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Structural and functional correlates of a mutation in the malignant hyperthermia-susceptible pig ryanodine receptor
Volume 301, number I. 49-53 FEI)S 10915 8 1992 Fcdcration or Europctin Biochsmicul Socktics 00115793/92/%5.00
April
1992
Structural and functional correlates of a mutation in the malignant hyperthermia-susceptible pig ryanodine receptor
Received
18 January
1992; revised
version received 24 February
1992
The rkclutel muscle ryunodinc rcccptor or milligntint hypcrthcnniu-sus~~ptiblc (MHS) pigs contains a mutation ut r&due 615 that is highly corrclutcd with various abnormalities in the rcgulution ol’sarcoplasmic reticulum (SRI Cu?’ ch;mncl uctivhy. In isolutcul SR membranes the Argaf3 to Cys”‘” rynnodinc rcccptor mutuiion is now shown to bc directly rcsponsiblc Ibr an altcrcd cryptic pcptidc map. due to the elimination of the Arp”” clcuv;~gc site, Furthsrmorc. trypsin tre;nmcnt rclcuscd 86-99 kDu ryunodinc rcccptor fragmsnts encompassing residue 615 from the SR nxmbrunes. WC conclu~Ic hi! the Y6-99 kD;I domain conttiining rcsiduc! 615 is near the cytoplasmie surfax of the ryanodinc receptor and likely near inlporlunl CG‘ rhitnncl regulatory sites. S;lrcopl;lsmic
1,
rciiculum:
Rynnodinc
rcccptor:
Maligntint
INTRODUCTION
The skeletal muscle sarcoplasmic reticulum (SR) ryanodine reccptor/Ca?’ channel is the pathway by which stored Ca2* is relcascd into the sarcoplasm during excitation-contraction coupling [I]. This homotetrameric complex of 565 kDa subunits, seen as a quatrcfbil structure in electron micrographs [I], has been sequenced from the cDNA [2]. Many reports of alterations in the activity of the porcine skeletal muscle ryanodine receptor (e,g. [3-61) have made it the most likely candidate gene for conferring susceptibility to the tnetabolic disorder malignant hypcrthermia (MH). In support of this hypothesis Fujii et al. [7] have recently identified a nucleotide base mutation (resulting in the replacement of Arg”” with Cys”‘“) in the ryanodine receptor cDNA from MH-susceptible (MHS) pigs. Thus, this natural mutation may be utilized to gain further insight inio the structure and function of a channel critical to the regulation of muscle contraction, In this report we have identified 86 and 99 kDa tryptic fragments of the normal and MHS pig ryanodine receptor that contain residue 615. Our results indicate that this domain is near both the surface of the protein and important channel regulatory sites.
Cor~c,sl,orlil~~~rlP&/w,ss: J.R. Mickclson, Dcpurtmcnt or Veterinary PathoBiology. University or Minncsotu. 295 An. Sci./Vct. Med.. St, Paul. MN 55108. USA. Fax: (I) (612) 625 0204.
hypcrthcrmin;
Mutation:
Calcium
rclcasc channel
2. MATERIALS AND METHODS Pictruin pigs homoeygous for the MH gene [S]. Yorkshire and Minnesota no. I pigs homozygous for the normal ycnc [8.9]. and Near-Pictrdin pigs derived from more than 4 generations ofbnckcross. ing hulothanc-n~g;rtivc pigs to Pictrains [9]. wcrc obtuincd from the closed herd muintuincd at the University of Minnesota Exprimcntal Farm. MH susceptibility wadctcrmincd bycxposurc to 3% halothanc (in 97% oxygen) by mask. with a positive test rcsponsc indicated by limb muscle rigidity in Icss thun 4 min. Trypsin trcptmcnt or hcuvy SR mcmbrana from MHS and normt-d pigs wus ut 37°C for IO min in a medium conttiining I M sucrose. 