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Veterinary
Immunology
and
Immunopathology
jo u r n al h om ep a ge :w w w . e l s e v i e r . c o m / l o c a t e / v e t i m m
Research
paper
Microsatellite
(GT)
n
polymorphism
at
3
′
UTR
of
SLC11A1
influences
the
expression
of
brucella
LPS
induced
MCP1
mRNA
in
buffalo
peripheral
blood
mononuclear
cells
Sivamani
Balasubramaniam, Subodh
Kumar, Arjava
Sharma, Abhijit
Mitra
∗GenomeAnalysisLaboratory,DivisionofAnimalGenetics,IndianVeterinaryResearchInstitute,Izatnagar,243122Bareilly,India
a
r
t
i
c
l
e
i
n
f
o
Articlehistory:
Received10September2012
Receivedinrevisedform8December2012 Accepted28December2012
Keywords:
Buffalo Chemokine MCP1 SLC11A1 Brucellosis PBMC
a
b
s
t
r
a
c
t
A(GT)nmicrosatellitepolymorphismat3′UTRofSLC11A1(solutecarrierfamily11A1)is associatedwiththenaturalresistancetobovinebrucellosis.ApleiotropiceffectofSLC11A1 onothercandidategenesinfluencingthehostresistanceincludingmonocyte chemotac-tic/chemoattractantprotein1(MCP1)isalsohypothesized.Inthepresentstudy,wereport thecloningandcharacterizationofthecompletecodingsequenceofbubaline(bu)MCP1 anditstissuedistributionatthetranscriptlevel.ThebuMCP1exhibitedashighas99%and >80%ofsequenceidentitieswiththebovineandotherdomesticanimalspecies homo-logues.ThebuMCP1mRNAwas abundantacrossthedifferenttissues:mostabundant inliverandmammarygland,moderateinovary,skeletalmuscleandtestis,andleastin uterus.Further,quantitativereal-timePCR(RTqPCR)analysisrevealedthatPBMCs carry-ingsocalledresistantGT13alleleproducedmoreMCP1mRNAendogenouslyaswellas
wheninducedwithbrucellaLPSsuggestingthepleiotropicrolesofSLC11A1inconferring resistanceagainsttheintracellularpathogensparticularlyagainstbrucellosis.However,the underlyingmolecularmechanismsbywhich3′UTRSLC11A1concomitantlyincreasesthe
productionofchemokineslikeMCP1areyettobeinvestigated.
© 2013 Elsevier B.V. All rights reserved.
1. Introduction
SLC11A1(solutecarrierfamily11A1),erstwhilereferred as NRAMP1 (NaturalResistance Associated Macrophage Protein1),regulatesthemacrophagefunctionandthereby confersresistancetoanumber ofantigenicallydifferent intracellularpathogensincludingBrucellaabortus(Bradley et al., 1979; Gros et al., 1981; Plant and Glynn, 1974; Skameneetal.,1982).A(GTn)microsatellite(wherenis 13,14,15,or16)polymorphism,locatedinthe3′UTRof bovineSLC11A1,hassignificantassociationwiththe natu-ralresistancetobrucellosis(AdamsandTempleton,1998). Thesusceptiblemousemacrophagecelllines(RAW264.7) transfected with the bovine SLC11A1 resistant allele
∗ Correspondingauthor.Tel.:+915812303382;fax:+915812303284.
E-mailaddress:[email protected](A.Mitra).
restrictedtheintracellularreplicationofB.abortus(Barthel etal.,2001).GTnpolymorphismhasalsobeenassociated withthebrucellosisresistanceinbuffalo(Borrielloetal., 2006;Capparelli etal., 2007;Ganguly et al.,2008).The pleiotropic effect of SLC11A1 on other candidate genes influencingthehostresistanceisalsohypothesized(Lang etal.,1997;Gazoulietal.,2008).Nevertheless,the inde-pendentrolesof someofthesecandidategenessuchas MCP1,TNF-alpha, TGF-beta,IL-10, IL-6 and IFN-gamma, E-selectin,L-selectin, and TLR4have alsobeenreported elsewhere(Eskraetal.,2003;Karaoglanetal.,2009;Rafiei etal.,2006,2007;Rezazadehetal.,2006).
