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A Comparative Genomics based evaluation on Evolutionary aspect of Cystinosin: the Protein defective in Cystinosis
Manish Dwivedi and Dwijendra K. Gupta
Abstract Lysosomes are intracellular sacs of various hydrolases enzymes which cause the degradation of the macromolecules inside the
cell. The hydrolytic digest products are then transported across the lysosomal membrane via specic membrane transporters proteins, to be either reused by the cell or excreted outwards. In this computer-aided effort, we reported a bioinformatics based sequence analysis of cystinosin proteins, using different bioinformatics tools like BLAST, ClustalW, MEGA4, BioEdit (version 7.0.0) etc. to analyze the cystinosin protein sequences with the prospects of molecular evolutionary relationship among 18 taxa as well as to explore the different analytics on sequence. In our study we find out the number of coserved domains and established a evolutionary tree and hydrophobicity profile along with the entropy plot. This protein is basically hydrophobic in nature as most of the positions were an above mean hydrophobicity with hydrophobic amino terminal and non hydrophobic carboxy terminal end in case of most of the organisms studied here. This approach help to reveal the molecular basis of the disease cystinosis and cell bilogic features with the subcellular localization of the protein cystinosin. This information also assist to predict the exact function of the cystinosin in the lysosomal membrane and enable us to understand the critical regions of cystinosin. Index Terms Bioinformatics, cystinosin, evolutionary, lysosomes.

1 INTRODUCTION
tion in mammalian cells. An imperfection in either the transportation or degradation process can result in an accumulationoftheundigestedsubstrateorthehydrolyt ic degradation product within the lysosomes, affecting thecellphysiologyandcausingadisordercalledaslyso somal storage disorder. Number of such disorders have been investigated and classified according to the mole cules deposited intralysosomally [1]. Most of these dis orders are caused by the defects in one of the lysosomal hydrolases, however, some of the lysosomal disorders arisesduetodefectsinoneofthemembranetransporters proteins [2].Cystinosis (MIM 21980) is one of such auto somal recessive disorder which is caused by defects in cystinosintransporterproteins. Cystinosin is a lysosomal membrane transporter protein but how it is targeted to this lysosome organelle, is still under investigation and any mutation in cystinosin lead to the cystinosis. This inherited lysosomal transport dis order,cystinosisisdistinguishedbydefectivetransportof cystine amino acid from the lysosomes to cytosol results intoaccumulationofintralysosomalcystineanditisthe most frequent inherited cause of the renal Fanconi syn drome. Several biochemical researches showed that the

