Supplementary information

Size: px

Start display at page:

Download "Supplementary information"

Alexandra Dalton
5 years ago
Views:

1 Supplementry informtion Unsturted liphtic lcohol s nturl lignd for mouse odornt receptor Keiichi Yoshikw, Hiroki Nkgw, Noki Mori, Hidenori Wtne nd Kzushige Touhr* Deprtment of Applied Biologicl Chemistry, The University of Tokyo, Tokyo , Jpn. Correspondence to e sent to : Kzushige Touhr, E-mil: ktouhr@mil.ecc.u-tokyo.c.jp 1

7.5 camp level (pmol/well) 5. Bsl ELG SLG SMG PG HG PPG MG Exocrine glnd extrct Bsl ELG SLG SMG PG HG PPG MG ATP 1.2 F rtio.5 Exocrine glnd extrct ELG SLG SMG PG HG PPG MG ATP F rtio.

2 7.5 camp level (pmol/well) 5. Bsl ELG SLG SMG PG HG PPG MG Exocrine glnd extrct Bsl ELG SLG SMG PG HG PPG MG ATP 1.2 F rtio.5 Exocrine glnd extrct ELG SLG SMG PG HG PPG MG ATP F rtio.5 1 min Supplementry Figure 1. camp or clcium responses of HEK293 cells to exocrine glnd extrcts. () Inherent camp responsiveness of HEK293 cells to exocrine glnd extrcts. HEK293 cells were incuted with exocrine glnd extrct solutions, nd every extrct cused n increse in intrcellulr camp levels even in the sence of trnsient OR expression (n=3). Group dt re reported s the men ± s.e.m. () Clcium responses of HEK293 cells to exocrine glnd extrcts. (top pnels) Pseudocolored imges of fur-2-loded HEK293 cells stimulted with exocrine glnd extrcts. The pseudocolors represent reltive vlues of the fluorescence rtio (F-rtio). (ottom) A representtive trce of extrct-responsive HEK293 cells. A different exocrine glnd extrct ws pplied t ech of the times indicted y rrowheds. ATP (1 µm) ws utilized s control for cell viility. Intrcellulr clcium increses were oserved with ll of the extrcts. These ckground responses hmpered evlution of n OR-medited response to potentil lignds in these crude extrcts. Other cell lines such s COS-7 nd CHO-K1 lso showed nonspecific responses to tissue extrcts. Extrct solutions used in the camp ssy nd the clcium imging contined 1-mg equivlent of glnd tissue per 1 µl uffer.

3 MOR23 Olfr288 c + CFTR Lyrl (µm) CFTR + RTP1 Lyrl (µm) CFTR δ-undeclctone (µm) CFTR + RTP1 δ-undeclctone (µm) MOR23 + CFTR + RTP1 + Gαolf + Ric-8B MOR23 + CFTR + RTP1 + Gαolf MOR23 + CFTR + RTP1 + Ric-8B MOR23 + CFTR + RTP1 MOR23 + CFTR Lyrl ( µm) + CFTR + RTP1 + Ric-8B Lyrl (µm) CFTR + RTP1 + Gαolf Lyrl (µm) CFTR + RTP1 + Ric-8B δ-undeclctone (µm) CFTR + RTP1 + Gαolf δ-undeclctone (µm) d Olfr288 + CFTR + RTP1 + Gαolf + Ric-8B Olfr288 + CFTR + RTP1 + Gαolf Olfr288 + CFTR + RTP1 + Ric-8B Olfr288 + CFTR + RTP1 Olfr288 + CFTR δ-undeclctone (µm) 1 e + CFTR + RTP1 + Ric-8B + Gαolf 2. µa 1 min Lyrl (µm) µa + CFTR + RTP1 + Ric-8B + Gαolf δ-undeclctone (µm) min OR RTP1 Adenylyl cyclse Gαolf ATP camp PKA Cl - P CFTR Supplementry Figure 2. Co-expression of RTP1 nd Gαolf gve the most efficient odornt responses in Xenopus oocytes. (, ) Representtive response profiles of Xenopus oocytes expressing MOR23 or Olfr288 with different comintions of RTP1, Ric-8B, nd Gαolf. A different concentrtion of the synthetic lignd for MOR23 (lyrl) or Olfr288 (δ-undeclctone) ws pplied t ech of the times indicted y rrowheds. (c, d) Dose-response curves of OR-expressing oocytes ginst incresing concentrtion of lignd under the conditions descried in Supplementry Figure 2 nd 2 (n=3 5). The verticl rs represent ± s.e.m. The response mplitude ws smller when oocytes expressed OR nd CFTR ( ) thn when oocytes co-expressed with RTP1 ( ). The ddition of Ric-8B incresed the mplitude slightly (). Oocytes expressing OR, RTP1, Gαolf nd CFTR showed the est OR-medited odornt responses ( ). Finlly, the ddition of Ric-8B to this comintion did not result in further enhncement ( ). (e) A schemtic digrm of the moleculr mechnisms involved in this ssy systems. The OR is efficiently trnslocted to the cell surfce y RTP1, nd n ctivted OR efficiently couples to Gαolf, resulting in n increse in intrcellulr camp level tht cuses Protein kinse A (PKA) ctivtion. CFTR is then phosphorylted y PKA, leding to Cl - efflux nd electricl responses.