Monocyte chemotactic/chemoattractant protein 1 (MCP1), a member of the C C chemokine family, is expressedin a variety of cell types including endothe-lium, fibroblast,epithelium,smooth muscle,mesanglial, astrocyte,monocyteandmicroglialcells(Deshmaneetal., 2009).However,monocytesarethemajorsourceofMCP1
(Yoshimuraet al.,1989).TheMCP1 hasbeenassociated withvariousinflammatory (Namikiet al.,2002; Spoettl et al., 2006; Ip et al., 2006) as well as few infectious diseases such as tuberculosis (Flores-Villanueva et al., 2005),paratuberculosis(Buzaetal.,2003)andbrucellosis (Eskraetal.,2003).
South and Southeast-Asiancountriespossess around 83% of the world buffalo (Bubalus bubalis) population ofwhich India alonecontributes 57%. Although numer-ically buffalo is just half the population of cattle, it contributes around 60% of the annual milk production of India (FAOSTAT, 2010).Further they are anecdotally referredassturdyandresistanttovariousinfectious dis-eases.A survey in India revealed 16.4% buffaloes were positiveforbrucellosis(Gwidaetal.,2010).However,the factthatonefifthofanimalsstillremainsero-negativeeven ina heavilyinfected herd(Borrielloetal., 2006) neces-sitatestheexploitationof thenaturalhostresistanceto brucellosisinbuffaloes.
SincethediscoveryofhumanMCP1(Yoshimuraetal., 1989) its homologues have been cloned in different speciesincludingrabbit(YoshimuraandYuhki,1991),rat (Yoshimuraetal.,1991),cattle(Wempeetal.,1991,1994), guineapig(Yoshimura,1993),pig(Hosangetal.,1994)and sheep(Dunphyetal.,2001).However,bubalineMCP1is yettobecharacterized.In thepresentstudy,wecloned andcharacterizedthecompletecodingsequenceof buba-lineMCP1andfurtherinvestigatedtheeffectofSLC11A1 genotypesonthedifferentialexpressionofMCP1inPBMCs whileinducedwithbrucellaLPS.
2. Materialsandmethods
2.1. Animals
Healthyadultfemalebuffaloes(n=5)belongingto Mur-rahbreed maintainedin theinstitute herdat Izatnagar (U.P.),India,wereincludedinthestudy.Alltheseanimals weretestedtobesero-negativeforRoseBengalBlueTest (RBPT),standardtubeagglutinationtest(STAT)and indi-rectELISAasdescribedbyKumaretal.(2005).Furtherthese animalshadnohistoryofabortionandvaccinationagainst brucellosis.
2.2. GroupingofanimalsonthebasisofGTngenotypes
Genomic DNA was isolated from the venous blood using standard phenol chloroform extraction method (Sambrooketal.,1989).Aregioncorrespondingto401bp of3′UTRofSLC11A1wasPCRamplifiedusingtheprimers (forward:5′-CGCTTCCTGTATGGGCTTC-3′andreverse:5′ -TCCAAGGTAGGTGTCCCCA-3′)asdescribedbyRajaravindra (2008).Theamplifiedproductswereresolvedbyagarose gel(1%)electrophoresisandvisualizedusingethidium bro-mide(EtBr)stainingunderUVlight.ThePCRproductswere subjectedtofragmentanalysisformicrosatellite genotyp-ing.Theanimalswereclassifiedintotwogroupsbasedon theirnumberofGTrepeatspresentatthe3′UTRofSLC11A1 gene viz., GT13 (GT13/GT13)constituting of twoanimals
homozygousforGT13alleleandNonGT13(GTn/GTn,where
n =/ 13) consistingofthree animalscarrying otherthan GT13allelesintheirhomozygousform.
2.3. IsolationofPBMC
Venous blood (10ml)was collectedaseptically from the healthy adultanimals. The peripheralblood mono-nuclearcells(PBMC)wereseparatedbydensitygradient centrifugationusingHisep(Himedia,India)following man-ufacturer’sinstructions.Briefly,8mlofPBS-diluted(1:1) bloodsampleswerelayeredover4mlofHisepin15mltube andcentrifugedat400×gfor30min.ThePBMCs aggre-gatedattheinterphasewerecollectedandwashedthree timeswithsterilePBS.