LYSOSOMES are major sites for the intracellular diges

lysosomal cystine transporter exclusively transports the cystineaminoacidthereforeitisdistinctfromtheplasma membranecystinetransportersproteins. This autosomal recessive disorder cystinosis represents threeallelicclinicalforms,dependingonseverityandage ofonset.Theinfantileform(MIM21980)usuallyemerges at 68 months of age with a proximal renal tubulopathy, can lead to death by 10 years of age due to renal failure and in the absence of renal transplantation (Gahl et al., 1995).Additionalclinicalsymptomsareretinalblindness, hypothyroidism, diabetes mellitus, swallowing difcultiesandneurologicaldeterioration,whichappears ultimatelyduetotheextensiveaccumulationofcystinein most tissues. The juvenile form (MIM 219900) is representedbyglomerularrenaldamage,whichapparent at around 1012 years of age and gradually leads to glo merular insufciency and corneal cystinecrystal deposi tion causing photophobia. Finally, the ocular non nephropathic form (MIM 219750) is solely characterized byamildphotophobiabutnorenalanomalies. Cystine is a byproduct of lysosomal protein hydrolysis, andisreducedtothedisuldecysteineaminoacidinthe cytoplasm. Cystinotic cells normally contains the en zymes involved in cyst(e)ine redox reactions, it has been postulatedthattheunderlyingmetabolicdisorderofcys tinosis is a imperfect lysosomal membrane cystine trans M. Dwivedi is a Doctoral scholar at the Center of Bioinformatics, Univer- port(Gahletal.,1995).Supportforthispostulatehasbeen provided by the investigation showing that cystine isra sity of Allahabad, Allahabad-211002 INDIA. Prof. Dwijendra K Gupta is Coordinator-Chair at the Center of Bioinforpidlyvanishedfromarticiallyloadednormallysosomes, matics, University of Allahabad, Allahabad-211002, INDIA. whereas egress of cystine from cystinotic lysosomes is almost nonexistent (Gahl et al., 1982b; Jonas et al.,
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1982a,b; Steinherz et al., 1982). Furthermore, carrier me diatedcystinetransporthasbeenobservedacrossthely sosomalmembranewhichisapprovedbytherepresenta tionofsaturationkineticsbythevelocityofcystineefflux fromnormallysosomes(Gahletal.,1982a)andthecoun ter transport exhibition of the cystine amino acid across the normal lysosomal membrane (Gahl et al., 1983) whicharetwoimportanthallmarksrepresentingthecar riermediatedtransport. The cystinosin protein containing 367 amino acids, is codedbythegeneCTNS,andpositionalcloningstrategy (Town et al., 1998) was used to identify this gene. The aminoterminalendoftheproteinishighlyglycosylated whereas carboxy terminal end carries a GYXXF lyso somal targeting motif comprising seven predicted transmembrane domains, a Nterminal region of 128 amino acid bearing seven Nglycosylation sites and a cytosolic Cterminus with 10 amino acid having a tyro sinebasedlysosomalsortingmotif(GYDQL). Atpresent,Homosapiensonlinedatabasescomprisessev eralcystinosinisoforms[isoformCRA_b(gi:EAW90495), cystinosisnephropathic(gi:AAH32850),cystinosisneph ropathicisoform1(gi:NP_001026851),unnamedprotein product (gi: BAF84708)]. Among these, presence of a longer transcript is designated by the partial expressed sequence tag sequences and complete sequences from humanspleenandtestis(gi:AAH32850andBAF84708), as a consequence of an alternative splicing of the ulti mate exon. Protein databases (XP_001089495.1, XP_854520.1, XR_021668.1) and nonhuman DNA also havesomesimilarisoforms,although thesealternativecystinosin sequences arenot supportedbyfunctionaldata. Although any defects in cystinosin proteins have been linked to themany forms of cytinosis (Shotelersuk et al., 1998; Town et al., 1998;Attard et al., 1999; Thoene et al., 1999),ithasnotyetbeenconcludedwhetherthiscystino sinproteinisdirectlyorindirectlyresponsibleforthede fective cystine transport across the lysosomal membrane in cystinotic cells. Lysosomal cystine transporter may be represented by Cystinosin itself. However, recently known transporters do not illustrate similarity with its sequence or predicted topology. On the basis of these facts, alternatively it has been suggested that cystinosin mightindirectlyaffectlysosomalcystineegress(Attardet al.,1999;Mancinietal.,2000). In this study, we addressed the evolutionary account of cystinosinwithrespectto18selectedorganismsasshown in table1 by using a strategy that exploited our recent knowledge of the Phylogenetic relationship of the mem braneproteins.Wealsoreportedthehydrophobicitypro file,entropyandconserveddomainwithinthecystinosin proteinfamily.

TABLE 1 CYSINOSIN SEQUENCES WITH THEIR LENGTH AND NCBI ACCESSION CODE

Length Organism Mus musculus Bos Taurus Homo sapiens Rattus norvegicus Caenorhabditis elegans Gallus gallus Canis familiaris Aedes aegypti Caenorhabditis briggsae Drosophila melanogaster Ornithorhynchus anatinus Caligus clemensi Trypanosoma cruzi Perkinsus marinus Schistosoma mansoni Ixodes scapularis Pediculus humanus corporis Culex quinquefasciatus NCBI Accession code gi|11967808|emb|CAC19455.1| gi|182639279|sp|A7MB63.1| gi|3036840|emb|CAA11021.1| gi|109491251|ref|XP_001080248.1| gi|32565006|ref|NP_872022.1| gi|50758292|ref|XP_415851.1| gi|73967295|ref|XP_548340.2| gi|157167697|ref|XP_001655585.1| gi|215275218|sp|A8WN56.1| gi|34223744|sp|Q9VCR7.2| gi|149641786|ref|XP_001508868.1| gi|225717886|gb|ACO14789.1| gi|71422082|ref|XP_812021.1| gi|239885524|gb|EER09461.1| gi|238665302|emb|CAZ36053.1| gi|240999715|ref|XP_002404776.1| gi|242006380|ref|XP_002424029.1| gi|170044802|ref|XP_001850022.1| (aa) 367 367 367 392 374 377 367 367 403 397 605 409 383 302 359 266 356 399