4 MOR31-2 Vleric cid (µm) µa n= Vleric cid (µm) I7 Octnl (µm) µa n= Octnl (µm) c mor-eg Eugenol (µm) µa Normlized response (% of 1 µm response) Eugenol (n=3) Vnillin (n=4) Lignd (µm) d M71 Acetophenone (µm) µa 1 min Acetophenone (n=3) Benzldehyde (n=4) Lignd (µm) Supplementry Figure 3. Functionl expression of mouse ORs in Xenopus oocytes. Oocyte responses to the synthetic lignds were mesured under conditions in which n OR ws co-expressed with RTP1, Gαolf, nd CFTR. Representtive current trces of OR-expressing oocytes to ech synthetic lignd nd dose-response curves re shown. The verticl rs represent ± s.e.m..

5 (2) (3) (4) (5) c C4 O O O O O O O O C5 C6 C7 (6) (7) O O O O C8 C1 (6) (8) (9) (5) (1) O O O O O O O O C8 C8 C8 C7 C7 5. (2) (3) (4) (5) (6) (7) (6) (8) (9) (5) (1) C chin length: C4 C5 C6 C7 C8 C1 ring: δ γ ester group: d (11) (1) (12) e 7 4 (13) OH 5 (14) 11 OH OH OH 6 OH (15) OH 7 (13) (16) OH 7 O O 5. (11) (1) (12) (13) (14) (15) doule ond: Z4 Z5 Z6 Z7 Z11 sturted 5. (13) (16) functionl group: -OH -OAc Supplementry Figure 4. The moleculr receptive rnge of Olfr288. A series of odornts ws selected s potentil gonists for Olfr288 sed on the chemicl structure of δ-undeclctone (5), (-c) nd Z5-14:OH (1), (d, e). Olfr288-expressing Xenopus oocytes were stimulted with ech odornt t 3 µm nd men response mplitudes were shown in grphs (± s.e.m., n=3). () Olfr288 ws responsive to lctones tht hd cron chin length more thn C6 (4)~(7), ut not to those with chin lengths shorter thn C5 (2)~(3). () A lctone with 5-memered ring (8) showed nerly identicl ctivity to tht with 6-memered ring (6). In contrst, n ester compound (9) tht hs similr structure to the γ-lctone (8), ut does not hve closed ring structure showed less ctivity. (c) A lctone-like compound with no ester group (1) ws inctive. (d) Olfr288 recognizes tetrdecen-1-ols with single doule ond t different positions ((Z)-4-, (Z)-5-, (Z)-6-, (Z)-7- nd (Z)-11-tetrdecen-1-ol: (1), (11)~(14), ut not corresponding sturted lcohol, tetrdecn-1-ol (15). (e) An cette derivtive of tetrdecen-1-ol (16) hd no ctivity.

