Site-directed mutagenesis of Arginine282 suggests how protons and peptides are co-transported by rabbit PepT1 The mammalian proton-coupled peptide transporter PepT1 is the major route of uptake for dietary nitrogen , as well as the oral absorption of a number of drugs , including β-lactam antibiotics and angiotensin-converting enzyme inhibitors . Here we have used site-directed mutagenesis to investigate further the role of conserved charged residues in transmembrane domains . Mutation of rabbit PepT1 arginine282 -LRB- R282 , transmembrane domain 7 -RRB- to a positive -LRB- R282K -RRB- or physiologically titratable residue -LRB- R282H -RRB- , resulted in a transporter with wild-type characteristics when expressed in Xenopus laevis oocytes . Neutral -LRB- R282A , R282Q -RRB- or negatively charged -LRB- R282D , R282E -RRB- substitutions gave a transporter that was not stimulated by external acidification -LRB- reducing pHout from 7.4 to 5.5 -RRB- but transported at the same rate as the wild-type maximal rate -LRB- pHout 5.5 -RRB- ; however , only the R282E mutation was unable to concentrate substrate above the extracellular level . All of the R282 mutants showed trans-stimulation of efflux comparable to the wild-type , except R282E-PepT1 which was faster . A conserved negatively charged residue , aspartate341 -LRB- D341 -RRB- in transmembrane domain 8 was implicated in forming a charge pair with R282 , as R282E/D341R - and R282D/D341R-PepT 1 had wild-type transporter characteristics . Despite their differences in ability to accumulate substrate , both R282E - and R282D-PepT1 showed an increased charge : peptide stoichiometry over the wild-type 1:1 ratio for the neutral dipeptide Gly-l-Gln , measured using two-electrode voltage clamp . This extra charge movement was linked to substrate transport , as 4-aminobenzoic acid , which binds but is not translocated , did not induce membrane potential depolarisation in R282E-expressing oocytes . A model is proposed for the substrate binding/translocation process in PepT1 . 1 Introduction The proton-coupled di - and tri-peptide transporter PepT1 -LRB- SLC15a1 -RRB- is the major route of uptake of dietary nitrogen from the intestine , and is also important along with the higher affinity gene product PepT2 -LRB- SLC15a2 -RRB- in the re-absorption of filtered peptides in the kidney -LRB- Daniel & Kottra , 2004 ; Meredith & Boyd , 2000 -RRB- . In addition , PepT1 is the route of entry of a wide class of orally bio-available pharmaceutically important compounds , including the β-lactam antibiotics , angiotensin-converting enzyme -LRB- ACE -RRB- inhibitors , antiviral and anticancer agents -LRB- Terada & Inui , 2004 -RRB- . Although these therapeutic compounds are not di - or tri-peptides , they are carried by virtue of their similar 3D shape to endogenous substrates , i.e. they are peptidomimetic , and modelling of the substrate-binding site from the features in common of this huge and diverse range of substrates has led to predictions concerning which parts of the PepT1 protein may be important . For example , a substrate template model has been developed by several groups -LRB- Bailey et al. , 2000 ; Bailey et al. , 2006 ; Biegel et al. , 2005 -RRB- which allows prediction of binding affinity for a potential substrate . The rabbit PepT1 is a 707 amino acid protein , with twelve transmembrane spanning domains -LRB- TMDs -RRB- as confirmed by epitope mapping -LRB- Covitz , Amidon , & Sadee , 1998 -RRB- . In the absence of a crystal structure , attempts have been made to computer model the PepT1 transporter itself -LRB- Bolger et al. , 1998 -RRB- , with site-directed mutagenesis used to test hypotheses generated by these models . One potential complication for these kind of studies is our recent report that PepT1 may form multimers in the plasma membrane -LRB- Panitsas , Boyd , & Meredith , 2006 -RRB- although it is not clear how the subunits interact . Site-directed mutagenesis has been a useful tool to identify functionally important residues in PepT1 . One such residue is arginine282 in the rabbit PepT1 sequence . The mutation of arginine282 to a glutamate produced a peptide transporter -LRB- R282E-PepT1 -RRB- that was no longer driven by a proton-gradient but behaved more like a facilitated peptide transporter , whilst simultaneously exhibiting peptide-gated currents that were proposed to be through a non-specific cation channel activity -LRB- Meredith , 2004 -RRB- . Residue 282 is located approximately halfway down the predicted transmembrane domain 7 -LRB- TMD7 -RRB- , and is either an arginine or a lysine in all cloned mammalian PepT1 sequences to date . The presence of a charged amino acid residue in a TMD , along with its conservation , suggested a functional role . Here , we have systematically investigated the role of arginine282 in rabbit PepT1 by making further mutations to determine the requirement for the charge and have identified an interacting residue , aspartate341 , located in putative TMD8 . Some of these data have been previously published in abstract form -LRB- Pieri , Boyd , & Meredith , 2004 -RRB- . 2 Materials and methods 2.1 Site-directed mutagenesis of the PepT1 gene Oligonucleotides were custom synthesised -LRB- Sigma-Genosys , UK -RRB- for the following sequences -LRB- residues in bold are changed from wild-type PepT1 -RRB- : - R282-PepT1 mutants forward : where xxx was CAA for R282Q , AAG for R282K , GAT for R282D , CAT for R282H , and GCG for R282A.-D341-PepT1 mutants forward : where xxx was CGC for D341R . Reverse primers for the PepT1 mutant PCR reactions were the reverse compliment of the forward primers . The site-directed PepT1 mutants were generated using the Quikchange protocol -LRB- Stratagene -RRB- , and the resulting constructs confirmed by DNA sequencing -LRB- Department of Biochemistry , University of Oxford , UK -RRB- . 2.2 cRNA synthesis and oocyte injection PepT1 constructs were linearised with XbaI -LRB- New England Biolabs , UK -RRB- and cRNA generated by in vitro transcription -LRB- T7 mMessage mMachine , Ambion , Cambridgeshire , UK -RRB- . X. laevis oocytes were obtained under MS222 anaesthesia -LRB- 0.2 % , w/v -RRB- in accordance with the UK Animals -LRB- Scientific Procedures -RRB- Act , 1986 , and maintained at 18 °C in modified Barth 's medium -LRB- 88 mM NaCl , 1 mM KCl , 0.82 mM MgSO4 , 2.4 mM NaHCO3 , 0.42 mM CaCl2 , 10 mM Hepes , 5 mM sodium pyruvate , 50 μg ml − 1 gentamicin -LRB- Fluka , Poole , UK -RRB- , adjusted to pH 7.6 with 1 M NaOH -RRB- . Transport measurements were performed at least 72 h after micro-injection of oocytes with 27nl cRNA -LRB- 1 μg/μl -RRB- , with medium changed daily . 