Molecular evolution of the enzymes involved in the sphingolipid metabolism of Leishmania: selection pressure in relation to functional divergence and conservation
© Mandlik et al.; licensee BioMed Central Ltd. 2014
Received: 13 December 2013
Accepted: 13 June 2014
Published: 21 June 2014
Selection pressure governs the relative mutability and the conservedness of a protein across the protein family. Biomolecules (DNA, RNA and proteins) continuously evolve under the effect of evolutionary pressure that arises as a consequence of the host parasite interaction. IPCS (Inositol phosphorylceramide synthase), SPL (Sphingosine-1-P lyase) and SPT (Serine palmitoyl transferase) represent three important enzymes involved in the sphingolipid metabolism of Leishmania. These enzymes are responsible for maintaining the viability and infectivity of the parasite and have been classified as druggable targets in the parasite metabolome.
The present work relates to the role of selection pressure deciding functional conservedness and divergence of the drug targets. IPCS and SPL protein families appear to diverge from the SPT family. The three protein families were largely under the influence of purifying selection and were moderately conserved baring two residues in the IPCS protein which were under the influence of positive selection. To further explore the selection pressure at the codon level, codon usage bias indices were calculated to analyze genes for their synonymous codon usage pattern. IPCS gene exhibited slightly lower codon bias as compared to SPL and SPT protein families.
Evolutionary tracing of the proposed drug targets has been done with a viewpoint that the amino-acids lining the drug binding pocket should have a lower evolvability. Sites under positive selection (HIS20 and CYS30 of IPCS) should be avoided during devising strategies for inhibitor design.
KeywordsEvolutionary biology Sphingolipid metabolism of Leishmania Functional divergence and conservedness Specificity determining positions Selection pressure Codon usage bias Relative synonymous codon usage Effective number of Codon GC content
Leishmania, a protozoan parasite is responsible for causing the infectious disease Leishmaniasis. Around 12 million people are affected by this disease worldwide. The sphingolipid metabolism of vertebrates, fungi and plants has been well documented. Sphingolipid metabolism in parasites like Leishmania plays an important role in maintaining the infectivity of the parasite. Sphingolipids form an integral component of the parasitic membranes . They are localized in the membrane micro domains and are involved in a wide array of signal transduction pathways  Parasites like Leishmania scavenge host sphingolipids and remodel them into parasite specific sphingolipids. The key enzymes involved in the sphingolipid metabolism are a) SPT (Serine pamitoyl transferase) b) SPL (Sphingosine 1-P phosphate) and c) IPCS (Inositol phosphorylceramide synthase) .
The sphingolipid metabolism of Leishmania and many other pathogens is highly conserved and offers a series of attractive drug targets for further inhibitor design. IPCS, SPT and SPL in Leishmania have been identified as important target proteins by biochemical network modeling.  We present the phylogenetic relationship among these key enzymes in Leishmania to obtain a comparative history of the related proteins. Role of selection pressure, assessment of the strength of purifying versus diversifying selection for all the three target proteins has provided an idea of the molecular evolution of target proteins.
Acquisition of sequences
Amino acid and coding sequences of the three enzymes (IPCS, SPL and SPT) in the sphingolipid metabolism were retrieved from NCBI, ENSEMBLE and UNIPROT databases. A total of 54 sequences for IPCS and SMS, 40 sequences of SPL and 78 sequences for SPT were used in this study (Additional file 1: Table S1A-E).
Sequence alignment, selective constraints and phylogenetic analysis
The sequences were aligned using CLUSTALW v2.0.9 using the default parameters. Larkin et al.  Phylogenetic tree reconstruction for the sphingolipid metabolism was done using MEGA 5 program by the Neighbour-Joining method with 10000 bootstrap resampling’s. Saitou and Nei [12, 13]. The evolutionary distances (Number of amino acid substitutions/site) were computed using the Poisson correction method.
To evaluate the potential functional divergence and to predict the amino acid residues accounting for the functional differences among the three enzymes, Type I functional divergence was estimated using DIVERGE 2.0. Gaucher et al.  Sequences were classified into three different groups (IPCS, SPL and SPT) using the P-distance method (Additional file 1: Table S2). For each pairwise comparison, the coefficient of evolutionary functional divergence (θ) and standard error were determined. Utilizing the coefficient of evolutionary functional divergence (θ) the sequences were subjected to significant functional divergence and likelihood ratio test (LRT). Based on the site-specific posterior probabilities, sites experiencing functional divergence in the subgroups were identified ,.