2.4. InductionofPBMCculture
FollowingtheadjustmentofconcentrationofPBMCto 5×106cells perml, thePBMCwereculturedin a25ml
tissuecultureflaskfor30–36husingRPMI1640cell cul-turemedium(Sigma,USA)supplementedwith10%heat inactivatedFetalCalfSerum(Sigma,USA)andantibiotics PenicillinG(100IU/ml)andStreptomycin(100g/ml).The PBMC of animals belong to both the genotype groups wereinducedwiththeLPSderivedfromB.abortus strain-99 (OIEManual,2004)ata finalconcentrationof0 and 20g/5×106cellsfor12h.
2.5. cDNAsynthesis
The total RNA was isolated from PBMCs using Tri-reagent (Sigma, USA) according to the manufacturer’s instructions. Theisolated RNAsample wastreatedwith DNase using DNA-freeTM DNase Treatment & Removal
Reagents(Ambion,USA).Theconcentrationsandpurities ofRNApreparationsweredetermined spectrophotomet-ricallyatOD260andOD280.ThetotalRNAwas reverse-transcribed using Reverse Transcription (RT) System (Promega,USA)followingthemanufacturer’sinstructions. BrieflythecDNAwassynthesizedfromapproximately2g totalRNAusingoligo-dTprimersandavianmyloblastosis virusreversetranscriptaseinafinalvolumeof20l.The resultantfirststrandofcDNAwasstoredat−20◦Cuntil use.
2.6. PCRamplificationofMCP1
A complete coding region of MCP1 was ampli-fied using a pair of MCP1 specific primers (forward: 5′-CCAACAGCTTCCACGCTGAAAC-3′ and reverse: 5′ -GCAGTTAGGGAAAGCCGGAAGA-3′). PCR amplification wascarriedoutinatotalvolumeof25lthatcontained 7.5pmolof each primer, 1lof cDNA, 10mM Tris–HCl (pH8.8),50mMKCl,2.0mMMgCl2,2.5mMofeachdNTPs
amplified products were resolved by agarose gel (1%) electrophoresis and visualized using ethidium bromide (EtBr)stainingunderUVlight.
2.7. Cloningandsequencing
The amplicon of MCP1 was cloned into pGEMT Easyvector(Promega,USA)followingthemanufacturer’s instructions.Positiverecombinantcloneswereidentified usingblueandwhitescreening.Further,thepresenceof the insert was confirmed by restriction digestion with EcoRIand PCR amplification of theinsert using recom-binant plasmidas a template. Thepositive clones were sequencedusinganautomatedsequencerusingSanger’s dideoxychainterminationmethodwiththestandardT7 andSP6sequencingprimers.
2.8. Publicsequenceused
In additiontothebuMCP1sequencedin thepresent study, coding sequences of the several mammalian speciesavailableinGenBankdatabasewerealsoincluded; Indian cattle(EU837194), taurinecattle(EU276059),pig (EU682328), horse (EU438774), dog (NM001003297), human (NM002982), crabeating macaque(AF276081), olive baboon (NM 001173538), pig tailed macaque (AY206693), rabbit (NM001082294), rhesus monkey (NM001032821),Sumatranorangutan(NM001132340), mouse (NM011333), rat (NM031530.1), guinea pig (NM001172926.1),andHispidcottonrat(AF165953).
2.9. Sequenceanalysis
The sequences were subjected to BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST).Thenucleotidesas wellasthededucedaminoacidsequenceswerealigned withthoseofavailablespeciesintheGenBankdatabase using the Clustal method of MegAlign Programme of Lasergene Software (DNASTAR, USA). Phylogenetic and molecular evolutionary analyses were conducted using
MEGA version4(Tamuraetal.,2007).Phylogenetictree was constructed using neighborhood joining method. Reliability of a tree obtained was tested by bootstrap-pingtestofphylogeny.Ratiosbetweensynonymousand non-synonymous substitutions (ω) along AA sites were estimatedwiththeprogramcodemlofthePAMLpackage, usingmodelM3,withK=3AAsiteclassesasinYangetal.
(2000).Signalpeptideofthesequenceswaspredictedby SignalP4.0serverhttp://www.cbs.dtu.dk/services/SignalP/ (Petersenetal.,2011).Theisoelectricpointandthe molec-ularweightofdeducedpeptidewerepredictedusingthe expasy tools (http://expasy.org/cgi-bin/protparam). The domain features of deduced buMCP1 were determined byScanPrositeofexpasytools.O-linkedglycosylation, N-linked glcosylation, phosphorylation sites and disulfide bonds werepredicted usingYinOYang 1.2 tool, NetNG-lyc1.0server,http://www.predictprotein.organdDiANNA webserver1.1,respectively.