6 mor-eg ELG SLG SMG PG HG Eugenol Eugenol PPG MG 1. µa MOR23 ELG SLG SMG PG HG Lyrl Lyrl PPG MG c I7 ELG SLG SMG PG HG Octnl Octnl PPG MG.2 µa 1. µa d M71 ELG SLG SMG PG HG AP PPG MG AP e MOR31-2 ELG SLG SMG PG HG VA PPGMG VA.2 µa 1. µa f.5.25 M71 g 2. MOR PPG MG PPG MG ELG SLG SMG PG HG ELG SLG SMG PG HG Supplementry Figure 5. Responses of OR-expressing oocytes to exocrine glnd extrcts. ( e) Representtive current trces from Xenopus oocytes expressing n OR, RTP1, Gαolf, nd CFTR. A series of extrcts of EGs were pplied to cells t the time indicted y rrowheds. Ech extrct contined 1-mg-tissue equivlent in 1 µl of ssy uffer. Synthetic odornts were used s positive controls t the eginning nd the end of the extrct series: eugenol ws used for mor-eg; lyrl for MOR23; octnl for I7; cetophenone (AP) for M71; vleric cid (VA) for MOR31-2. (f-g) Summry of response mplitude (µa) of OR-expressing oocytes tht responded to extrcts. Dt shown re the men of experiments (M71; n=3, MOR31-2; n=6).

7 Ultr-filtrtion M.W. 3 D. >3 UL Size (M.W.) PPG >3 <3 Extrct UL Preputil glnds (PPG) Methnol Extrction Seprtory funnels <3 EtOAC 1. µa UL EtOAc Phse Wter <3 UL <3 frction Wter 1. µa c <3 frction ODS regin Sep-Pk ODS Methnol solution MeOH (%) UL <3 UL 1. µa Methnol: 1 % 5 % 9 % Supplementry Figure 6. Chemicl properties of ctive component(s) in the PPG extrct. () Size frctiontion of the PPG methnol extrcts y ultr-filtrtion t moleculr-weight cutoff of 3, D. (i.e., >3, nd <3, frctions). The flow-through <3, frction elicited response from Olfr288-expressing oocytes, indicting tht the ctive compound(s) hd moleculr weight lower thn 3, D.. () Liquid-liquid extrction (i.e., ethyl cette (EtOAc) nd wter phses) of the <3, frction. The resulting ethyl cette lyer contined the ctivity. This result indicted tht the ctive compound(s) ws reltively lipophilic. (c) Frctiontion of the <3, frction on crtridge column with ODS resin tht dsors compounds vi hydrophoic interctions. Of the three distinct frctions (elutes in 1, 5, or 9% methnol solution), only the elute in 9%-methnol solution contined the ctive compound(s).

8 Intensity (µv) c e g ( x1) Frction E Z5-14:OH Z6-14:OH Z7-14:OH Z8-14:OH Z9-14:OH Z11-14:OH Retention time (min) (Z)-5-tetrdecen-1-ol (Z5-14:OH) (Z)-6-tetrdecen-1-ol (Z6-14:OH) (Z)-7-tetrdecen-1-ol (Z7-14:OH) (Z)-8-tetrdecen-1-ol (Z8-14:OH) (Z)-9-tetrdecen-1-ol (Z9-14:OH) (Z)-11-tetrdecen-1-ol (Z11-14:OH) Frction E d f h Supplementry Figure 7. GC-MS nlysis of uthentic tetrdecen-1-ols. () GC chromtogrms from the ctive frction (Frction E) nd uthentic tetrdecen-1-ols, ech of which hs doule ond t different position. Smples were loded onto n Rxi-1ms column, nd the elutes were detected using frme ioniztion detector. ( h) Mss spectr of purified nd uthentic tetrdecen-1-ols. First, the mss spectrum of frction E ws identified y compring it to ll the cndidtes in the NIST lirry dtse to determine the est mtch with the highest spectrl similrity. As result, the mss spectr of frction E ws 93% similr to tht of (Z)-7-tetrdecen-1-ol in the NIST lirry dtse. However, the retention time nd mss spectrum of uthentic (Z)-7-tetrdecen-1-ol differed from those of frction E. Other tetrdecen-1-ol compounds which ech hd doule ond t position 6, 8, 9, or 11, respectively, were tested, ut none hd retention time tht perfectly mtched tht of frction E. The retention time of (Z)-8-tetrdecen-1-ol ws nerly identicl to tht of frction E, ut slight differences (<.4 s in retention time) were oserved. To determine doule ond position precisely, we performed dimethyl disulfide derivtiztion of frction E (Supplementry Figure 8).

