Molecular Evolutionary Studies of Sequences for NR1subunit of NMDA Receptor from Diverse Organisms SOUMYA.P1, MEDHA S RAJADHYAKSHA2, MANJULA MATHUR3 1: Dept. of Bioinformatics, Bharathiar University, Coimbatore-641046 2: Sophia College for Women, Bhulabai Desai road, Mumbai-400 026 3: Mol. Biol. Division, Bhabha Atomic Research Centre, Mumbai -400085 Abstract:-We have done sequence analysis of NR1 subunit of NMDA receptor taking the sequence data from NCBI. Phylogenetic analysis of nucleotide sequences by Distance method, Maximum Parsimony method and Maximum Likelihood gave us three clusters consisting of vertebrates, invertebrates and mollusk leading us to infer that the mollusks are a class apart from other invertebrates and vertebrates. This could be related to the structural organization of neurons for a faster conduction rate, where the new role of NMDA receptor besides excitatory synaptic transmission needs further investigation. The presence of NR1 subunit like amino acid sequence in Arabidopsis thaliana suggests that cell to cell signaling by excitatory amino acids existed before the divergence of plants and animals during organic evolution. Understanding the regulatory role of NR1 subunit in Ca++ permeability and selective conduction of ions through channel pores in plants and other organisms would enhance the research on biochemistry of brain functions. Key-words: - NMDA receptor, NR1 subunit, Cluster analysis, Molecular Evolution, Excitatory synaptic transmission, Distance method, Maximum parsimony method, Maximum likelihood method. 1 Introduction: Ionotrophic glutamate receptors (iGluR’s) (Sprengel et al 1995) are known to be highly conserved and reported in diverse life forms. They mediate excitatory synaptic transmission in neurons of the animals while their function in plants such as Arabidopsis thaliana is under investigation. Vertebrate GluR's are pharmacologically classified as 1)AMPA(- amino-3-hydroxy-5-methyl-4-iso xazolepropionic acid 2) Kianate 3) NMDA (Nmethyl-D-aspartic acid) receptors. Of these NMDA receptors display three unique properties: 1-NMDA receptor depends on simultaneous binding of glutamate from synaptic vesicles and glycine from the extra cellular fluids which can modulate the receptor responses. (Johnson and Ascher 1987; Kleckner and Dingledine 1998). 2-For activation of NMDA receptor the membrane should be depolarized and the blockage of ion pore should be removed. (Mac Donald et al., 1982; Myer et al 1984; Nowak et al 1984) 3-NMDA receptor is highly permeable to calcium which can further activate several pathways in the post synaptic neuron. NMDA receptor has been implicated in several brain functions such as memory, neuronal differentiation and synaptic plasticity during development. Further it is also implicated in neurotoxical changes in several diseases. It is therefore a molecule that deserves attention. Like other iGluR’s, NMDA receptor too is a tetrameric protein (Hollamnn and Heinemann, 1994; Laube et al; 1998) containing two subunits NR1 and NR2 with several subtypes of each being reported. NR1 is generated from a single gene through utilization of alternate RNA splicing, (Anantharam et al., 1992; Durand et al., 1993; Hollmann et al 1993; Sugiharal et al., 1992) while NR2 subunit is encoded by four separate genes (2A/2B/2C/2D) (Ikeda et al., 1992; Ishii et al., 1993; Kutsuwada et al., 1992; Meguro et al., 1992; Monyer et al., 1992). NR1 and NR2 will form functional receptor complex with varying properties depending on the subunit composition. The NMDA receptor protein like other non NMDA receptors has four distinct structural domains 1- The amino terminal region forming the extra cellular part of the receptor. 2- Tran membrane helices TM1, TM3, TM4 and a pore forming P region. 3- Two segments S1 and S2 which connect the transmembrane receptor segments (O’Hara et al., 1993; Stern-Bach et al., 1994) and S P S1 M1 M2 M3 4- Intracellular carboxy terminal domain involved in the protein-protein interactions. While the NR1 gene is conserved through the animal world, their subtypes and expression in different neurons is because of alternative RNA splicing and may have functional significance. A cluster analysis of sequences of conserved gene provides not only phylogenetic information but also reflects on the evolution of function. NR1 is therefore a good candidate gene to study the same. S2 M4 Fig: 1 the general structure of NR1 subunit. . S Fig 2: A typical vertebrate NR1 subunit is shown below. Red represents Signal peptide, blue is S1 and S2 domains, Black are TM regions, violet represents highly variable regions across the sequence 2 Materials and Methods: 2.1 Sequences: 2.2.1 Alignment: The nucleotide sequences of NR1 subunit used in our study were retrieved from the database of NCBI (National Centre for Biotechnology Information) http://www.ncbi.nlm.nih.gov. Table 1 list all these nucleotide sequences with their species of origin, accession numbers, number of nucleotides, number of amino acids coded by them and the symbols used in the phylogenetic trees. First we started our search using key word NR1 subunit of NMDA receptor. Later for retrieving more sequences we did BLAST search with rat NR1 subunit as query sequence. With BLAST search we got 100hits from various species. Of these we selected complete mRNA sequences of NR1subunit from 12 different species starting from Protozoan to Mammalians with an exceptional hit from a plant viz., Arabidopsis thaliana that we included in our studies. TABLE 1: The list of NR1 subunit mRNA sequences used in our study NR1 genes Accession Numbers Length Of mRNA Coding Regions AA Symbol In tree Homo Sapiens NM_007327 5137bp 1094-3910 (2817) 938 Human Rattus norvegicus Mus Musculus Gallus galus Anas platyrhynchos Xenopus laevis Apteronotus leptorhychua Aplysia californica Drosophila melanogaster Anopheles gambiae Apis Mellifera Caenorapditis elegans Arabidopsis thaliana NM_017010 NM_008169 AY510024 D83352 X941556 AF060557 AY234809 NM_169059 XM_321646 AY331183 AF318613 NM_116503. 