2.3 Transport experiments Zero-trans uptake of -LSB- 3H -RSB- - d-Phe-l-Gln -LRB- 17.4 Ci/mmole , custom synthesised , Cambridge Research Biochemicals , Stockton-on-Tees , UK -RRB- was performed as previously described -LRB- Meredith , 2004 -RRB- . Briefly , 5 oocytes were incubated in 100 μl of uptake medium -LRB- 95 mM NaCl , 2 mM KCl , 1 mM CaCl2 , 0.42 mM MgCl2 , 10 mM Tris/Hepes pH 7.4 or Tris/Mes pH 5.5 -RRB- with tracer -LRB- 0.4 μM -RRB- -LSB- 3H -RSB- - d-Phe-l-Gln . After incubation , the oocytes were washed sequentially five times in 1 ml of ice-cold 120 mM NaCl solution , lysed individually with 100 μl 2 % -LRB- w/v -RRB- SDS and liquid scintillation counted . As a control non-injected oocytes were also incubated in uptake medium with -LSB- 3H -RSB- - d-Phe-l-Gln as above . The affinity of wild-type and mutant PepT1 were assessed by competition studies with 0.4 μM -LSB- 3H -RSB- - d-Phe-l-Gln and Gly-l-Gln present in the uptake medium in concentrations from 0 to 2 mM using the protocol above , and the Ki calculated using the method of Deves and Boyd -LRB- 1989 -RRB- . Efflux studies were performed as previously described -LRB- Meredith , 2004 -RRB- , with the exception that the extracellular trans-stimulant Gly-l-Gln was used at 5 mM , and an efflux time-course was performed . Diethylpyrocarbonate -LRB- DEPC -RRB- inhibition of PepT1 was performed using a similar protocol to that of Terada , Saito , and Inui -LRB- 1998 -RRB- . Briefly , PepT1 oocytes were preincubated with 1 mM DEPC for 15 min at pHout 5.5 in the absence or presence of the PepT1 substrates Gly-l-Gln , N-Acetyl-Phe -LRB- Meredith et al. , 2000 -RRB- , and l-Ala-Tyramine -LRB- custom synthesised -RRB- and the non-substrate Tyr -LRB- all 5 mM -RRB- . The oocytes were then washed in uptake medium before uptake assays were performed as detailed above . 2.4 Electrophysiology Measurements of membrane potential were made by impaling oocytes with a single glass microelectrode -LRB- Intra 767 amplifier , WPI , Stevenage , Hertfordshire , UK -RRB- perfused in uptake medium -LRB- as above -RRB- with or without 0.4 μM d-Phe-l-Gln -LRB- synthesised in house -RRB- , 0.6 mM Gly-l-Gln -LRB- Sigma , Poole , UK -RRB- or 10 mM 4-aminobenzoic acid -LRB- 4-AMBA , Sigma -RRB- . Two-electrode voltage clamp -LRB- TEVC -RRB- was performed by placing oocytes in a 0.1 ml recording chamber and perfusing with uptake solution -LRB- pH 5.5 or 7.4 -RRB- at a rate of 15 ml/min . Oocytes were impaled by two agarose-cushioned microelectrodes filled with 3 M KCl -LRB- 0.5 -- 2.0 MΩ -RRB- and voltage-clamped at − 60 mV using a Geneclamp 500B amplifier and PCLAMP 8.1 software -LRB- Axon Instruments , CA , USA -RRB- . The holding potential was stepped from − 60 mV over the range of − 150 to +50 mV in 10 mV steps , each pulse lasting 100 ms , and returning to − 60 mV in between test voltage pulses . Typically traces were filtered at 1 kHz during recording and digitized at 0.5 -- 5 kHz using the DigiData 1200 interface -LRB- Axon Instruments , CA , USA -RRB- . All experiments were carried out at room temperature . 2.5 Data analysis All data are expressed as mean ± S.E.M. , except for Fig. 6 where the error bars represent the maximum range of stoichiometry values when taking into account the errors for the uptake data and the currents . In order to calculate the apparent charge : substrate stoichiometry , the peptide induced current that was measured by two-electrode voltage clamp at the oocyte resting membrane potential was divided by the radiolabelled dipeptide uptake in oocytes from the same preparation . This value was normalised to 1:1 for wild-type PepT1 , the accepted stoichiometry for a neutral dipeptide -LRB- Fei et al. , 1994 ; Steel et al. , 1997 -RRB- . 2.6 Statistical analysis Statistical analyses were performed using one-way ANOVA with differences considered significant if p < 0.05 when data were compared to the wild-type control , as detailed in the text and/or figure legend . 3 Results 3.1 pH dependence of d-Phe-l-Gln uptake into R282 mutants Fig. 1 shows the pH dependence of d-Phe-l-Gln uptake into oocytes expressing mutant PepT1 transporters where R282 has been changed into number of amino acid residues . Of the residues tested , R282K - and R282H-PepT1 behaved like the wild-type PepT1 , in that the initial rate of uptake -LRB- 1 h incubation time -RRB- of dipeptide was significantly faster -LRB- p < 0.05 , one-way ANOVA -RRB- at an external pH of 5.5 than at 7.4 . The other amino acid substitutions tested , R282E - -LRB- as previously reported , Meredith , 2004 -RRB- , R282D - , R282A - and R282Q-PepT1 all gave the same initial rate of uptake at pH 5.5 and 7.4 , indicating that transport by these mutants is not stimulated by an inwardly directed proton gradient . These changes can not be ascribed to changes in the affinity of the mutant PepT1 proteins for their substrate , as the Ki of Gly-l-Gln inhibiting 0.4 μM -LSB- 3H -RSB- - d-Phe-l-Gln was unchanged in the mutants -LRB- Fig. 2 -RRB- . 3.2 Can the R282 mutant PepT1 transporters concentrate substrates ? An earlier finding was that , unlike the wild-type , the R282E-PepT1 mutant was unable to concentrate substrate even in the presence of an inwardly directed proton gradient -LRB- Meredith , 2004 -RRB- . The ability to concentrate substrate was therefore tested for the other R282 mutants -LRB- Fig. 3a and b shows representative time-course experiments at pHout 5.5 and 7.4 , respectively -RRB- , and the mean accumulation levels are shown for 8 h uptakes in Fig. 3c for pHout 5.5 . In contrast to R282E-PepT1 , all were found to be able to concentrate peptide well above the equilibrium level when the external pH was 5.5 -LRB- an oocyte was assumed to have a volume of 1 μl , Petersen & Berridge , 1996 ; Yao & Tsien , 1997 -RRB- . In the absence of the proton driving force -LRB- pHout 7.4 , Fig. 3d -RRB- a similar level of intracellular d-Phe-l-Gln concentration was reached by all the mutant PepT1 transporters , including R282E-PepT1 if the incubation time was increased to 24 h -LRB- accumulation of 2.3 ± 0.4-fold compared to 3.1 ± 0.5-fold for wild-type PepT1 -RRB- . Although at 24 h incubation times we observed that cell survival can be a limiting factor , there was no statistical increase in the accumulation for the wild-type PepT1 at pHout 5.5 or 7.4 between 8 and 24 h incubations , nor between 8 and 24 h for the wild-type PepT1 at pHout 7.4 -LRB- 8.0 ± 1.4 vs. 8.3 ± 1.0 , 2.5 ± 0.5 vs. 3.1 ± 0.5 and 1.0 ± 0.4 vs. 1.5 ± 0.3 respectively , all p > 0.05 , one-way ANOVA -RRB- . 3.3 Rates of d-Phe-l-Gln efflux from R282 mutants Fig. 4 shows the rates of efflux of -LSB- 3H -RSB- - d-Phe-l-Gln from oocytes expressing either wild-type PepT1 , R282 PepT1 mutants or non-injected controls . All of the PepT1 constructs showed a significantly faster efflux than the non-injected oocytes -LRB- one-way ANOVA , p < 0.05 -RRB- , whilst R282E-PepT1 was significantly faster than the wild-type -LRB- one-way ANOVA , p < 0.05 -RRB- as previously described -LRB- Meredith , 2004 -RRB- . Interestingly , R282E-PepT1 showed a faster efflux than all the other R282 mutants -LRB- one-way ANOVA , p < 0.05 -RRB- , which were not significantly different to the wild-type -LRB- p = 0.14 -RRB- . 