9 Intensity c d (x1 6 ) Frction E - DMDS Z5-14:OH - DMDS Frction E Frction E - DMDS 2.2 Solvent Retention time (min) CH3S M + = 36 OH Z7-14:OH - DMDS CH3S 173 (x1 6 ) 7 M + = 36 OH SCH3 161 SCH Temp. ( o C) Supplementry Figure 8. Dimethyl disulfide (DMDS) derivtiztion of frction E nd uthentic tetrdecen-1-ols. () TICs for frction E (lck), the control rection solvent (green), nd the derivtiztion product of frction E (mgent). Blck nd mgent rrowheds indicte tetrdecen-1-ol in frction E nd pek specificlly oserved in the product (lso shown in inset figure), respectively. () A mss spectrum of the derivtiztion product of frction E (mgent rrowhed in Supplementry Figure 8). This mss spectrum showed moleculr ion t 36 corresponding to the DMDS derivtive of tetrdecen-1-ol nd two intense frgment ions t 133 nd 173. This frgmenttion pttern corresponded to tetrdecen-1-ol with one doule-ond etween C5 nd C6. (c) A mss spectrum of the DMDS derivtive of uthentic (Z)-5-tetrdecen-1-ol (Z5-14:OH). (d) A mss spectrum of the DMDS derivtive of uthentic (Z)-7-tetrdecen-1-ol (Z7-14:OH).

10 M71 AP Z5-14:OH (µm) AP MOR31-2 Z5-14:OH (µm) VA VA 1. µa.5 µa Supplementry Figure 9. Z5-14:OH dose not ctivte M71 or MOR31-2. A representtive trce of Xenopus oocytes expressing M71 or MOR31-2 on stimultion with Z5-14:OH. 1 µm of cetophenone (AP) or vleric cid (VA) were used s positive control. These finding indicted tht the ctive compound(s) in the PPG extrcts tht ctivted MOR31-2 nd M71 (in Supplementry Figure 5) ws not Z5-14:OH.

11 12 week Z5-14:OH Supplementry Figure 1. Mss spectrum corresponding to the pek tht represent Z5-14:OH in the voltile constituents of dult mle urine, relted to Figure 3. (left) The mss spectrum from dult mle urine. (right) Mss spectrum from uthentic Z5-14:OH.

12 Intensity ( 1 7 ) Z5-14:OH (µm) Retention time (min) Pek re ( 194) ( 1 3 ) stndrd mle urine y=1327x-7.9 R 2 = Z5-14:OH (µm) Supplementry Figure 11. Quntittive nlysis of urinry Z5-14:OH y GC-MS. We used GC-MS nlysis to quntify the Z5-14:OH concentrtion in mle mouse urine. Urine from PPG-ectomized mles ws spiked with vrious concentrtions of uthentic Z5-14:OH. The hedspce urinry voltiles were dsored using SPME nd nlyzed using GC-MS. TIC peks corresponding to Z5-14:OH were shown in left figure. From this TIC, the extrcted ion chromtogrms for 194 were nlyzed, nd pek res corresponding to Z5-14:OH were clculted. The re of ech extrcted ion chromtogrm for 194 tht corresponded to the exogenous Z5-14:OH in the urine smples ws linerly correlted with concentrtion of the dded Z5-14:OH (right; lck squre, n=3), nd the detection limit ws pproximtely.1 µm. Bsed on this stndrd curve, we clculted the concentrtion of Z5-14:OH in urine smples. The vlues from five dult mle mouse specimens re shown on the stndrd curve (mgent circle). The verge concentrtion of Z5-14:OH in urine smples from five dult mle mice ws.37± 3 µm.

Shm + oil Mle Cstrted + oil Cstrted + testosterone c + Oil Femle + Testosterone Z5-14:OH (µm).75.5.25 Mle Shm + oil N.D. Cstrted + oil d Z5-14:OH (µm) 2. 1.5 1. Femle.5 N.D. Cstrted + Oil + Testosterone + testosterone Supplementry Figure 12.

13 Shm + oil Mle Cstrted + oil Cstrted + testosterone c + Oil Femle + Testosterone Z5-14:OH (µm) Mle Shm + oil N.D. Cstrted + oil d Z5-14:OH (µm) Femle.5 N.D. Cstrted + Oil + Testosterone + testosterone Supplementry Figure 12. Z5-14:OH is testosterone-dependent urinry voltile. () PPGs from mle mice re shown; (left) shm-operted, (center) cstrted, (right) cstrted with susequent testosterone tretment. Testosterone ws injected dily for 2 weeks. Scle rs, 1 mm. PPGs weighed less in cstrted mle mice thn in shm-operted controls. The differences in PPG size etween cstrted mice nd shm-operted mice were eliminted when cstrted mles were given testosterone for two weeks. () The concentrtions of Z5-14:OH in mle mouse urine under severl conditions s indicted (n=6). Quntifiction ws performed using the stndrd curve shown in Supplementry Figure 11. (c) PPGs from femle mice with or without 3-week testosterone tretment. Scle rs, 5 mm. (d) The concentrtions of urinry Z5-14:OH in femle mouse (n=5). Administrtion of testosterone to femle mice drmticlly incresed the weight of the PPG nd led to detectle levels of urinry Z5-14:OH. The verticl rs represent ± s.e.m. N.D.: not detected.