2.10. ExpressionofbuMCP1invarioustissuesusing semi-quantitativePCR
TotalRNAwasextractedfromapproximately100mg ofeachtissue includingliver,mammary gland, skeletal-muscle, ovary, testis and uterus were collected upon slaughter from apparently healthy adult buffalo from the local abattoir. Two micrograms of total RNA from eachsamplewasreversetranscribedasdescribedabove. Exactly1lof cDNAofalltissueswassubjectedtoPCR usingMCP1PCRprimersasdescribedunderSection2.6. GAPDH was used as an internal control to assess the variationsintheefficienciesoftheRTstepamong differ-entexperimentalsamples.A496bpfragmentofGAPDH was amplified independently using a pair of primers (forward: 5′-CAAGGTCATCCATGACAACTTTG-3′; reverse: 5′-GTCCACCACCCTGTTGCTGTAG-3′).For amplification of GAPDHgene, thesame reactionconditions and compo-nentsasdescribedfortheamplificationofbuMCP1gene wereused.InordertochecktheDNAcontaminationfor eachRNAsample,acontrolreactionwassetupinwhich reversetranscriptaseenzymewasomittedduringcDNA synthesis.ThenumberofcyclesusedtoamplifythecDNAof boththegeneswaskeptidentical.EachPCRmixture(8l) waselectrophoresed through EtBr stained 1.0% agarose gel and photographed using gel-documentation system (Syngene,USA).ThesizeofPCRproductswasverifiedby comparing with a DNA Ladder (SM #1173; Fermentas, USA).TheconcentrationsoftheGAPDHandbuMCP1mRNA weredeterminedbydensitometryanalysis(TanandNg, 2008)usingImageJsoftware(ImageJ,version1.46g,NIH, USA).Inbrief,thegelimagetobeanalyzed(TIFformat)is openedwithImageJandconvertedto8bitformat.Thenthe backgroundissubtractedusingrollingballradiusoption. Thebandsareselectedbyrectangletoolandthelanesare plottedusingplotlanesoption.Oncetheplottedlanesare selectedthroughlineandwandtoolstheareaisdisplayed correspondingtotheirbandintensityandsize.The rela-tiveexpressionwasdeterminedasarbitraryunits,defined astheratio ofmRNAlevel of MCP1to the correspond-ingGAPDH mRNAlevel [value=(area ofMCP1)/(area of GAPDH)].
2.11. ExpressionofMCP1indifferentgenotypesof SLC1A1usingquantitativereal-timePCR(RTqPCR)
The total RNA was isolated from the induced and uninducedPBMCsofdifferentgenotypegroups andwas subsequently reverse transcribed as described above. The gene-specific real-time primers for MCP1 (forward 64F: 5′-CTCGCTCAGCCAGATGCAA-3′ and reverse 173R: 5′-TTGCTGCTGGTGACTCTTCTGT-3′)andendogenous con-trolGAPDH(forwardF:5′-CTCCTGCACCACCAACTG-3′and reverseR:5′-CGCCAGTAGAAGCAGGGA-3′)weredesigned using Primer Express (Ver3.0) onthe basis of available cDNAsequencesintheGenBank.
amplifiedat50:50nmol/landGAPDHat100:50nmol/lof forwardandreverseprimer combinations.ThePCR effi-ciency(E)forMCP1 andGAPDH wasfoundtobe101.8 and100percentages,respectively.
Then, theRTqPCRwascarried out oncDNA samples usingAppliedBiosystems(ABI7500)real-timePCRSystem. AllPCRreactionswereperformedin20lvolumein dupli-cates.The reaction mixture contained1× SYBR® Green MasterMix(AppliedBiosystems,USA)5lofcDNA(1:10 diluted)templateandgenespecificprimersalongwithone notemplatecontrolforeachgene.Thethermo-cycling con-ditionswere:50◦Cfor2min,95◦Cfor10min,followedby 40cyclesof95◦Cfor15sand60◦Cfor1min.The disso-ciationreactionwascarriedout underfollowingcycling conditions.Theywere1cycleof95◦Cfor15s,60◦Cfor 1min,95◦Cfor15sand60◦Cfor15s.TheobservedC
T val-ueswereusedtocalculatetherelativequantitation(RQ) usingthecomparativeCTmethod(LivakandSchmittgen,
2001).