20 nrM Tris-HCI. pH 7.0. I mg/SR protcinlml. .nnd thr indiuttcd trypsin conccnmtion [IO]. SR protcins were clcctrophorcscd on 3-1246 gradicm polyucrylamidc gels and analyzed on immunoblots with shop anti-ryanodinc receptor antisera and horerddish pcroxidug conjugilted rabbit anti-sheep I@ sccondury untiboditi [IO]. Sheep antibod. its spLxific ror the MHS and normal ryanodinc rcccptor tryplic fragmcnts wcrc uftinity-purified otT of Immobilon strips [I I ] containing the indicotcd ryunodinc rcccptor tryptic fragments. Anti-ryanodinc ruccptor antibody beads wrrc prcparcd by coupling sheep antibodies (allinity-purified from a rabbit ryanodinc rcccptor column) to CNBr-uctivuted Sepharox 46. For immunoprccipitution orthc 86 kDti ryunodinc rcccplor friigmcnt. trypsin truatcd SR mcmbranes were solubilired in the prcscncc of I M NaCI. 2% CHAPS. 1% phosphatidylcholine. SO mM Tris-HCI. pH 7.4. and a proteass inhibitor cocktuil. The sc?lublc extract was then added to an aliquot of unti-ryanodinu receptor immunobadds nnd incubated at 4’C for 4 h. Spcriticdlly bound ryanodinc rcccptor fragments wurc ululd from the immunobuads by addition or clcctrophorcuis butTcr containing 5% 2.murcnptocthanol and 2% SDS. The clutcd protein was l?nctioniad 011 polyacrylamidc gels ;md iI*:: Nexrminal squcncc obtained from trims& onlo lmmobilon mcnlbrdnes [II]. Conditions For the polymrrvsc chain reaction amplificaion of s 74 bp ryanodine receptor scquencc from porcine gcnomic DNA were as described by Fujii ct nl. [7]. Squcnrc of the PCR product was dctcrmined by the didcoxy chain termination method 1131.l’ollowing bluntend ligarion into the S/WI site of pWucscrip1. Dot blot hybridization
49
Volume
301, number
FEBS WETTERS
I
of “I’-lnbrid
oligonucleotidc probes to the PCR producls WIS iIlso ns described by Fujii CLaI. [7], with 5’.TGGCCGTGCGCTCCAAC-3’ for the C 1843 ;IllOk and S’-GGCCGTGTGCTCCAA-3’ for the T I813 ullclu.
3. RESULTS The immunoblot shown in Fig. 1A indicates the presence of the high M,. ryanodine receptor in the korcinc heavy SR preparations prior to trypsin treatment. In addition ro the intact ryanodinc receptor two lower M, components were also observed (Fig. IA). likely due to a partial proteolysis of this protein during SR mem-
A
6
I234
C
I 2
3 4
S
6
7 6
8 IO II 12
MNMNN
99 66
1234
6678
9lOlll2
Fig. I. Tryptic immunopcptide maps of the MHS and normal SR ryunodine receptor. i-icavy SR vcsiclcs from 3 different MHS and Z difkrcnt normal pigs were incubaicd in the ubscncc of trypsin (A) or in the presence of VSII./OUStrypsin conccntraiions (B) for IO min at 37°C. Panel 6: luncs I-4. trypsin 10 SR wcighl ratio of l:QO: lanes S-8, trypsin to SR ratio of 1:?40; lanes 9-12. lrypsin IO SR ratio of I: 160. Pancl C: I MHS and I normal SR preparation wcrc trcatcd with trypsin (1:?40). alkr which they wcrc diluted wiih AI equal volume of 0.3 M sucrose und centrifuged at 150,000 x g for 30 min. Sumplcs 01 trypsin-trcatcd SR (lanes l-4), und the rcsukrnt supcrntamt (lanes S-8) and pellets (Iiines 9-l?) were analyzed, Lana 1.2.5.6.9.10 IO min trypsin incubation: lanes 3,4.7.X.1 l,l:! - 5 min trypsin incubnlion. All samples were unalyzcd for ryanodinc rcccp~or immunorcxtivc fragments as described in section 2. The positions of molccul:~~ weight markers arc indicaicd 011 the lcli, and the posiiions of the 93 ;lJd 86 kDu ryanodinc rcccptor fragmcms arc indicutcd by the sin& arrowheads.