3.4 Apparent transport stoichiometry -LRB- charge to substrate -RRB- using two-electrode voltage clamp As well as uptake being pH independent and non-concentrative , the electrophysiological characteristics of R282E-PepT1 were strikingly different to the wild-type -LRB- Meredith , 2004 -RRB- . Since R282D-PepT1 is similarly pH independent but does accumulate substrate , the apparent proton to peptide stoichiometry was examined , where the uptake of 0.4 μM d-Phe-l-Gln was compared to the peptide-induced current in the same batch of oocytes . The current at the oocyte resting membrane potential -LRB- − 27.0 ± 1.1 mV at pHout 5.5 , and − 36.8 ± 2.2 mV at pHout 7.4 , n = 12 -RRB- was taken to represent the membrane potential under uptake conditions , as the addition of 0.4 μM substrate does not produce a detectable change in membrane potential -LRB- Fig. 5 -RRB- . The ratio of proton to neutral dipeptide co-transported through wild-type PepT1 is 1:1 -LRB- Fei et al. , 1994 ; Steel et al. , 1997 -RRB- , yet in R282E - , R282D - and R282A-PepT1 the apparent stoichiometry is substantially higher -LRB- 4 , 5 and 5 respectively at pHout 5.5 , Fig. 6 -RRB- . 3.5 Is the extra current measured dependent on substrate transport ? In an attempt to see if the extra current carried by R282E-PepT1 was dependent on substrate translocation rather than simply substrate binding , the non-translocated PepT1 substrate 4-aminobenzoic acid -LRB- 4-AMBA , Darcel , Liou , Tome , & Raybould , 2005 ; Meredith et al. , 1998 -RRB- was used . As found for wild-type PepT1 , 4-AMBA failed to induce a depolarisation in R282E-PepT1 oocytes at 10 mM , three times its Ki -LRB- Meredith et al. , 2000 -RRB- , in contrast to the known transported substrate Gly-l-Gln , also at three times Ki -LRB- Fig. 5 -RRB- . 3.6 Identification of an interacting residue for R282 The conservation of a positively charged amino acid in a transmembrane domain -LRB- TMD7 -RRB- , and the results above , strongly suggested that the presence of a positive charged residue was necessary for wild-type-like PepT1 transport function . In TMD8 , predicted to be at approximately the same level in the membrane , there is a conserved aspartate -LRB- D341 -RRB- , and it was an appealing hypothesis that the two oppositely charged side chains might be forming a charge pair . To test this , double mutants were made , R282E/D341R - and R282D/D341R-PepT 1 , to swap the charges over . As can be seen in Fig. 7 , these double mutants showed the same pH dependence of influx as the wild-type transporter , providing strong evidence to support the hypothesis . Both R282E/D341R - and R282D/D341R-PepT 1 were also able to concentrate substrate like the wild-type -LRB- data not shown -RRB- . 3.7 Diethylpyrocarbonate inhibition of PepT1 function Preincubation of wild-type PepT1-expressing oocytes with 1 mM diethylpyrocarbonate -LRB- DEPC -RRB- for 15 min completely inhibited the PepT1 mediated dipeptide uptake measured over 1 h , as shown in Fig. 8 . This inhibition by DEPC was largely prevented by the presence of the known PepT1 substrates Gly-l-Gln and N-Acetyl-Phe -LRB- Meredith et al. , 2000 -RRB- , but not by the amino acid non-substrate Tyr . Interestingly , despite being a good PepT1 substrate -LRB- Ki approximately 0.1 mM , data not shown -RRB- , a modified peptide lacking a carboxyl terminus -LRB- l-Ala-Tyramine -RRB- only partially prevented DEPC inhibition -LRB- Fig. 8 -RRB- . 4 Discussion The R282K mutation of rabbit PepT1 is not only the most conservative one regarding the charge , but in a number of species , including dog , rat and mouse , lysine is the naturally occurring residue at this position . Therefore it was not surprising to find that this mutant behaves like the wild-type rabbit PepT1 -LRB- pig , sheep , rhesus and crab-eating monkeys and human also have R282 -RRB- . The finding that R282H also behaved like the wild-type was interesting , as histidine has a side-chain that can be titrated over the range used in the experiments -LRB- pK of ∼ 6 in free solution -RRB- . Our findings could be interpreted in several ways , including the possibility that only at pHout 5.5 is there the formation of a positive charge by side-chain titration that gives a stimulation of uptake over that seen at pHout 7.4 . A second possibility is that the protein environment surrounding H282 is such that its side chain pK is shifted away from 6 and it is therefore always protonated , and thus behaves more like an arginine or lysine . This effect has been shown for example in the enzyme protein tyrosine kinase -LRB- Tishmack , Bashford , Harms , & Van Etten , 1997 -RRB- , where histidine residues had a pK as high as 9.2 when analysed by NMR . In the original study on R282E-PepT1 , it was concluded that the uptake of peptide by the mutant transporter was uncoupled from the movement of protons , and that in addition to acting as a facilitated peptide transporter , R282E-PepT1 also displayed a peptide-gated non-specific cation conductance -LRB- Meredith , 2004 -RRB- . However , it is possible that this conclusion needs updating in the light of the current findings that there are R282 mutants that , like R282E-PepT1 , are not pH stimulated , yet are still able to accumulate substrate above equilibrium when an inwardly directed proton gradient is imposed . For a transporter to be able to accumulate substrate above equilibrium , an energy source must be involved , in this case the proton electrochemical gradient . Therefore , the mutant PepT1 proteins that can accumulate substrate but do not show pH stimulation -LRB- R282A and R282D -RRB- must still be coupled to the movement of protons down their electrochemical gradient . The lack of pH stimulation could be attributed to the fact that during the transport cycle these specific mutants have a different rate limiting step to the wild-type , and that for these mutants that step is not pH dependent . It has already been reported that R282E-PepT1 has a faster rate of efflux than wild-type , consistent with an uncoupling of peptide uptake from the proton driving force -LRB- Meredith , 2004 -RRB- ; the finding here that the rates of efflux for the other R282 mutants are not different from that of the wild-type is in agreement with the hypothesis that they are still proton-coupled , as shown by their ability to accumulate substrate above the extracellular concentration at pHout 5.5 but not 7.4 . The simplest hypothesis was that in R282E-PepT1 , the extra