2.12. Nitricoxideestimation
Thenitricoxide(NO)productionbyPBMCsafter stimu-lationwithB.abortusLPSwasmeasuredusingcolorimetric assaykit(CaymanChemicals,ItemNo.780001,USA) fol-lowingthemanufacturer’sinstructions.
2.13. Statisticalanalysis
Gene expression fold change values obtained from RT-qPCR were log transformed to normalize the data. Subsequently,differencesbetweenthegroups forMCP1 expressionandNOproductionwerestatisticallyanalyzed byonewayANOVA(SPSS16.0).Allnumericaldatawere expressedasamean±SE.
3. Results
3.1. SequenceanalysisofbuMCP1
A405bpfragmentofbuMCP1cDNA(Fig.1), compris-ing of the complete coding sequence (CDS) of 300bp, wasclonedandsequenced.Blastanalysisoftheobtained sequencerevealed>80%identitywiththeMCP1sequences of domestic animal species available in the GenBank. Accordingly,thebuMCP1sequencewassubmittedinthe GenBankundertheAccNo.HQ889748.
3.2. MCP1expressioninvarioustissues
TheMCP1mRNAwasthemostabundantintheliver andmammaryglandtissues.TheexpressionofMCP1was moderateintheovary,skeletalmuscleandtestis,andwas theleastintheuterus(Fig.2).
3.3. DifferentialexpressionofMCP1inPBMC
ThedifferentialexpressionofMCP1 wasobservedin thePBMCofdifferentgenotypegroups(Fig.3).Therewere fivefoldsmore(P<0.01)endogenousexpressionofMCP1 mRNAinthePBMCofGT13animalscomparedtoNonGT13
Fig.1.Amplificationof405bpfragmentofMCP1usingrecombinant plas-midasatemplate(lane1)andrestrictionenzyme(EcoRI)digestionof recombinantplasmid(lane2)showingthereleaseoftheMCP1insertof 424bp.LaneM:100bpDNAladder.
LPSinductionfurtherpotentiatedtheMCP1mRNA expres-sionby 2.5times(P<0.01) inthePBMCsof GT13 group
(Fig.3)withnoeffect(P=0.511)onNonGT13group.
3.4. NOproduction
TheamountofNOproductiondidnotdiffermarkedly inthePBMCofdifferentgenotypicgroups.
4. Discussions
4.1. CharacterizationofbuMCP1
We have cloned theCDS (300bp) of buMCP1 which encodes MCP1 peptide comprising of 99 amino acids havingthemolecularweightof11.1Kdaandpredicted iso-electricpHof9.5.Likebovine,swine,equineandprimates,
Fig.3.ExpressionofbuMCP1inGT13(n=2)andNonGT13(n=3)
geno-typegroupsuponinductionwithBrucellaLPSatconcentrationsof0and 20g/5×106cells.
thesizeofthebuMCP1CDSisconserved.However,CDS islongerindog,rodentsandrabbit.TheMCP1peptideof dogpossessestwoadditionalaminoacidsatthepositions of93and94.Inrodents(guineapig-120,mouse-148, rat-148andHispidcottonrat-150aminoacids)andrabbit(125 aminoacids),excessaminoacidsarepresentatthecarboxyl terminalofthepeptide.TheMCP1proteinishighly con-servedattheNterminusamongdifferentspecieswhereas Cterminusremainsmuchmorevariable(YaoandTsirka, 2010).
ThebuMCP1proteinpossessesa signalpeptideof23 aminoacidswhichispredictedtobecleavedbetweenAla andGlnresiduestoyieldthematurebuMCP1peptide(76 aminoacids)of24thto99thaminoacidresidues.Thesignal peptidelengthandthefirstfouraminoterminalresidues (QPDA) of themature peptide are conserved in all the species(Yoshimura,1993)(exceptguineapigwhichhas Gatthefourthplace)analyzedunderthepresentstudy. Thepredictedsecondarystructureofpeptideshowsaround 39.4%helical,18.2%strandand42.4%loopwithaC-terminal ␣helicalstretch.