4213bp 3031bp 3820bp 3874bp 4060bp 4102bp 3331bp 4099bp 2901bp 3160bp 3078bp 747 bp 266 -3082 (2817) 94 – 2910 (2817) 7– 2904 (2898) 463 – 3360 (2898) 109 -2823 (2715) 29 – 2929 (2901) 204 -2852 (2649) 237 – 3230 (2994) 1 – 2901 ( 2901) 131 – 2992 (2682) 1-3078 (3078) 1-747 ( 747) 938 938 965 965 904 966 882 997 966 893 937 248 Rat Mouse Chick Duck Frog Fish Seahare Drosophila Anopheles Honey bee Celegans Arabidopsis 2.2 Methodology: DAMBE (Data Analysis in Molecular Biology and Evolution), (Xia. 2000) an integrated software package for analyzing molecular sequence data was used in our study. We downloaded the DAMBE through the internet from the website: http://web.hku.hk/~xxia/software/software.htm mRNA sequences were aligned using the inbuilt clustal W program in DAMBE with default parameters using BLOSUM matrix. First the nucleotide sequences were uploaded as fasta format files and translated into amino acid sequences to check their quality. Then the aligned amino acids are compared against the unaligned nucleotide sequences. This procedure ensures that no frame shifting indels are introduced as an alignment artifact. A tree topology was generated with aligned sequences. Multiple alignment of amino acid sequences of all NR1 sequences was also carried out using MULTALIN program (Corpet 1988). We used Risler substitution matrix for aligning NR1 sequences. Subsequently we took NR1 subunit proteins of Drosophila, human and sea hare as representative species from the three different clusters of invertebrates, vertebrates and mollusks. We used BLOSUM substitution matrix, for alignment that gave us a good alignment of conserved domains in the three amino acid sequences. Alignment results of these three amino acid sequences of NR1 are further discussed below. 2.2.2 Cluster Analysis: . 1) Distance method, 2) Maximum parsimony method and 3) Maximum likelihood methods inbuilt in DAMBE software package were used for phylogenetic analysis. DAMBE calculated genetic distances using nucleotide sequences and the resulting matrix were used for phylogenetic reconstruction using Kimura’s (Kimura 1980) distance method. This method operates on genetic distance matrix based on K80 substitution model. The results are shown in Figure 1. For Maximum parsimony (Baldi and Brunak 1998) analysis, the parsimony algorithm DNAPARS implemented in DAMBE was used with default parameters choosing seahare as the out group. The results are shown in Figure 2. This method assumes that the substitutions are rare, uniform and the substitution rate is constant over time in different lineages. For Maximum likelihood analysis DNAML program implemented in DAMBE was used with the nucleotide substitution model F84 that includes both frequency parameters as well as rate-ratio parameters. The results are shown in Figure 3. The underlying assumption in this method is that the substitutions occur independently in different sites and different lineages by a stationary Markov process. 3 Results and Discussion: 3.1 Neuronal Conduction : The origin of every thought or action is a co -ordination of nerve impulses that travel through the neurons. These impulses are generated by the movement of electrically charged inorganic molecules through the neural membranes with the help of channels embedded in them. These channels are large proteins and their properties are determined by the genome, itself the result of evolution. The fundamental property of this membrane is that it is semi-permeable i.e. it allows some charged molecules, known as ions, to pass through more easily than others. Among these ions that play an important role in the nervous system, potassium (K+), which is positively charged, is the one that passes most easily through a neural membrane in its resting state and is involved in the process of synaptic transmission. The process that enables a nerve impulse to pass from one neuron to another is called synaptic transmission. This transmission is effected by neurotransmitters, which bind to the specific receptors. It is through variations in the amount of neurotransmitters released, the receptors available, and the affinity between the two that the synapses undergo changes and enable learning. Synaptic transmission is an omnipresent mechanism that is the source of the brain’s great plasticity. This synaptic plasticity is the key role of NMDA receptors and is crucial for memory. NMDA receptor is highly permeable to Ca++ which activates intracellular signals that can modify the synaptic function (Dunn et. Al. 1999). 