inward charge movement associated with peptide uptake collapsed the membrane potential , which is known to be the major driving force for proton-coupled peptide uptake -LRB- Temple & Boyd , 1998 -RRB- , hence the apparent lack of substrate accumulation . Therefore by extension one would expect R282D - and R282A-PepT1 to have the same charge coupling as the wild-type , as they too accumulate substrate , but this was not the case : both R282E - , R282D - and R282A-PepT1 showed the same increased charge : peptide apparent stoichiometry , i.e. substantially larger than the wild-type . This stoichiometry itself showed pH dependence , with a lower value of around 2 for the mutants at pH 7.4 , suggesting that the current is either carried by protons or is a pH-sensitive phenomenon . There was no difference in the uptake in the absence or presence of sodium , either for R282E-PepT1 -LRB- Meredith , 2004 -RRB- or R282D-PepT1 -LRB- data not shown -RRB- . The finding that the R282E/D341R - and R282D/D341R-PepT 1 double mutants -LRB- the latter being a charge pair reversal of the naturally occurring residues in rabbit PepT1 -RRB- had the same characteristics as the wild-type protein strongly suggests that these two residues do interact in the 3D protein -LRB- Pieri et al. , 2004 -RRB- as previously proposed -LRB- Meredith , 2004 -RRB- . During the preparation of this manuscript , Kulkarni et al. -LRB- 2007 -RRB- reported findings consistent with R282 and D341 forming a charge pair in human PepT1 . The initial hypothesis that in R282E-PepT1 repulsion between E282 and D341 allowed the movement of extra ions through the protein when a peptide is transported was not supported however by the finding that the single D341R mutant also behaved like the wild-type , as one might have thought that R282 and R341 would repel in much the same way as E282 and D341 . That D341R-PepT1 behaved like wild-type suggests that although R282 and D341 seem to form a charge pair , in rabbit PepT1 residue 341 being negatively charged is not crucial to PepT1 function ; interestingly , in human PepT1 the D341R single mutant had reduced function -LRB- Kulkarni et al. , 2007 -RRB- . The reason for this difference between rabbit and human PepT1 is not clear . The observation that the non-transported PepT1 substrate 4-AMBA did not induce a depolarisation in R282E-PepT1-expressing oocytes clearly shows that the charge movement is linked to substrate binding and translocation and not to binding alone . One explanation for the increase in proton -- peptide stoichiometry is that in wild-type PepT1 the presence of a positively charged residue deep in the binding pocket at position 282 repels proton movement through the transporter protein during the translocation step . The data in Fig. 6 are consistent with this , as the apparent stoichiometry is lower at pHout 7.4 than it is at pHout 5.5 , indicating that the proton electrochemical gradient is involved . In the case of mutations where R282 was replaced with a non-positively charged amino acid , the rate-limiting step of the transport cycle must be insensitive to extracellular pH , hence the lack of stimulation when the pHout is dropped from 7.4 to 5.5 . Kinetic analysis of peptide transport by PepT1 in rat renal brush border membrane vesicles -LRB- Temple & Boyd , 1998 -RRB- showed that at pHout7 .4 / pHin7 .4 , it was the protonation of the carrier protein that was the rate-limiting step -LRB- Temple , Bailey , Bronk , & Boyd , 1996 -RRB- , whereas at pHout5 .5 / pHin7 .4 it was the return of the unloaded carrier . The rate of peptide uptake by R282E-PepT1 -LRB- corrected for protein expression in the intact oocyte plasma membrane by luminometry , Panitsas et al. , 2006 -RRB- is the same as for the wild-type at pHout 5.5 -LRB- data not shown -RRB- , but , unlike for the wild-type , is not slower at pHout 7.4 . This indicates that the mutations to arginine282 that abolish pH sensitivity -LRB- R to E , D , A , or Q -RRB- are affecting the rate limiting step , so when pHout is 7.4 the rate limiting step has the same magnitude as that at pHout 5.5 , hence the lack of sensitivity to changing pHout . The rate limiting protonation of the outward-facing carrier protein at pHout 7.4 , the first step in the transport cycle -LRB- Temple et al. , 1996 -RRB- , was proposed by us -LRB- Bailey et al. , 2000 ; Meredith & Boyd , 1995 -RRB- and others -LRB- e.g. Steel et al. , 1997 ; Uchiyama , Kulkarni , Davies , & Lee , 2003 -RRB- to be protonation of histidine57 -LRB- H57 -RRB- . In the R282-PepT1 mutants , except for R282K and R282H , the lack of a positively charged residue might result in a conformational change of the protein that changes the local environment and increases the pKa of H57 , such that it is more easily protonated at a higher pHout . Thus at pHout 7.4 the rate limiting step is no longer the protonation of H57 , but the return of the unloaded carrier , as it is at pHout 5.5 , hence the similar transport rates at pHout 5.5 and 7.4 . A model for how proton -- peptide transport might occur is shown in Fig. 9 : the empty PepT1 is primed by protonation of H57 , followed by the binding of a zwitterionic peptide , with the N-terminal co-ordinated at E595 and the C-terminal at H57-H + -LRB- Meredith et al. , 2000 -RRB- . Although the binding of the substrate C-terminal to His57-H + is in disagreement with the conclusion of Terada et al. -LRB- 1998 -RRB- , it is supported by the finding that N-Acetyl-Phe , a known PepT1 substrate which does not have a free amino terminal -LRB- Meredith et al. , 2000 -RRB- , can protect PepT1 against inhibition by DEPC . Additionally , the finding that significant DEPC inhibition is still evident when oocytes were co-incubated with the PepT1 substrate l-Ala-Tyramine which lacks a carboxyl terminus further adds to the notion that H57-H + binds the carboxyl terminus of the substrate -LRB- Fig. 8 -RRB- . H57-H + then donates its proton to the C-terminal carboxyl group , and the transporter undergoes a conformational change that leads to the breaking of the salt bridge between R282 and D341 , re-orientating the binding site to be inward facing as simultaneously the protonated N-terminal of the substrate binds to D341 , neutralising the charge , and the R282 charge is stabilised by Y167 -LRB- the chemical properties of this tyrosine have been shown to be essential , Yeung et al. , 1998 -RRB- . The peptide molecule is then released into the cytoplasm , whereby it returns to the zwitterionic state by releasing the proton from the carboxyl terminal . The transporter then undergoes the reverse conformational change to re-orientate the binding site to outward facing , and R282 reforms the salt bridge with D341 . As R282E-PepT1 can not accumulate substrate , the implication must be that the