50
April
1992
brane preparation. Fig. I B (lanes l-4) shows an immunoblot of trypsin-treated SR membranes, where ryanodine receptor doublet fragments of M,. approximately 99 kDti were detected in MHS SR. while normal SR contained ryunodinc receptor doublet fragments of both 99 and 86 kDa. The figure also shows that the use of higher trypsin concentrations resulted in the loss of these components (lanes S-12). Following centrifugation of trypsin-digested MHS and normal SR for 30 min at 150,000 x g, the 99 and 86 kDa ryanodine receptor fragments were detected in the resulting supcrnatant (Fig. IC, lanes 5-8) and absent from the pellet (lanes 9-12). The 99 and 56 kDa ryanodinc receptor fragments could not be differentiated from the numerous other SR protein fragments on Coomassie blue-stained gels (not shown). When purified ryanodinc receptor preparations (isolated by the method of Lai et al. [I]) were treated with trypsin we were unable to detect the 86 and 99 kDa fragments on immunoblots, although numerous other fragments were detected (not shown). Affinity-purified antibodies to the 99 and 86 kDa ryanodinc receptor fragments were prepared and utilized to st;h irnmunoblo~s. Fig. 2C shows that antibodies to the normal 99 kDa tryptic fragments recognized the 86 kDa normal fragments. as well as the 99 kDa MHS fragments. Similarly, antibodies to the 86 kDa normal fragments recognized both the 99 kDa normal and the 99 kDa MHS fragments (Fig. 2D), ;md antibodies to the 99 kDa MHS fragments recognized both the 99 and 86 kDa normal fragments (Fig. 2E). Thus, the 86 and 99 kDa tryptic fragments appear to arise from the same domain of the ryanodine receptor. Antibody beads were used to immunoprecipitate the 86 kDa normal ryanodinc receptor fragment from trypsin-treated normal SR. The major immunoprecipitated component detected on Coomassie blue-stained gels had a M,. of 86 kDa. and was recognized on immunoblots by the anti-ryanodine receptor antibody (data not shown). The N-terminal sequence of this fragment indicated significant homology to residues 617-627 01 the rabbit ryanodine receptor (Fig. 3A), just downstream from a potzn!ir:l trypsin cleavage site at Arg”“. DNA corresponding to this region of the porcine ryanodine receptor gene wits then amplified by the polymerase chain reaction and sequenced (Fig. 3B). The nucleotide sequences bctwecn the PCR primers were identical to those determined by Fujii et al. [7], with a C in position 1843 of the normal Yorskhirc DNA and a T in position 1843 of the MHS Pietrain DNA, translating into the At@’ to Cys”” mutation. Dot blots of the 74 bp porcine DNA PCR product were hybridized to either the Cl 843 (normal) or Tl843 (MHS) ryanodine receptor oligonucleotide probes (Fig. 4). This confirmed that all pigs from the MHS Pietrain herd utilized for our previous studies of ryanodinc receptor alterations [5,6,9,10], contained only the T1843 allele, while the normal Yorkshire and Minnesota no.
Volume 301, number 1
April 1992
FEBS LETTERS
D Ml-l N MH N
MH
N
E
Ml-l N
Ml-l N MHN
Mr
228
I IO
99 86
70
t
44
Fig. 2. Immunoblol analysis of the 99 and 8G kDs rynnodinc receptor tryptic fragments. MHS and normal heavy SR membranes were incubated in the ;ibscncc (A) or presence of trypsin ut it I:320 weight ratio for IO min (B-E). Antibodies utilized to slain the transfers were either the sheep ;mti.ryanodil:c rcccptor antiserum (A and B), or sheep antibodies that hud been affinity purified from immunoblotsof the ryanodinc rL=ptor uyptic Itagments: (C) anti-normal 99 kD:l antibodi& (D). anti-normal 86 kDa antibodies: (E). anti-MHS 99 kDa amibodics.
I herds conrained the Cl843 ullelc (Fig. 4A). We also examined families of pigs in which the oligonucleotide
hybridization results could be correlated with the results of the halothane-challenge test for MH susceptibility
(A) 615 Arg Set
Rabbit
Pig
86 kD Fragment
X
620 Asn Gln Asp Lou lie Asn Gln Asp
X
lie
625 Thr Glu Asn Leu Leu Prc X
Glu Asn Leu Leu Pro
(B) 1843 :Ek8
AAT GGT G
Normal Ml-is
ATT ACT GAG 1869
B TG GCC GTG CGC TCC AAC CAA GAT CTC Val Ala Val Arg Ser Am Gln Asp Leu TG GCC GTG TGC TCC AAC CAA GAT CTC Val Ala Val Cys Ser Am Gln Asp Leu 615
620
Fig. 3. N-Tcrminul sequcncc of the 86 kDa ryanodinc rcccptor tryptic fragment and DNA scqucncc of the ryanodinc rcccplor PCR product. (A) Normd SR was incubutcd with trypsin (I: 160 for IO min at 37”C), the 86 kDa normal ryanodinc rcceplor fragmcnl was immunoprccipitatcd. and 7 Amino acid rcsiducs whichcould not bc dctcrmincd the N-terminal scqtmce wus dctcrmined from an Immobilon transfer us dcscribcd in section _. are rcprcscntcd with an X. Scquencc and numbering scbcmc of the rabbit skulctul muscle ryanodinc rcccl%or [7] is provided. iBi MHS Pictrain and normal Yorkshire gcnomic DNA vxrc utilized for the production of the 74 bp ryanodine rcccplor DNA scgmcnt as dcscribcd in s&lion 2. Thusc PCR products wcrc ligated into pBlucscript and scqucnccd. Partial scquencc of the PCR primers (covcrcd with solid lines) and the ryanodinc rcccptor cDNA scque~w nuclcotidc numbcling system detcrmincd by FLjii CI al. [7] arc provided.