3.2 Cluster Analysis of NMDA receptor in various organisms: While a high percentage identity was seen among all organisms, according to our analysis carried out by three different methods in DAMBE the mRNA sequences of NR1 subunit grouped into three clusters. The phylogenetic trees obtained by DAMBE are shown in Fig. 1, 2 and 3. Cluster one includes essentially invertebrates viz., Drosophila, Anopheles, Honeybee and C. elegans which appear early in the evolutionary tree. Cluster two included the vertebrates viz., Human, Rat, Mouse, Duck, Chick, Frog and Fish which appear much later in the evolution. Very interestingly NR1 subunit of Seahare which is a Mollusc is clustering independent of the two clusters of vertebrates and invertebrates. . A. thaliana NR1 in our analysis is clustering with invertebrates in Figure 1 and 3 but differently in Figure 2. To study the details of functional evolution of NR1, we further did multiple alignment of amino acid sequences of one organism from each cluster viz. human, drosophila and seahare. The Tran membrane regions are integral part of the membrane. The TM1, TM2 and TM3 domains were found to be highly similar while variability is found in N-terminal extra cellular domains and C-terminal intracellular domains of NR1 sequence. The Domain names were used as such to represent human amino acid sequences in all the alignment pictures shown below. TM1 REGION: This region is about 21aminoacids long and is conserved across the phyla. There are however four regions where variations are seen. Substitution mutations seemed to have occurred in these sites during the evolution. TM3 REGION: This region spans across the membrane and is 21 amino acids long. This region is highly conserved with no variable sites across the phyla. TM4 REGION: This is the most variable segment of the protein. Apart from first 6 amino acids which are identical in all phyla, the region is highly variable , though not variable in actual size. S2 DOMAIN: This region is found in between TM3 and TM4 and is of 20 residues long. This along with S1and TM regions plays a role in ligand binding. Consensus are low in this region. EXTRACELLULAR DOMAIN: This region is of TM2 REGION: This is the main segment of the pore complex and does not span across the membrane but is associated with the cytoplasmic side. Except few point mutations this region is totally conserved. Strong functional constraints of this region are reflected in its very low variability. 540 residues long and is the N-terminal part of the protein. This region contains the S1 segment of ligand binding pocket. High variability is observed in this region .The glycosylation sites were mainly concentrated in this zone, which may thought to have some functional importance. INTRACELLULAR REGION: The size of this domain varies from 50-197 amino acids and is the C-terminal domain. There is no consensus in this segment. This segment is not involved in ligand binding and is suggested to be involved in species specific protein protein interactions. We have come to two conclusions from these inferences 1: Regions of the protein that are functionally involved in ligand binding or in the pore formation are highly conserved. 2: Variations are restricted to amino and carboxy terminal regions which are known to be spliced variably. Though these variable regions are not involved directly in ligand binding or calcium channel function, these regions of the molecule are important in differential control of NMDA receptor mediated responses. The variability in NR1 is being generated by differential splicing. The splicing of C-terminal region has been shown to be different in teleost fish and human and it has been proposed that evolution of this molecular strategy predates divergence of mammals and teleost fish [Dunn et al., 2003]. Our studies demonstrate that variability in the C-terminal is essentially responsible for the three clusters with invertebrates, vertebrates and mollusks forming the 3 diverged groups. This suggests that mollusks had an evolutionary history of neuronal development independent of other invertebrates. Interestingly mollusks are known to diverge separately from invertebrates and vertebrates in their strategy of increasing conduction efficiency of their neurons. Unlike other invertebrates they have big neurons with larger axons. Vertebrates on the other hand have evolved better conduction by developing myelination. This strictly reinforces the divergence of mollusks nervous system from other invertebrates that may also reflect in its molecular mechanism of processing post-synaptic response to NMDA stimulation and deserves to be investigated in detail. Especially divergence in the C-terminus of NR1 subunit suggests that differential protein protein interactions are possible and need to be investigated Figure 3 : Phylogenetic tree obtained by Distance method. The symbols represent data as given in table1. Figure 4 : Phylogenetic Tree obtained by Maximum Parsimony method. Figure 5 : Phylogenetic Tree obtained by Maximum Likelihood method-DNAML 3.3 Role of NMDA like receptors in Plants: Plants possess NMDA like receptors generically referred to as GluR’s that bind to glutamate (Joanna Chiu et al 1998). During our studies, BLAST search of the database with rat NR1 subunit, gave us one NMDA like receptor from Arabidopsis thaliana. Though it is much smaller compared to other NR1 sequences, we have included it in our analysis. The role of NR1 subunit like protein in plants is very interesting. As NMDA receptors are subfamily of GluR’s and are important in ion conduction further investigation is suggested. GluR’s are demonstrated to be involved in the synergistic action of glutamate and glycine to control ligand gated calcium intake (Christian Dubos et al., 2003). The presence of glutamate receptors in plants suggests that NMDA receptor like molecules have evolved even before the divergence of plants and animals. Similar inferences were drawn by Joanna Chiu et al 1999 on the basis of theoretical analysis of GluR sequences. They stated that the signaling by excitatory amino acids in human brain has evolved from a primitive signaling mechanism that existed prior to the divergence of plants and animals. 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