movement of peptide is no longer coupled to the movement of the protons , whereas in all the other mutants coupling must be maintained . If for R282E-PepT1 the pKa of H57 was raised to the point that it was no longer favourable for it to donate its proton to the carboxyl terminus of the substrate , then peptide transport would no longer be proton-coupled , and this would explain the failure of R282E-PepT1 to accumulate substrate . Intriguingly , it can be seen in Fig. 6 that both R282D - and R282A-PepT1 appear to carry approximately one more charge per substrate peptide than R282E-PepT1 , which is consistent with the hypothesis that one of the charges carried is coupled with the substrate . In conclusion , the arginine at position 282 in rabbit PepT1 plays an intriguing role in the function of the transporter , with mutations to different residues revealing that a positive residue is required for pH dependence , whilst only R282E-PepT1 can not concentrate substrate above equilibrium ; this is despite other mutations , most notably R282D-PepT1 , having a similarly increased charge : peptide stoichiometry . As previously proposed , R282 -LRB- TMD7 -RRB- forms a charge pair with D341 -LRB- TMD8 -RRB- , with R282E/D341R-PepT 1 showing normal transport characteristics . Further biological testing or a crystal structure of PepT1 will be required to establish the validity of the model proposed .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

epithelia
membrane transport
nutrient absorption
protein structure–function
site-directed mutagenesis


Stable RNA markers for identification of blood and saliva stains revealed from whole genome expression analysis of time-wise degraded samples Human body fluids such as blood and saliva represent the most common source of biological material found at a crime scene . Reliable tissue identification in forensic science can reveal significant insights into crime scene reconstruction and can thus contribute toward solving crimes . Limitations of existing presumptive tests for body fluid identification in forensics , which are usually based on chemoluminescence or protein analysis , are expected to be overcome by RNA-based methods , provided that stable RNA markers with tissue-specific expression patterns are available . To generate sets of stable RNA markers for reliable identification of blood and saliva stains we -LRB- 1 -RRB- performed whole-genome gene expression analyses on a series of time-wise degraded blood and saliva stain samples using the Affymetrix U133 plus2 GeneChip , -LRB- 2 -RRB- consulted expression databases to obtain additional information on tissue specificity , and -LRB- 3 -RRB- confirmed expression patterns of the most promising candidate genes by quantitative real-time polymerase chain reaction including additional forensically relevant tissues such as semen and vaginal secretion . Overall , we identified nine stable mRNA markers for blood and five stable mRNA markers for saliva detection showing tissue-specific expression signals in stains aged up to 180 days of age , expectedly older . Although , all of the markers were able to differentiate blood/saliva from semen samples , none of them could differentiate vaginal secretion because of the complex nature of vaginal secretion and the biological similarity of buccal and vaginal mucosa . We propose the use of these 14 stable mRNA markers for identification of blood and saliva stains in future forensic practice . Introduction Human body fluids such as blood and saliva are the most common sources of biological trace material found at a crime scene . Reliable tissue identification in forensic casework is important as it provides crucial insights into crime scene reconstruction and can thus contribute towards solving crimes . Blood stains are routinely tested in forensic practise using various methods including the tetrabase -LRB- 4,4-bis -LRB- dimethylamino -RRB- diphenylmethane -RRB- test -LSB- 1 -RSB- , the Kastle -- Meyer phenolphthalein test , the tetramethylbenzidine test -LSB- 2 -RSB- , the orthotolidine test -LSB- 3 -RSB- , or the luminol -LRB- 3-aminophthalhydrazide -RRB- chemoluminescence test -LSB- 4 -RSB- , with the latter especially appropriate for detecting blood stains after cleaning attempts -LSB- 2 , 5 -RSB- . All these presumptive -- thus indicative but not identifying -- tests take advantage of the peroxidase-like activity of the heme unit of the hemoglobin molecule in human blood . Therefore , false-positive results can be caused by the presence of strong oxidants , such as chlorine-containing detergents or by true peroxidases -LRB- e.g. , from plants -RRB- -LSB- 6 -RSB- . Saliva stains are usually detected in forensic practise via an enzymatic amylase test using Phadebas -LSB- 7 -RSB- or with a recently developed enzyme-linked immunosorbent assay-based method -LSB- 8 -RSB- . However , because of amylase degradation , the time window for the successful performance of such tests can be limited -LSB- 9 -RSB- . Furthermore , no amylase assay can distinguish between salivary amylase and amylases from other tissues -LRB- pancreatic , urinary , etc. -RRB- ; therefore , the tests for saliva identification are only presumptive -LRB- similar to existing blood identification tests -RRB- . On the other hand , methods for identification and quantification of mRNA are already well established , although mostly outside the forensic field . These methods make massive multiplex gene expression profiling possible -- among many other applications -- for the discovery of tissue-specific mRNA markers . The major concern of using mRNA markers for forensic applications is their assumed high susceptibility to degradation . However , recent studies using a few selected genes demonstrated that it is possible to isolate total RNA of sufficient quality and quantity from biological stains that are several months or even years old -LSB- 10 -- 12 -RSB- . It has also been suggested , although with limited evidence so far , that different types of mRNA seem to follow different rates of degradation -LSB- 13 -RSB- . It is assumed that the degradation process of mRNA is influenced by many external and internal factors , including structural peculiarities like the presence of AU-rich elements -LRB- ARE motifs -RRB- , protein binding properties , and cellular localization -LSB- 14 , 15 -RSB- . However , detailed knowledge on the molecular reasons for differences in RNA degradation between different types of RNAs as well as between mRNAs of different genes is currently lacking and further investigations are sorely needed . Although a small number of mRNA markers has been tested for tissue identification in forensic science -LSB- 16 -- 19 -RSB- , no systematic study has yet been performed . In addition , the identification of candidate