51
FEB.5 LETTERS
Volume 301. number 1 Pielmin
YorkWe 34
12 A
5
6
7
Minn. ryl 6
9
IO
II
I2
tf843 Cl843
6
Cl843
l
*
-1
”
l Orn!O
8
00
---++-+-2
3
4
5
6
7
8
9
IO
II
Fig. 4. Dot blot oliyonuclcotidc hybridization anslysis or the 74 bp ryanodinc receptor PCR product. (A) Four dilkrcnt MHS Pictrnin. normal Yorkshire. and normal Minnesota no. I pigs wcrc nnalyzcd, (B) Near-Pietrain p;urnis;tnJ their progeny. Thr figure rcprcscnt the autoradiogrum that rcsultcd I’rom hybridiz;llion to the ‘2P-l;lbclcd Cl843 ;md Tl843 oligcnuclcotidss as dcscribcd in section 1. The response 0T CilCll pig to lhc h;ilotll;inc-chiill~i~~~ ltsl 1’or MI-I is :llso given. with + indicating ;I positive lulo~i~a~~c rcsponsc ond - indicating il nc_u;~tivt huloiliiinc rceponse.
(Fig. 46). In this example the Ilalotlli~ne-negati\le NcnrPietrain parents had copies of each ryanodine receptor allele (i.e. were heterozygotes) and the progeny of this mating demonstrated all three possible genotypes. Interestingly, one heterozygotc pig (individual no. 6) reacted positively in the halothunc-challenge test. That all homozygotcs for the Tl843 allele were MHS (in agreement with Fujii et al. [7]), while heterozyyotes based on the oligonuclcotide hybridization assay could be either positive or negative in the halotha~ic-ch~~IIe~7gP test (Fig, 4B), has been a consistent finding in the 8 Near Pietrain pedigrees examined to date. This appears to indicate ambiguities associated with applying the in vivo challenge test for the identification of hctcrozygates. 4. DISCUSSION This study confirms our previous observation of the generation of 99 kDa MHS and 86 kDa normal porcine SR ryanodine receptor tryplic fragments [IO], and demonstrates that a normal 99 kDa ryanodine receptor fragment can also be detected (Fig. 16). Affinity- purified antibodies to each of thcsc fragments recognized the other two (Fig. 21, indicating that these three fragments were all derived from the same domain of the ryanodine receptor, The 86 kDu normal ryanodine receptor tryptic fragment had residue 616 as its N !erminus (Fig. 3A) and the DNA sequences of the MHS and normai ryanodinc receptors in this region confirmed the ArghlS to Cysh15mutation described by Fujii ct al. [7] (Fig. 3B). These data thus indicate that the Arg”15to Cys mutation in the MHS pig ryanodinc rcccptor climinutes
April 1992
an accessible trypsin cleavage site at residue 615, such that the normal 86 kDa fragment cannot be formed. That these 99 and 86 kDa fragments were not observed when purified MHS and normal ryanodine receptors were subjected to proteolysis may indicate an altered conformation of this molecule in the detcrgent-solubiliced purified state. Our results suggest that Arg”” may be on the surface of the native ryanodine receptor molecule, as it is accessible to trypsin. The 86-99 kDa tryptic fragments are released from the SR (Fig. lC), indicating that they are derived from the cytoplasmic foot region [I .2]. Arg”15 may also be located near important regulatory sites, as the M HS and normal porcine ryanodine receptors demonstrate differences in Ca?’ regulation of Ca’+ release [3-51. channel gating [B], and [fH]ryanodine binding activity [5,9]. To date none of the various ligand binding sites (e.g. Ca’+, Mg?“. ATP. calmodulin [I]) which regulate this channel have been identified. Further study of the MHS porcine ryunodine receptor mutation should help define structure/function relationships within this Ca” channel protein. .I~~k/,trll~l~~~/~c~rrrc,r/.~: The authors wish to hunk Dr. Clivc Sl;myhtcr 01 the Howard Huphss Mcdicul loslitutc. University or TL’XIS Southwcstcrn Medical Center, Ibr protein-suqucnciii~ cxpcrtisc. This work was supported by pr;lnts rrom 111~’Muscular Dystrophy Association ol’Amcric;i ;II~ the Niltiunill Pork Producers COUIIC~I (lo J.R.M. und C.F.L.). NIH GM 31382 (to C.F.L.). ;mrl by the University or.Minncsotil Agricultural Experiment Stiltion (C.F,L. und W.E,R,), K.P.C. is UII Invcstiyutor ol’ The Ho\vurd Hughes Mcdicul Institute.