Thededucedaminoacidsequenceofbu-MCP1 exhib-itedthesequenceidentityof100%and 99%withthatof Indianandtaurinecattle.Alignmentreportofthededuced aminoacids(Fig.4)oftheBovidaespeciesindicatedonly oneaminoacidchangeat69thpositionhavingValinIndian cattleandbuffalo,andIleintaurinecattle.Theother domes-ticanimalspeciesincludingpig,horseanddogexhibited 83.8,79.8and73.7%identities,respectively.Ataminoacid level, the Cercopithecidae family of primates exhibited 72.7% identity whereas theHominidae family exhibited 70.7%only.Rodentsexhibitedtheleastidentities(50–55%) withthatofbuMCP1peptide.
Thephylogenetictreeconstructedbasedonnucleotides (Fig.5)anddeducedaminoacids(notshown)sequences revealedsimilartrend.buMCP1formedasinglecladwith Indian and taurine cattle.The nextnearer branchesare formedbypig(87.3%)andhorse(84.3%).Theorder car-nivora including dog and American mink showed the identity ofaround 83% withthebuMCP1.The primates formedaseparatecladwiththeidentityofaround81–83%.
Theprimatecladisfurtherseparatedintotwobranches; Cercopithecidaefamily (crab eatingmacaque, pigtailed macaque and rhesus monkey) and Hominidae family (human and orangutan). The order rodentia including mouse,guineapig,ratandHispidcottonratexhibitedthe identityof65to73%.ThedN/dSanalysisrevealedthat24 outof99aminoacidsshoweddN/dSratioof>1indicating positiveselectionpressure.Amongthemeightaminoacids atpositionsof20,45,49,69,71,88,89and99receivedthe highestscorerangingfrom0.983to1.0.
Structure-function studies demonstrated the impor-tanceoftheN-terminalregionofMCP1inreceptorbinding andactivation(Clark-Lewisetal.,1991).TherodentMCP1 C-terminusisdecoratedextensivelywithO-linked glyco-sylation sites (Zhang et al., 1996), which promotes the formationofdimerorhigher orderaggregatesofMCP1. Itisthencleavedbyplasminduringcellinjuryandthus abrogatesthedimer,makethemeasilydiffuseinthecell and bindefficiently CCR2.The plasmin-regulated extra-cellular cleavage step is a functional characteristic for otherchemokinessuchascCAF(chickenchemotacticand angiogenicfactor)andthehemofiltrateCCchemokine1 (Vakilietal.,2001;Martins-Green,2001).Forprimatesand domesticanimals,theMCP-1geneseemstohaveevolved tolosethisregulatorycleavagestepsincetheylackthe C-terminalextension(YaoandTsirka,2010).Yoshimuraand Yuhki(1991)demonstratedthatthesequencemotifYXR (positions51–53)dictatesthereceptor specificityofthe MCP1molecules.Weandothers(Hosangetal.,1994)found the52ndresidueXtobeabasicinallthespecieswiththe exceptionofguineapig.
The34–76aminoacidsofbuMCP1peptiderepresent thesignaturesequenceofsmallcytokineC Csubfamily. TheconservedCysresidueswereobservedatthepositions of34,35,59and75 inallthespeciesincludingrodents andtheywerepredicted(DiANNAwebserver1.1)toform disulfidebridgesatCys34–59andCys35–75.TheYinOYang 1.2toolrevealedthemostprobablesitesofO-linked gly-cosylationfor Thrat thepositions of19, 55and 96and
Serat4and56.However,wecouldnotdetectany puta-tiveN-linkedglycosylationsitesas reportedbyDunphy et al. (2001) in ovine.Ruggiero et al. (2003) proposed thatdifferentialglycosylationmayallowhighlyeffective short-livedMCP1orless-effectivelong-livedMCP1tobe obtained, and may thus represent a novel mechanism of adaptation to pathological versus physiological con-ditions.APROSITE databasesearchofpredicted protein identifiedtwoputativecAMPandcGMP-dependent pro-teinkinasephosphorylationsites,threeproteinkinase-C phosphorylationsitesand a tyrosine kinase phosphory-lation site. WhereasDunphy et al.(2001) reportedone putativecAMP/cGMP kinasesite andtwoproteinkinase Csitesinsheep.