markers in previous studies was based on a mixed literature and database search , apparently without strict criteria of selection , considering only a limited number of genes and tissues , and not taking into account RNA degradation levels . Furthermore , expressed sequence tags databases , which were used previously , like the Cancer Genome Anatomy Project -LSB- 18 -RSB- , are expected to provide heavily biased information on candidate genes because of the nonrandom character of representation of clone libraries . To find stable mRNA markers for body fluid identification in forensic practice , we performed a systematic and comprehensive whole-genome gene expression analysis on time-wise degraded blood and saliva stains using the Affymetrix U133 plus2 GeneChip . This expression array contains > 54,000 mRNA probe sets , which encompass most , if not all , known and predicted human genes . Tissue-specific expression patterns of the most promising candidate genes from the array analyses were further confirmed using the GNF SymAtlas expression database -LSB- 20 -RSB- , which covers about 100 human tissues , and finally verified by quantitative real-time polymerase chain reaction -LRB- PCR -RRB- in blood and saliva as well as in other body fluids relevant for forensic casework , i.e. , semen and vaginal secretion . Materials and methods Sample collection Aliquots of 5 ml of whole blood and saliva were collected from each of five healthy volunteers -LRB- four men and one woman -RRB- of western European genetic origin under informed consent before their inclusion in the study . Native blood was collected without anticoagulation treatment to avoid disturbing effects of anticoagulation reagents on gene expression . In each sample , 75 cotton swabs were immersed . Special care was taken to shorten the time between collection and swab absorption to avoid blood coagulation . After complete absorption of the fluids , swabs were left until dry on a bench top at room temperature . When dry , the swabs were stored in dust-free nonhumid conditions -LRB- but subjected to normal daylight -RRB- for different time intervals . Swabs were visually inspected and sorted out to ensure similar liquid content between individual swabs . After 0 , 1 , 3 , 7 , 14 , 21 , 57 , and 180 days , swabs were stored at − 80 °C until RNA isolation . For the time interval 0 days , samples were frozen immediately after drying . Semen and vaginal secretion samples were collected from one male and one female individual absorbed with cotton swabs and dried overnight before RNA isolation . RNA isolation RNA was isolated using the Qiagen RNeasy kit -LRB- Qiagen Benelux B.V. -RRB- according to the manufacturer 's instructions with minor modifications . These included cutting up the cotton swab into 1 × 1-mm pieces and soaking them in RLT buffer for 1 h at 4 °C before the extraction . Trial experiments to lengthen this incubation time up to 24 h did not reveal any improvement in respect to RNA quantity and quality -LRB- data not shown -RRB- . Microarray hybridization and gene expression data analysis Before hybridization to Affymetrix U133 plus2 .0 GeneChip arrays -LRB- Affymetrix , Santa Clara , CA -RRB- , RNA isolated from blood and saliva stains was amplified using the Ambion MEGAscript T7 two-cycle amplification kit -LRB- Applied Biosystems , The Netherlands -RRB- . Amplification , labeling , hybridization , washing , and scanning were performed by the microarray core facility of the Erasmus MC Center for Biomics according to Affymetrix specifications . Background subtraction and probe signal summarization were calculated according to the robust multiarray analysis algorithm -LSB- 21 -RSB- using the R Bioconductor software -LSB- 22 -RSB- ; the resulting log2 signal values were back-transformed to linear scale . Presence/absence calls for individual probe sets were calculated with the mas5calls function of the Bioconductor mas package . Because the constant global mean assumption does not hold true for arrays hybridized to differentially degraded RNA samples , the normalization of the signal intensities between samples was performed using the nonhuman control genes present on Affymetrix arrays -LRB- spiked-in probes -RRB- . Normalization factors for each array were inferred from the average signal intensities of bioB , bioC , bioD , and Cre control probe sets . Analysis of differential gene expression was performed using the significance analysis of microarrays -LRB- SAM -RRB- algorithm -LSB- 23 -RSB- implemented in the TM4 software -LSB- 24 -RSB- . In the saliva dataset , we selected only genes with signal intensities above 50 -LRB- which is below the usually applied background threshold in expression array experiments -RRB- that had a signal intensity below 50 in the blood dataset . The selection of blood-targeted genes was done in a similar manner but with different criteria , the lower intensity limit in blood was set to 1,000 to reasonably restrict the number of candidates . Real-time PCR First strand cDNAs were synthesized with SuperScript ® III RTS First-Strand cDNA Synthesis Kit -LRB- Invitrogen BV , The Netherlands -RRB- using total RNA as a template . The primers were designed with Primer3 software -LSB- 25 -RSB- so that forward and reverse primers were complementary to different exons of the respective genes and most closely located to the 3 ′ - end of the corresponding RefSeq cDNA -LRB- Electronic Supplementary Material Table S1 -RRB- . Real-time PCR reactions with the SuperScript ® III Platinum ® SYBR ® Green One-Step qPCR Kit -LRB- Invitrogen BV -RRB- were performed on an ABI 7300 PCR machine -LRB- Applied Biosystems , The Netherlands -RRB- using the following parameters : initial denaturation at 94 °C for 10 min , followed by 45 cycles of denaturation at 94 °C for 15 s , and a final annealing/elongation at 60 °C for 30 s. Melting profiling and agarose gel electrophoresis were used to confirm the specificity of the primers and the absence of DNA contamination . Quantification of the amplified cDNA yield in comparative blood and saliva PCRs was done by the standard curve method . PCR experiments with semen and vaginal secretion were quantified using delta Ct -LRB- dCt -RRB- method . In both cases , GAPDH gene was used as an endogenous control to normalize the amplification signal between the samples from different tissues and individuals . Time points were compared to each other without normalization : Assuming the temporal degradation of all RNA molecules , no internal control gene could be used , and the only proper way to normalize RT-PCR signals was to use the same amount of template in each reaction . We found that this requirement holds true for our experiments because the GAPDH expression variability between different samples from the same tissue