REFERENCES [I] Lai. F.A.. Erickson. H.P.. Roussc;n~. E.. Liu. Q.-Y. and Mcissncr. G. (19Y8) NilLure 331. 315-319. [?I T;lkcshimil. H,. Nishinnux. S.. bkllSl~lllUlO. T.. Ishkh. l-l.. KillI~;Iw;I. K.. Miniunino. N.. Miltsuoa Ii.. Ucdti. M.. H:III;IO~;I. M.. Hirosc. T. iInd Numa. S. (1989) Nmurc 339. 439-445, 1.11Ndson. T.E. (1983) J. Clin. Invest. 72. 862-870. [A] Kim. D.H,, Srekr. F.A.. Ohnishi. ST.. Ryan. J.F.. Roberts. J., Allen. P.D.. Mcszlros. LG.. Antoniu. 13.and Ikcmolo. N, (19S4) Biochim. Biophys. Actu 775. 320-327, [5] Mickclson. J.11.. Gullilnt. E.M.. Liticrcr, L.A., !ohson, K.M., Rcmpcl, W.E, and Louis, C.F. (19%) J. Biol. Chcni. 263. 93lO9315, [O] Fill. M.. Coronudo. R.. Mickclson. J.R.. Vilvcn. J.. Mu. J.. J;icobson. B./z, and Louis, CF. i 1990) Biophys. J, 50. 471--175. F.. DcLcdn. S,. Khannu. V.K.. 171 Fujii. J.. Otsu. K.. Zorznto. Wcilcr. J,E.. O’Brien. P,J. and MucLcnnan. D.H. ( 1991) Science 253. 448-45 I. 181 Rcik, T.R.. Rcmpcl. W.E.. McCir;~th. C.J. und Addis. P.B. ( 1983) J. An. Sci. 57, X26-831, W.E.. Johnson. K.M.. [91 Mickclso~~.J.R.. GulliLnt. E.M.. Run@. Littcrcr. L,A,, Jitcobson. B.A. illId Louis. C,F. (1989) At11. J. Physiul. 257. C7X7-C794. J-R.. Louis. C.F. and Cumpbsll, [lOI Ktmdson, C %I.. Mirkelson, K.P. (1990) J. Biol. Chcm. 265. 2421-2414. D. ( 1998). in: Antibodies, A I.uhor;lt~ry [l II Hurlow, E. and Lax, M;mual. Cold Spring Hurbor Luborutory. p. 498. Matsuduira. P. (1987) J. Biol. Chum. 262. 10035-10038. ii:; Tubor. S. i\nd RIchardsol:, CC. (1989) J. Biol. Chem. X4,6447M58.
April
1992
Structural and functional correlates of a mutation in the malignant hyperthermia-susceptible pig ryanodine receptor
Received
18 January
1992; revised
version received 24 February
1992
The rkclutel muscle ryunodinc rcccptor or milligntint hypcrthcnniu-sus~~ptiblc (MHS) pigs contains a mutation ut r&due 615 that is highly corrclutcd with various abnormalities in the rcgulution ol’sarcoplasmic reticulum (SRI Cu?’ ch;mncl uctivhy. In isolutcul SR membranes the Argaf3 to Cys”‘” rynnodinc rcccptor mutuiion is now shown to bc directly rcsponsiblc Ibr an altcrcd cryptic pcptidc map. due to the elimination of the Arp”” clcuv;~gc site, Furthsrmorc. trypsin tre;nmcnt rclcuscd 86-99 kDu ryunodinc rcccptor fragmsnts encompassing residue 615 from the SR nxmbrunes. WC conclu~Ic hi! the Y6-99 kD;I domain conttiining rcsiduc! 615 is near the cytoplasmie surfax of the ryanodinc receptor and likely near inlporlunl CG‘ rhitnncl regulatory sites. S;lrcopl;lsmic
1,
rciiculum:
Rynnodinc
rcccptor:
Maligntint
INTRODUCTION
The skeletal muscle sarcoplasmic reticulum (SR) ryanodine reccptor/Ca?’ channel is the pathway by which stored Ca2* is relcascd into the sarcoplasm during excitation-contraction coupling [I]. This homotetrameric complex of 565 kDa subunits, seen as a quatrcfbil structure in electron micrographs [I], has been sequenced from the cDNA [2]. Many reports of alterations in the activity of the porcine skeletal muscle ryanodine receptor (e,g. [3-61) have made it the most likely candidate gene for conferring susceptibility to the tnetabolic disorder malignant hypcrthermia (MH). In support of this hypothesis Fujii et al. [7] have recently identified a nucleotide base mutation (resulting in the replacement of Arg”” with Cys”‘“) in the ryanodine receptor cDNA from MH-susceptible (MHS) pigs. Thus, this natural mutation may be utilized to gain further insight inio the structure and function of a channel critical to the regulation of muscle contraction, In this report we have identified 86 and 99 kDa tryptic fragments of the normal and MHS pig ryanodine receptor that contain residue 615. Our results indicate that this domain is near both the surface of the protein and important channel regulatory sites.