SDP (Specificity determining positions) analysis
SDP’s determine the differences in functional specificity within the protein family [17, 18]. The input set of sequences was divided into 4 groups (IPCS, SMS, SPL and SPT) containing 34, 20, 40 and 78 sequences respectively. SDP’S were predicted by SDPfox and SDPpred and the Z scores were calculated for each alignment column.
Selection pressure assessment of the protein families
The selected clades from functional divergence were submitted as nucleotide alignment in fasta format to the Selecton server for analysis of the non-synonymous (dN) versus synonymous substitution (dS) ratio . To study the effect of selection pressure on the conservedness of the three proteins, the protein MSA was submitted to the CONSURF server, which classifies the residues based on their conservation in the MSA .
Selection pressure assessment at the codon level
Selection pressure on the codon was estimated for the targeted organism Leishmania. Protozoan coding sequences were obtained from GenBank (release 137). In order to normalize codon usage within datasets of differing amino acid compositions, relative synonymous codon usage (RSCU) values were calculated. The reference set consisted of highly expressed genes pertaining to the lipid metabolism as reported by  Codon Adaptation Index (CAI) was calculated using CAI Calculator 2 . Other species non-specific indices like ENc (Effective Number of Codon) and Fop (Frequency of optimized codons) were calculated using DAMBE software ) and CodonW respectively . GC3s values were calculated using CodonO webserver . Coding sequences of elongation factors were retrieved from GeneDB. A comparison of the CAI values of the elongation factors with the CAI values of IPCS, SPT and SPL was made to determine the codon usage bias in each of the three genes.
Results and discussion
Phylogenetic tree construction for the sphingolipid metabolism
Specificity determining positions [SDP]
Gene duplication, deletion events along with the evolutionary selection pressures alter the basic biochemical properties of a protein like ligand binding, protein-protein interactions and the specificity towards the substrate. The amino acid positions which vary only in certain subgroups and alter the specificity of a protein are called the Specificity determining positions (SDP’s). Alignment positions, accounting for such functional specificity of the three enzymes in L. major were mapped and analyzed for their conservation. A total of 34 positions were identified as SDP’s in all the four groups (Additional file 1: Table S3).
Selection pressure over the protein families
Codon usage bias indices for IPCS, SPL and SPT genes
CAI values of IPCS, SPL and SPT genes in comparison with CAI values of the highly expressed elongation factors of Leishmania
eEF1B beta 1 (LmjF.34.0820)
The key enzymes (IPCS, SPL and SPT) of the sphingolipid metabolism of L.major were studied in relation to other members of the same protein family. The present study supports the classification of sphingolipid metabolism with a high bootstrap value on the internal branches. To derive sufficient information about the kind of evolutionary pressure the three enzymes are being subjected to, functional divergence and conservation at the level of both amino acids and codons was studied. Functional divergence analysis indicated that PAP2c family diverged from SPL and SPT. Amino acids accounting for functional specificity (SDP’s) of the three enzymes in L.major were mapped. Along with the functional divergence, the selection pressures over the protein families were assessed. The effect of selection pressure was more predominant at the codon level as indicated by the CUB indices. IPCS gene showed a lower codon usage bias as compared to SPL and SPT genes. At the protein level, the effect of purifying selection was largely predominant and this accounted for the functional conservation of the three drug targets.
Inositol phosphoryl ceramide synthase
Serine palmitoyl transferase 1
Serine palmitoyl transferase 2
Specificity determining positions
Relative synonymous codon usage
Effective number of codon
Frequency of optimized codons
Codon adaptation index
Multiple sequence alignment
Codon usage bias.
Vineetha Mandlik acknowledges the financial support as Senior Research Fellow of Department of Biotechnology, Government of India. The work was supported by Department of Biotechnology, Government of India (BT/PR6037/GBD/27/372/2012). The authors would also like to thank Dr. Shekhar C. Mande for supporting the Bioinformatics and High Performance Computing Facility at National Centre for Cell Science, Pune, India.
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