was relatively low -LRB- CV < 25 % , data not shown -RRB- , which is probably because of approximately the same amount of blood or saliva absorbed with cotton swabs during material collection . Results and discussion Microarray expression data As expected , hybridization signals demonstrated high variability between individuals ; however , the most striking differences were observed between the different tissues . Signal intensities in blood samples were on average about five times higher than in saliva -LRB- 174.2 ± 1.9 in blood samples vs 26.9 ± 0.7 in saliva ; Wilcoxon test rank sum p < 0.001 -RRB- . In addition , at the time-point zero , the number of the probe sets called as present according to the Affymetrix algorithm was , on average , more than three times higher in blood than in saliva -LRB- 30.2 % ± 0.9 vs 9.3 % ± 0.6 ; t test p < 0.001 -RRB- . The SAM test with stringent parameters -LRB- false discovery rate was set to 0 % -RRB- showed that , both in blood and saliva experiments , no genes demonstrated significant expression differences in a time range of 0 -- 57 days of stain storage . Only few genes -LRB- 37 and 10 significantly differential genes for saliva and blood , respectively -RRB- appeared to be differentially expressed at 180 days in comparison to other time points . This suggests that in dried blood and saliva , mRNA molecules remain relatively stable for a long period . Recent studies of Heinrich et al. -LSB- 26 -RSB- also revealed poor correlation between RNA degradation and postmortem time intervals . Selection of tissue-specific markers The initial selection of tissue-specific genes was performed using the normalized signal intensities of microarray hybridizations averaged across the five biological replicates at the zero experimental time point . About 500 apparent saliva-specific and 1,000 apparent blood-specific candidate genes were selected . Further refinement of tissue-specific gene sets was achieved by probing the selected candidates against the GNF SymAtlas tissue database -LSB- 20 -RSB- after excluding all cell lines from the database retaining only tissues and organs for the analysis . Genes were selected only if they were highly and exclusively expressed in the target tissue -LRB- s -RRB- based on the GNF SymAtlas database . For blood , target tissue in the database was defined as whole blood ; while for saliva , the target tissues were salivary gland , tongue , trachea , and tonsils . The selection criteria were as follows : high expression -LRB- signal intensity > 1,000 -RRB- in target tissue and low expression -LRB- signal intensity < 200 -RRB- in nontarget tissues . Using these criteria and combining data from expression array experiments as well as GNF SymAtlas database verification , we identified six saliva-targeted genes and 15 blood-targeted genes that were highly expressed only in target tissues -LRB- or respective organs -RRB- but not , or nearly not , in the nontarget tissues -LRB- Electronic Supplementary Material Figure S1a , S1b , S1c -RRB- . RT-PCR confirmation of tissue-specific markers To confirm the microarray results , real-time PCR experiments were designed for the 21 best candidate markers selected from array hybridizations and database searches and performed using RNA extracted from aged blood and saliva stains , also providing a method suitable for forensic applications . In agreement with the array results , all 21 markers analyzed showed good amplification in the target tissue but no or only marginally detectable amplification in the nontarget tissue -LRB- Fig. 1a -- c -RRB- . Among the candidate markers , only the PPL gene that was targeted for saliva demonstrated significant expression overlap with blood and therefore was excluded from further experiments . Our results demonstrate that , irrespective of the stain storage time , sufficient RT-PCR amplification was observed in all samples , even in the samples from the longest storage time tested -LRB- 180 days -RRB- , indicating marker stability over long periods of sample storage time . The only exception was the CCR2 gene for which no amplification was detected in the blood stains stored for 180 days and was therefore excluded from further analyses . A plausible explanation for this peculiarity could be the location of the PCR-amplified region , which is more than 1 kb distant from the 3 ′ end of the mRNA because of the very long untranslated region of CCR2 . Apparently , the degree of degradation of the CCR2 mRNA after 180 days of sample storage was too high to allow its efficient reverse transcription using the oligo -LRB- dT -RRB- method that targets the 3 ′ end of the molecule . This observation highlights the necessity to design PCR primers for the most 3 ′ - proximal part of the mRNA molecule for successful amplification of cDNA fragments in degraded samples . Fig. 1a , b RT-PCR results for blood-targeted genes in blood and saliva stains . c RT-PCR results for saliva-targeted genes in saliva and blood stains . Genes were selected based on expression microarray results and GNF SymAtlas database . Expression values for each time point were averaged across three male and three female RNA samples ; no gender-specific expression differences were detected -LRB- t test p < 0.05 -RRB- . B indicates blood ; S indicates saliva ; samples were processed after complete drying of blood and saliva at 0 , 21 , 57 , and 180 days , respectively Expression of the candidate markers in other body fluids For additional confirmation of the tissue-specificity , we tested by RT-PCR the expression patterns of our candidate RNA markers in other body fluids that might be observed in a forensic case , i.e. , vaginal secretion and semen . According to the GNF SymAtlas database , all our markers targeted for blood and saliva are not expressed in testis nor in uterus tissues . In agreement , our dedicated RT-PCR experiments revealed that two of the saliva-targeted mRNA markers -LRB- SPRR3 and SPRR1A -RRB- show no detectable expression in semen -LRB- after 50 RT-PCR cycles -RRB- , and the remaining three -LRB- KRT4 , KRT6A , and KRT13 -RRB- show vast overexpression in saliva compared to semen -LRB- ddCt > 15 , Fig. 2a -RRB- , keeping with the assumption of high saliva specificity of the five proposed mRNA markers . SPRR1A and SPRR3 genes both encode cornified envelope precursor proteins and are predominantly expressed in oral and esophageal epithelia , where they are strictly linked to keratinocyte terminal differentiation -LSB- 27 -RSB- . Keratins 4 , 6A , and 13 are known as one of the major structural proteins of oral mucosa -LSB- 28 , 29 -RSB- . Fig. 2a RT-PCR for saliva and blood-targeted genes in semen stains . b RT-PCR for saliva and blood targeted in vaginal secretion stains . Delta Ct -LRB- dCt -RRB- values were