Cor~c,sl,orlil~~~rlP&/w,ss: J.R. Mickclson, Dcpurtmcnt or Veterinary PathoBiology. University or Minncsotu. 295 An. Sci./Vct. Med.. St, Paul. MN 55108. USA. Fax: (I) (612) 625 0204.
hypcrthcrmin;
Mutation:
Calcium
rclcasc channel
2. MATERIALS AND METHODS Pictruin pigs homoeygous for the MH gene [S]. Yorkshire and Minnesota no. I pigs homozygous for the normal ycnc [8.9]. and Near-Pictrdin pigs derived from more than 4 generations ofbnckcross. ing hulothanc-n~g;rtivc pigs to Pictrains [9]. wcrc obtuincd from the closed herd muintuincd at the University of Minnesota Exprimcntal Farm. MH susceptibility wadctcrmincd bycxposurc to 3% halothanc (in 97% oxygen) by mask. with a positive test rcsponsc indicated by limb muscle rigidity in Icss thun 4 min. Trypsin trcptmcnt or hcuvy SR mcmbrana from MHS and normt-d pigs wus ut 37°C for IO min in a medium conttiining I M sucrose. 20 nrM Tris-HCI. pH 7.0. I mg/SR protcinlml. .nnd thr indiuttcd trypsin conccnmtion [IO]. SR protcins were clcctrophorcscd on 3-1246 gradicm polyucrylamidc gels and analyzed on immunoblots with shop anti-ryanodinc receptor antisera and horerddish pcroxidug conjugilted rabbit anti-sheep I@ sccondury untiboditi [IO]. Sheep antibod. its spLxific ror the MHS and normal ryanodinc rcccptor tryplic fragmcnts wcrc uftinity-purified otT of Immobilon strips [I I ] containing the indicotcd ryunodinc rcccptor tryptic fragments. Anti-ryanodinc ruccptor antibody beads wrrc prcparcd by coupling sheep antibodies (allinity-purified from a rabbit ryanodinc rcccptor column) to CNBr-uctivuted Sepharox 46. For immunoprccipitution orthc 86 kDti ryunodinc rcccplor friigmcnt. trypsin truatcd SR mcmbranes were solubilired in the prcscncc of I M NaCI. 2% CHAPS. 1% phosphatidylcholine. SO mM Tris-HCI. pH 7.4. and a proteass inhibitor cocktuil. The sc?lublc extract was then added to an aliquot of unti-ryanodinu receptor immunobadds nnd incubated at 4’C for 4 h. Spcriticdlly bound ryanodinc rcccptor fragments wurc ululd from the immunobuads by addition or clcctrophorcuis butTcr containing 5% 2.murcnptocthanol and 2% SDS. The clutcd protein was l?nctioniad 011 polyacrylamidc gels ;md iI*:: Nexrminal squcncc obtained from trims& onlo lmmobilon mcnlbrdnes [II]. Conditions For the polymrrvsc chain reaction amplificaion of s 74 bp ryanodine receptor scquencc from porcine gcnomic DNA were as described by Fujii ct nl. [7]. Squcnrc of the PCR product was dctcrmined by the didcoxy chain termination method 1131.l’ollowing bluntend ligarion into the S/WI site of pWucscrip1. Dot blot hybridization
49
Volume
301, number
FEBS WETTERS
I
of “I’-lnbrid
oligonucleotidc probes to the PCR producls WIS iIlso ns described by Fujii CLaI. [7], with 5’.TGGCCGTGCGCTCCAAC-3’ for the C 1843 ;IllOk and S’-GGCCGTGTGCTCCAA-3’ for the T I813 ullclu.
3. RESULTS The immunoblot shown in Fig. 1A indicates the presence of the high M,. ryanodine receptor in the korcinc heavy SR preparations prior to trypsin treatment. In addition ro the intact ryanodinc receptor two lower M, components were also observed (Fig. IA). likely due to a partial proteolysis of this protein during SR mem-
A
6
I234
C
I 2
3 4
S
6
7 6
8 IO II 12
MNMNN
99 66
1234
6678
9lOlll2
Fig. I. Tryptic immunopcptide maps of the MHS and normal SR ryunodine receptor. i-icavy SR vcsiclcs from 3 different MHS and Z difkrcnt normal pigs were incubaicd in the ubscncc of trypsin (A) or in the presence of VSII./OUStrypsin conccntraiions (B) for IO min at 37°C. Panel 6: luncs I-4. trypsin 10 SR wcighl ratio of l:QO: lanes S-8, trypsin to SR ratio of 1:?40; lanes 9-12. lrypsin IO SR ratio of I: 160. Pancl C: I MHS and I normal SR preparation wcrc trcatcd with trypsin (1:?40). alkr which they wcrc diluted wiih AI equal volume of 0.3 M sucrose und centrifuged at 150,000 x g for 30 min. Sumplcs 01 trypsin-trcatcd SR (lanes l-4), und the rcsukrnt supcrntamt (lanes S-8) and pellets (Iiines 9-l?) were analyzed, Lana 1.2.5.6.9.10 IO min trypsin incubation: lanes 3,4.7.X.1 l,l:! - 5 min trypsin incubnlion. All samples were unalyzcd for ryanodinc rcccp~or immunorcxtivc fragments as described in section 2. The positions of molccul:~~ weight markers arc indicaicd 011 the lcli, and the posiiions of the 93 ;lJd 86 kDu ryanodinc rcccptor fragmcms arc indicutcd by the sin& arrowheads.