2 MATERIALS AND METHODS

In order to search cystinosin family members we per formedBLAST[3]byusingblastpprogramintheprotein database at NCBI [4].Homo sapiens cystinosin proteins

gi|3036840|emb|CAA11021.1| amino acid sequence was selected as query. From the hits 18 sequences, each from differentspecies(organism)wereselectedforfurtherstu dies.AllthesequencesweretakeninFASTAformat.The sequenceswereexaminedindividuallyandalignedusing CLUSTALW[5].Bioeditversion7.0.0.[6]wasusedforma nualeditingandanalysisofsequences.KyteJandDoolittle [7] method was used to plot hydrophobicity profile. En tropyisthencalculatedas: H(l)=f(b,l)ln(f(b,l)) where H(l) = the uncertainty, also called entropy at posi tion l, b represents a residue (out of the allowed choices forthesequenceinquestion),andf(b,l)isthefrequencyat which residue b is found at position l. The information content of a position l, then, is defined as a decrease in uncertainty or entropy at that position.As an alignment improves in quality, therefore, the entropy at each posi tion (especially conserved regions) should decrease, which gives a measure of uncertainty at each position relative to other positions. Maximum total uncertainty will be defined by the maximum number of different characters found in a column.A window of defined size that was 13 is moved along a sequence, the hydropathy scores are summed along the window, and the average (the sum divided by the window size) is taken for each positioninthesequence.Themeanhydrophobicityvalue wasplottedforthemiddleresidueofthewindow.Eisen berg et. al. method [8] was used to plot hydrophobic mo mentprofilewithawindowsizeof13residueshavingsix

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residuesoneithersideofthecurrentresidueandrotation angle,=100degrees. H={[Hnsin(n)]^2+[Hncos(n)]^2} Where H is the hydrophobic moment, Hn is the hydro phobicity score of the residue H at position n, =100 de grees, n is position within the segment, and each hydro phobic moment is summed over a segment of the same definedwindowlength.

Segment Length: 38 Segment Length: 83 Average entropy (Hx): 0.9289 Average entropy (Hx): 0.6944 Consensus: Consensus:
469 PVDLNDVFFSLHAVAATLITILQCCFYERGGQRVSWPA 506
543 WLDFLYCFSYIKLAITLIKYFPQAYMNFRRKSTVGWSIGNILLDFTGGSLSLLQMFLQAYNNDDWTLIFGDPTKFGLGVFSIF 625

3 RESULTS AND DISCUSSION

3.1Multi ple seq uen ceal ignme nt:
Fig. 2. Four conserved domains obtained with their length.

The Multiple alignment of cystinosin proteins (Figure 1) 3.3 Entropy pl ot resulted into an alignment having 680 positions. By statistical analysis of multiple aligned sequences it was An entropy plot, measure of the lack of the information observed that phenylalanine, isoleucine, leucine, serine, valineandglycinearethemostfrequentlypresentamino acidswithfrequencypercentageof7.80,8.40,10.95,7.95, 9.39 and 6.26 respectively. While within conserved sites glycine, lysine, asparagine, proline, glutamine and tyrosine are the most frequently occuring amino acids withfrequencypercentageof19.10,15.45,7.73,7.73,15.24 content and the amount of and 11.37 respectively. The multiple aligned sequence of cystinosin protein was found with No. of conserved variability, was generated for all the aligned positions sites=27, No. of parsimony informative sites= 293, No. of (Figure 3). The plot shows that entropy rarely touches a scale of two, showing minimal entropy at several singletonsites=101andno.ofvariables400. positionsfromposition400toposition650whereentropy 3.2 Conse rve d regio n search Aconservedregionsearchresultedintofourregionsfrom rarely crosses a scale of one, which is a sign of better position 374 to 421 (segment length = 48), 429 to 447 alignment in the region. Any position before 375 doesnt (segment length = 19), 469 to 506 (segment length = 38) showmuchconservedness. and543to625(segmentlength=83)withanaverage en 3.4 Hy dr op ho bi city profile a nd hy dr op ho b tropyof1.0170,0.8865,0.9289,and0.6944respectivelyas i c mo me nt A hydrophobicity profile plot shows that mean hydro phobicityoftheproteinformostofthepositionsisinall