calculated as follows : dCt = Ct -LRB- candidate gene -RRB- − Ct -LRB- endogenous control , GAPDH gene -RRB- . Low dCt values correspond to high expression level of the specific mRNA . Gray bars correspond to the samples from target tissues for selected genes -LRB- either blood or saliva -RRB- ; black bars correspond to samples from nontarget tissues -LRB- either vaginal secretion or semen -RRB- . Dotted bars represent the cases were amplification was not detected after 50 cycles , in this case , the expression values were arbitrary set to Ct value of 25 -LRB- plot maximum -RRB- For the 14 blood-targeted genes , we observed no detectable amplification in semen for nine genes -LRB- CASP1 , AMICA1 , C1QR1 , ALOX5AP , AQP9 , C5R1 , NCF2 , MNDA , ARHGAP26 -RRB- , keeping with the assumption of high blood specificity of the respective mRNA markers . These genes encode the proteins with important functions in different types of blood cells . They are known to be highly or even specifically expressed in peripheral leukocytes -LRB- AQP9 , NCF2 , CASP1 , C5R1 , C1QR1 , ALOX5AP -LSB- 30 -- 35 -RSB- -RRB- and myelocytes or hematopoietic cells -LRB- MNDA , ARHGAP26 , AMICA1 -LSB- 36 -- 38 -RSB- -RRB- . However , five genes demonstrated only slightly differential or even comparable expression in blood and semen -LRB- CD36 , CCR1 , PF4 , BIN2 , and ALOX5 -RRB- , not expected given the information provided by the GNF database , and were therefore excluded from the final list of blood-specific markers . Thus , our microarray-based genome-wide approach to find tissue-specific mRNA markers identified the genes that are functionally relevant for the target tissues . Furthermore , and not surprisingly , RT-PCR of all saliva - and blood-targeted markers in samples from vaginal secretion revealed gene expression at a level comparable to that in blood and saliva samples -LRB- Fig. 2b -RRB- . The natural occurrence of blood cells in vaginal secretion most likely explains the expression of our blood-targeted markers in vaginal secretion , whereas the high biochemical and histological similarity of oral and vaginal epithelia -LSB- 40 -RSB- makes the similarity of gene expression patterns between both tissues plausible . It should be pointed out that mRNA markers previously claimed to be useful for the identification of vaginal secretion such as HBD-1 -LSB- 18 -RSB- and MUC4 -LSB- 18 , 19 -RSB- are known to be abundant also in oral epithelial cells and the salivary transcriptome -LSB- 41 -- 43 -RSB- . Furthermore , Nussbaumer et al. -LSB- 19 -RSB- ruled out the potential to differentiate saliva and vaginal secretion using solely MUC4 . Our results , together with previous findings , suggest that establishing mRNA markers expressed exclusively in vaginal secretion could be a challenging if not impossible task . Comparison with previously suggested mRNA markers Interestingly , tissue-specific genes , as identified here , do not overlap with the ones previously suggested for blood and saliva stain identification -LSB- 18 , 19 -RSB- . This could be explained by the experimental setup and the systematic -LRB- but not ad hoc -RRB- approach of this study , namely , the degraded biological material analysed and the Affymetrix microarray platform applied . In contrast to previous studies , we restricted our marker ascertainment to those genes , which retained structural mRNA integrity during the stain dry-out process as well as the subsequent long-term storage of 180 days . This allows future application of detection of these markers in forensic stains of unknown age , at least up to an age of 6 months , but expectedly longer . Furthermore , our saliva-specific candidate genes were derived from mouth and pharynx epithelial cells , unlike the previously suggested STATH and HTN3 genes that are expressed in the salivary gland -LSB- 18 -RSB- . Secreted mRNAs that are abundant in fresh saliva are more prone to fast degradation by extracellular RNAses -LSB- 39 -RSB- ; they are therefore not expected to be present in dried stains , explaining why they were not detected by the relatively low-sensitive microarray hybridization method used in this study . The SPTB and PBGD genes , previously proposed as blood-specific markers -LSB- 18 -RSB- , do not demonstrate any overexpression relative to other tissues in whole blood according to the GNF SymAtlas database -LRB- data not shown -RRB- . Conclusions In summary , whole-genome expression analysis in time-wise degraded samples from blood and saliva stains in combination with RT-PCR verification of various forensically relevant body fluids has resulted in the identification of stable tissue-specific mRNA markers from five genes for saliva -LRB- SPRR3 , SPRR1A , KRT4 , KRT6A , and KRT13 -RRB- and nine genes for whole blood -LRB- CASP1 , AMICA1 , C1QR1 , ALOX5AP , AQP9 , C5R1 , NCF2 , MNDA , and ARHGAP26 -RRB- . For the first time , mRNA markers were ascertained considering almost the entire human transcriptome and based on experimental data of genome-wide gene expression as well as considering the degradation stability of mRNAs . We could demonstrate that the candidate genes identified here provide informative mRNA markers for blood and saliva identification for stains up to 180 days of age . We would like to propose their application in forensic case work -LRB- with the potential practical limitation of coamplification in vaginal secret -RRB- for stains of at least 6 months of age . However , we expect that the proposed mRNA markers will successfully identify older blood and saliva stains -LRB- respective experiments are currently in progress -RRB- . Finally , we would like to remark that tissue identification in forensics should be performed in a reciprocal way ; so that a tissue is identified because of the presence of markers specific for the relevant tissue together with the absence of markers specific for all other tissues in question . Clearly , more research should be dedicated towards finding the most suitable markers for tissue identification in forensics . Electronic supplementary material Below is the link to the electronic supplementary material . Table S1 RT-PCR primers for the genes amplified -LRB- DOC 79 kb -RRB- . Figure S1a GNF Symatlas expression profiles of blood-specific genes -LRB- GIF 15 kb -RRB- High resolution image file -LRB- TIF 82 kb -RRB- Figure S1b GNF Symatlas expression profiles of blood-specific genes -LRB- GIF 14 kb -RRB- High resolution image file -LRB- TIF 77 kb -RRB- Figure S1c GNF Symatlas expression profiles of saliva-specific genes -LRB- GIF 12 kb -RRB- High resolution image file -LRB- TIF 74 kb -RRB-

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O B I O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

body fluid identification
gene expression
blood
saliva
rna markers