50
April
1992
brane preparation. Fig. I B (lanes l-4) shows an immunoblot of trypsin-treated SR membranes, where ryanodine receptor doublet fragments of M,. approximately 99 kDti were detected in MHS SR. while normal SR contained ryunodinc receptor doublet fragments of both 99 and 86 kDa. The figure also shows that the use of higher trypsin concentrations resulted in the loss of these components (lanes S-12). Following centrifugation of trypsin-digested MHS and normal SR for 30 min at 150,000 x g, the 99 and 86 kDa ryanodine receptor fragments were detected in the resulting supcrnatant (Fig. IC, lanes 5-8) and absent from the pellet (lanes 9-12). The 99 and 56 kDa ryanodinc receptor fragments could not be differentiated from the numerous other SR protein fragments on Coomassie blue-stained gels (not shown). When purified ryanodinc receptor preparations (isolated by the method of Lai et al. [I]) were treated with trypsin we were unable to detect the 86 and 99 kDa fragments on immunoblots, although numerous other fragments were detected (not shown). Affinity-purified antibodies to the 99 and 86 kDa ryanodinc receptor fragments were prepared and utilized to st;h irnmunoblo~s. Fig. 2C shows that antibodies to the normal 99 kDa tryptic fragments recognized the 86 kDa normal fragments. as well as the 99 kDa MHS fragments. Similarly, antibodies to the 86 kDa normal fragments recognized both the 99 kDa normal and the 99 kDa MHS fragments (Fig. 2D), ;md antibodies to the 99 kDa MHS fragments recognized both the 99 and 86 kDa normal fragments (Fig. 2E). Thus, the 86 and 99 kDa tryptic fragments appear to arise from the same domain of the ryanodine receptor. Antibody beads were used to immunoprecipitate the 86 kDa normal ryanodinc receptor fragment from trypsin-treated normal SR. The major immunoprecipitated component detected on Coomassie blue-stained gels had a M,. of 86 kDa. and was recognized on immunoblots by the anti-ryanodine receptor antibody (data not shown). The N-terminal sequence of this fragment indicated significant homology to residues 617-627 01 the rabbit ryanodine receptor (Fig. 3A), just downstream from a potzn!ir:l trypsin cleavage site at Arg”“. DNA corresponding to this region of the porcine ryanodine receptor gene wits then amplified by the polymerase chain reaction and sequenced (Fig. 3B). The nucleotide sequences bctwecn the PCR primers were identical to those determined by Fujii et al. [7], with a C in position 1843 of the normal Yorskhirc DNA and a T in position 1843 of the MHS Pietrain DNA, translating into the At@’ to Cys”” mutation. Dot blots of the 74 bp porcine DNA PCR product were hybridized to either the Cl 843 (normal) or Tl843 (MHS) ryanodine receptor oligonucleotide probes (Fig. 4). This confirmed that all pigs from the MHS Pietrain herd utilized for our previous studies of ryanodinc receptor alterations [5,6,9,10], contained only the T1843 allele, while the normal Yorkshire and Minnesota no.
Volume 301, number 1
April 1992
FEBS LETTERS
D Ml-l N MH N
MH
N
E
Ml-l N
Ml-l N MHN
Mr
228
I IO
99 86
70
t
44
Fig. 2. Immunoblol analysis of the 99 and 8G kDs rynnodinc receptor tryptic fragments. MHS and normal heavy SR membranes were incubated in the ;ibscncc (A) or presence of trypsin ut it I:320 weight ratio for IO min (B-E). Antibodies utilized to slain the transfers were either the sheep ;mti.ryanodil:c rcccptor antiserum (A and B), or sheep antibodies that hud been affinity purified from immunoblotsof the ryanodinc rL=ptor uyptic Itagments: (C) anti-normal 99 kD:l antibodi& (D). anti-normal 86 kDa antibodies: (E). anti-MHS 99 kDa amibodics.
