactions were studied. The docked clusters were then viewed in PyMol and SPDBV.

RESULTS AND DISCUSSION:

The HADDOCK results provided the best structures or solution. The Z score indicates how many deviations from the average a particular cluster is located in terms of score (the more the negative the better). The lower the energy is better.

Table 2.Energy profile of before and after mutation (Ser279Pro) of BHA.

Energies/parameters	Binding energy before mutation (kcal/Mol)	Binding energy after mutation (kcal/Mol)
Z score	-1.2	-1.5
RMSD from the overall lowest energy structure	7.3 +/-0.1	1.2 +/-0.8
Van der Waals energy	-29.4 +/-4.4	-34.8 +/-4.9
Electrostatic energy	-127.4 +/-26.5	-184.1 +/-6.3
Restraints violation energy	58.4 +/- 3.00	39.5 +/-10.53
Haddock score	-66.6 +/-2.7	-89.9 +/- 2.4

Figure 1.The graphical representation of the energy before and after mutation

Unmutated A chain of BHA (2GK1_A) docking with GM2A (2AF9):

HADDOCK clustered 397 structures in3 cluster(s) out of which top 10 were shown, which represents 99.25% of water-refined models generated. Figure 1 shows the cluster 2 was the best solution with Z score of -1.2 out of 3 clusters. The electrostatic and Vander Waals energy were recorded as -127.4 and -29.4 respectively and the lowest RMSD value 7.3 (Table 2).

The unmutated A chain of Hex-A (2GK1_A) interaction with the GM2A (2AF9) shows that, the GSEP₂₈₃ alpha loop of 2GK1_A is interacting with 2AF9. We also found the amino acids Met0, Glu58, Asn136 and Ala60 of 2AF9 were interacting with the 2GK1_A GSEP₂₈₃ loop (Figure 2). The TYR137 of GM2A (2AF9) plays a major role in lipid transfer from the membrane to the Hex-A (2GK1_A), confirmed from the result obtained and also highlighted that its present on the surface.

Figure2. Partial GSEP₂₈₃loop(right) interaction with 2AF9 (left)

Mutated A chain of BHA (2GK1_A) docking with GM2A (2AF9)

HADDOCK clustered 379 structures in 4 cluster(s) out of which top 10 were shown, which represents 94.75% of water-refined models generated. Figure 1 shows that the cluster 1 was the best solution with Z score of -1.5 out of 4 clusters. The electrostatic and Vander Waals energies were-184.1 and -34.8 respectively (Table 2).

Figure 3.Partial GSEP₂₈₃loop (right) interaction with 2AF9 (left)

The mutated 2GK1_A interaction with the 2AF9 when viewed in SPDBV and PyMol we observed that the GSEP₂₈₃loop is partially interacting with the 2AF9 (the GM2A) as compare to the unmutated 2GK1_A docking results. We also analysed that this docking result are better than the unmutated 2GK1_A docking with 2AF9 as the Z score is -1.5. We found that the Gly280 and Ser281 of GSEP₂₈₃loop are the only one interacting with 2AF9 amino acids Asn136 and Glu58 and Tyr137 respectively (Figure 3). The Tyr137 interaction with the Ser281 is a weak hydrogen bond.

CONCLUSION:

This study is the evidence to research done earlier reporting that GSEP₂₈₃ alpha loop of Hex-A protein binds to the GM2Activator. From the above results we can hypothesise that Glu282 of GSEP₂₈₃alpha loop plays a crucial role in the functioning of Hex-A and GM2Actiavtor in degradation of gangliosides and that the Tyr137 is restricted after mutation to play its role in lipid transfer since it is interacting with Ser281(BHA) and results in TaySach’s disease. This in silico model also demonstrates for the first time that the GSEP₂₈₃ loop of Hex-A protein interact with Met0, Asn136, Glu58, Ala60 of GM2Activator. Our study opens the gate for further research regarding mutation, interactions and functioning involved in Tay-sach’s disease. It can also help in the study of drug related to TaySach’s disease

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Received on 11.06.2017 Modified on 07.07.2017

Research J. Pharm. and Tech 2017; 10(11): 3899-3902.

DOI: 10.5958/0974-360X.2017.00708.9