4.2. MCP1expressionandNOproductionbetween genotypegroups
Fig.4. AlignmentreportofdeducedaminoacidsequenceofbuMCP1withthatofotherspecies.Thedotsindicatethesameresidueswiththatofbuffalo. Thedoubleedgedarrow(↔)indicatesthesignalpeptideofbuMCP1.TherectanglecoveringtheQPDAatthepositionsof24–27residues(nexttothesignal peptide)indicatestheconservedN-terminalmaturepeptideinallthespecies.Thesmoothrectanglesatthepositionsof34–35;59and75indicatethe conservedcysteineresidues.Thethickrectangle(51–53)indicatestheconservedresidueswhichdictatethereceptorspecificity.Thesmoothrectangle encirclingthe80–91residuesindicatestheC-terminal␣helix.Twosmallellipticalcurvesatthepositionsof41and52indicatecAMPandcGMPdependent
proteinkinasephosphorylationsites.Threesmallsmoothrectanglesindicate(at40,56and96)theproteinkinasecphosphorylationsite.Onesmalltriangle (at81)indicatestyrosinekinasephosphorylationsite.
The mRNAexpression acrossvarious tissues of buffalo, observedinthepresentstudy,issuggestiveofitsrolein mountinganimmuneandinflammatoryresponseagainst infection(Rodríguez-Sanabriaetal.,2010).
Host-resistancetointracellularpathogensismediated through a complex process. SLC11A1 is known to play a majorrole in conferring resistance against intracellu-larpathogens (Skamene etal.,1982).The SSCAanalysis (Adams and Templeton, 1998) and transfection study (Barthel et al., 2001) proved the association of 3′UTR
Fig.5. Phylogenetictreeconstructedonthebasisofnucleotidesequence ofbuffaloMCP1genewiththatofotherspecies.
andNOthanthosefromNonGT13animalswhileinduced
withbrucellaLPS(Gangulyetal.,2008;Rajaravindra,2008). It is alsobelieved thatSLC11A1 mightexert pleiotropic effects oninnate immune functions by modulating the productionofchemokines,cytokinesandtheformationof reactiveoxygenandnitrogenspecies(Valdezetal.,2008). Accordingly,weintendedtoevaluatewhetherPBMCsfrom buffaloes having socalledresistant GT13 alleleproduce
moreMCP1whencomparedtoNonGT13.Wedemonstrated
that PBMCs carrying GT13 allele produced more MCP1
mRNAendogenouslyaswellaswheninducedwith bru-cellaLPS.However,wecouldnotdetectanychangesinthe NOproductionbetweengenotypegroups.Itmightbedue tothefactthatthePBMCculturewasinducedwith bru-cellaLPSonlyfor12h.Severalstudies(Wangetal.,1994; López-urrutiaetal.,2000)alsorevealedthatthe measur-ablechangeofNO productionbetweendifferentgroups couldbeobserved during24–48h. But,as weare more interested inthemRNAlevelof MCP1only, wedidnot extendthePBMCcultureforlongerduration.Howeverit willbeinteresting toseetheNO levelat differenttime intervals.
5. Conclusion
In conclusion this is the first ever study reporting the cloning, characterization and expression profile of buMCP1. The microsatellite polymorphism at 3′UTR of SLC1A1significantly influences theMCP1 expressionin PBMC suggesting the pleiotropic roles of SLC11A1 in conferringresistanceagainsttheintracellularpathogens particularlyagainstbrucellosis.However,theunderlying molecularmechanismsbywhich3′UTRSLC11A1 concomi-tantlyincreasestheproductionofchemokines(MCP1)are yettobeinvestigated.
Acknowledgements
Thisworkwascarriedoutundertheprojectsupported byIVRI(ICAR)IRCResearchGrantgiventoAMasa Princi-palInvestigator.FinancialAssistanceprovidedtoSBinthe formofInstitute(IVRI)PhD fellowshipisduly acknowl-edged.TheauthorsaregratefultoDr.A.K.STomar(Senior Scientist)ofLivestockProduction&ManagementSection, IVRIforprovidingbloodsamples,toDr.D.K.Singh (Prin-cipalScientist,DivisionofVeterinaryPublicHealth,IVRI) andtoDr.RajeshRathore(SeniorScientist,CADRAD,IVRI) forprovidingthefacilitiestoserotypetheanimals.
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