OB Brin OE c Dorsl Medil Nsl cvity.1 mv.1 sec d Numer of ctivted glomeruli 1 8 6 4 2 Supplementry Figure 13.

14 OB Brin OE c Dorsl Medil Nsl cvity.1 mv.1 sec d Numer of ctivted glomeruli Supplementry Figure 13. Z5-14:OH induces electricl responses in the olfctory epithelium nd ctivtes severl glomeruli in the nteroventrl OB. () Schemtic illustrtion of the mouse min olfctory system. In the olfctory epithelium (OE), ctivtion of ORs leds to depolriztion of olfctory sensory neurons (OSNs). Ech OSN expresses only one type of functionl OR, nd xons of OSNs expressing the sme OR converge onto typiclly two glomeruli in one side of the olfctory ul (OB). () A representtive electroolfctogrm (EOG) response of the mouse olfctory epithelium (OE) to vpor phse of Z5-14:OH. (c) Representtive c-fos immunohistochemistry in the OB. c-fos, n immedite erly gene product, ws used s n indictor of glomerulr ctivtion to exmine responses to Z5-14:OH in the OB. (left) An OB section of mice stimulted with Z5-14:OH. (middle) An enlrged view of the region indicted y the dshed squre in the left figure. Two rrowheds indicte ctivted glomeruli surrounded y c-fos-positive juxtglomerulr cells. (right) An OB section from n unstimulted mouse. Scle rs, 2 µm. (d) The numer of ctivted glomeruli ginst Z5-14:OH stimultion. Activted glomeruli were counted from one side of the OB from ech of seven nimls. Expression of c-fos ws induced in juxtglomerulr cells of 4-7 glomeruli which generlly resided within the nteroventrl OB. No c-fos induction ws oserved in the OB of mice without odor stimultion. These results indicted tht Z5-14:OH is detected y the mouse olfctory system through few different ORs.

15 c d time (sec) Investigtion ** 1 * 75 Intct Cst. Investigtion time (sec) 5 25 Z5-14:OH Vehicle Investigtion time (sec) C14:OH Vehicle Investigtion time (sec) Z5-14:OH Wter Cst. urine + Supplementry Figure 14. Z5-14:OH enhnces urine ttrctiveness to femle mice. Rw dt of the odor preference test shown in Figure 3e-f. Two dots connected with line shown in grphs indicte tht mount of time tht mouse spent investigting ech test smple within period. () Urine from cstrted mles vs urine from intct mles (n=2). () Cstrted mle urine vs cstrted mle urine spiked with Z5-14:OH (1. µm; n=2). (c) Cstrted mle urine vs cstrted mle urine spiked with C14:OH (1. µm; n=18). (d) Wter vs wter spiked with Z5-14:OH (1. µm; n=16). Asterisks indicte significnt difference etween two smples (Pired t-test, *P<5, **P<1).

16 Supplementry Tle 1. Effects of cstrtion nd testosterone tretment on ody weight, PPG weight, nd Z5-14:OH concentrtion in urine. Prmeters Mle Femle Shm+oil Cstrted+oil Cst. +testosterone +Oil +Testosterone Body weight (g) 22.98± ± ± ± ±.16 PPG wight (mg) 92.2± ± ± ± ±2.2 Z5-14:OH (μm).42±7 <.1.37±3 < ±.2 Ech vlue represents ± S.E. of six specimens from mles or five from femles. 1

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:1.138/nture1794 BR EPFs BRI1? ERECTA TMM BSKs YDA PP2A BSU1 BIN2 pbzr1/2 BZR1/2 MKK4/5/7/9 MPK3/6 SPCH Cell growth Stomtl production Supplementry Figure 1. The model of BR nd stomtl signling pthwys.