Fig. 1. M u l t i p e p r o t e i n s .

sequence

alignment

of

Cystinosin

Fig. 3. E n t r o p y ( H x ) P l o t .

shown in figure 2. This conservation has already been upheld by minimal entropy shown by positions 374 to 625.
Region1:Position374to421Region2:Position429to447 SegmentLength:48SegmentLengths:19 Averageentropy(Hx):1.0170Averageentropy(Hx):0.8865 Consensus Consensus:
374 NQTDPRIRFLVIHSRILSIISQVIGWIYFVAWSVSFYPQVILNWRRKS 421

429 FDFLALNLTGFVAYSVFNI 447

Region 3: Position 469 to 506

Region 4: Position 543 to 625

thespeciesisaroundzero,occassionalyitturnstobepos itive or negative (Figure 4). Nterminal domain and C terminal domain is nonhydrophobic in Ornithorhynchus anatinus.Maximumhydrophobicityisobservedfrompo sitions 240 to 270 and between 370 to 630 positions in Aedes aegypti, Drosophila melanogaster, Schistosoma mansoni and Perkinsus marinus. Nterminal domains are more hy drophobic while cterminal domains are non hydrophobicinthemostoftheorganisms.Alongchainof the protein in the Ornithorhynchus anatinus from position 1230 shows nonhydrophobicity. Culex quinquefasciatus andTrypanosomacruziexhibitshighjumpinhydrophobic

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ity from position 240 to 260 and 500 to 540 respectively. These proteins are basically of hydrophobic in nature as most of the positions are across show the above mean hydrophobicity in the case of most of the organisms studiedhere.

3.5 P hylo ge ne y
The phylogenetic trees were constructed by using Neighbour joining method (Figure 56). The tree shows different organisms on tree nodes branched on the basis

Fig. 4. K y t e profile plot.

and

Dolittle

scale

mean

hydrophobicity

of their cystinosin proteins. Perkinsus marinus and Trypa nosoma cruzi makes a totally diverged branch from the across family of organisms with special reference to maintreeamongtheselectedproteins.Nodeforinverte mammals.Thestudyestablishedanoverallframeworkof brates is supported by lower bootstrap values while the information for the family of Cystinosin proteins, which node for vertebrates (Homo sapiens, Mus musculus, Gallus mayfacilitateandstimulatethestudyofthisgenefamily gallus) is supported by very high bootstrap value i.e. across all organisms. This approach help to reveal the 100%. Branches corresponding to partitions reproduced molecular basis of the disease cystinosis and cell bilogic inlessthan50%bootstrapreplicatesarecollapsed.There features of the protein cystinosin and their subcellular wereatotalof223positionsinthefinaldataset.Thistree localization. This information also assist to predict the gives an idea about the evolutionary order of Cystinosin exact function of the cystinosin in the lysosomal proteins.Thisphylogenydoesnotseemtobecompletely membrane and can allow a better undersatanding of the consistent with the current view of taxonomy perhaps criticalregionsofcystinosin. due to use of a specific protein rather than complete genomes.

Fig. 6. Bootstrap original phylogenetic tree of Cystinosin proteins created by Neighbour-joining method showing bootstrap support values on the nodes.

AUTHORS CONTRIBUTION

CONCLUSION

Thisstudypresentsthefirstcomparativegenomicsstudy and evolutionary analysis of the cystinosin proteins

Authorsperformedthemethod,evaluationandprepared thedatasetsandconceptualizedthework.

ACKNOWLEDGMENT
MDisthankfultoDepartmentofScienceandTechnology, New Delhi for a research fellowship. The work has been supportedbyaDBTBIFGranttoDKGunderitsBTISNet scheme and University Grants Commission Innovative GrantSceme.

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M. Dwivedi, Senior Research Fellow, Center of Bioinformatics, University of Allahabad,INDIA, a paper published in BIJIT (ISSN No. 0973-5658),currently involved in the membrane protein characterization by using the IgYs and Bioinformatics Tools, Member of Indian Science Congress Association and International Academy of Physical Science. Prof. Dr. Dwijendra K. Gupta, Professor is Cordinator-Chair at Center of Bioinformatics, and former Head, Department of Biochemistry at University of Allahabad.