I herds conrained the Cl843 ullelc (Fig. 4A). We also examined families of pigs in which the oligonucleotide
hybridization results could be correlated with the results of the halothane-challenge test for MH susceptibility
(A) 615 Arg Set
Rabbit
Pig
86 kD Fragment
X
620 Asn Gln Asp Lou lie Asn Gln Asp
X
lie
625 Thr Glu Asn Leu Leu Prc X
Glu Asn Leu Leu Pro
(B) 1843 :Ek8
AAT GGT G
Normal Ml-is
ATT ACT GAG 1869
B TG GCC GTG CGC TCC AAC CAA GAT CTC Val Ala Val Arg Ser Am Gln Asp Leu TG GCC GTG TGC TCC AAC CAA GAT CTC Val Ala Val Cys Ser Am Gln Asp Leu 615
620
Fig. 3. N-Tcrminul sequcncc of the 86 kDa ryanodinc rcccptor tryptic fragment and DNA scqucncc of the ryanodinc rcccplor PCR product. (A) Normd SR was incubutcd with trypsin (I: 160 for IO min at 37”C), the 86 kDa normal ryanodinc rcceplor fragmcnl was immunoprccipitatcd. and 7 Amino acid rcsiducs whichcould not bc dctcrmincd the N-terminal scqtmce wus dctcrmined from an Immobilon transfer us dcscribcd in section _. are rcprcscntcd with an X. Scquencc and numbering scbcmc of the rabbit skulctul muscle ryanodinc rcccl%or [7] is provided. iBi MHS Pictrain and normal Yorkshire gcnomic DNA vxrc utilized for the production of the 74 bp ryanodine rcccplor DNA scgmcnt as dcscribcd in s&lion 2. Thusc PCR products wcrc ligated into pBlucscript and scqucnccd. Partial scquencc of the PCR primers (covcrcd with solid lines) and the ryanodinc rcccptor cDNA scque~w nuclcotidc numbcling system detcrmincd by FLjii CI al. [7] arc provided.
51
FEB.5 LETTERS
Volume 301. number 1 Pielmin
YorkWe 34
12 A
5
6
7
Minn. ryl 6
9
IO
II
I2
tf843 Cl843
6
Cl843
l
*
-1
”
l Orn!O
8
00
---++-+-2
3
4
5
6
7
8
9
IO
II
Fig. 4. Dot blot oliyonuclcotidc hybridization anslysis or the 74 bp ryanodinc receptor PCR product. (A) Four dilkrcnt MHS Pictrnin. normal Yorkshire. and normal Minnesota no. I pigs wcrc nnalyzcd, (B) Near-Pietrain p;urnis;tnJ their progeny. Thr figure rcprcscnt the autoradiogrum that rcsultcd I’rom hybridiz;llion to the ‘2P-l;lbclcd Cl843 ;md Tl843 oligcnuclcotidss as dcscribcd in section 1. The response 0T CilCll pig to lhc h;ilotll;inc-chiill~i~~~ ltsl 1’or MI-I is :llso given. with + indicating ;I positive lulo~i~a~~c rcsponsc ond - indicating il nc_u;~tivt huloiliiinc rceponse.
(Fig. 46). In this example the Ilalotlli~ne-negati\le NcnrPietrain parents had copies of each ryanodine receptor allele (i.e. were heterozygotes) and the progeny of this mating demonstrated all three possible genotypes. Interestingly, one heterozygotc pig (individual no. 6) reacted positively in the halothunc-challenge test. That all homozygotcs for the Tl843 allele were MHS (in agreement with Fujii et al. [7]), while heterozyyotes based on the oligonuclcotide hybridization assay could be either positive or negative in the halotha~ic-ch~~IIe~7gP test (Fig, 4B), has been a consistent finding in the 8 Near Pietrain pedigrees examined to date. This appears to indicate ambiguities associated with applying the in vivo challenge test for the identification of hctcrozygates. 4. DISCUSSION This study confirms our previous observation of the generation of 99 kDa MHS and 86 kDa normal porcine SR ryanodine receptor tryplic fragments [IO], and demonstrates that a normal 99 kDa ryanodine receptor fragment can also be detected (Fig. 16). Affinity- purified antibodies to each of thcsc fragments recognized the other two (Fig. 21, indicating that these three fragments were all derived from the same domain of the ryanodine receptor, The 86 kDu normal ryanodine receptor tryptic fragment had residue 616 as its N !erminus (Fig. 3A) and the DNA sequences of the MHS and normai ryanodinc receptors in this region confirmed the ArghlS to Cysh15mutation described by Fujii ct al. [7] (Fig. 3B). These data thus indicate that the Arg”15to Cys mutation in the MHS pig ryanodinc rcccptor climinutes
April 1992
an accessible trypsin cleavage site at residue 615, such that the normal 86 kDa fragment cannot be formed. That these 99 and 86 kDa fragments were not observed when purified MHS and normal ryanodine receptors were subjected to proteolysis may indicate an altered conformation of this molecule in the detcrgent-solubiliced purified state. Our results suggest that Arg”” may be on the surface of the native ryanodine receptor molecule, as it is accessible to trypsin. The 86-99 kDa tryptic fragments are released from the SR (Fig. lC), indicating that they are derived from the cytoplasmic foot region [I .2]. Arg”15 may also be located near important regulatory sites, as the M HS and normal porcine ryanodine receptors demonstrate differences in Ca?’ regulation of Ca’+ release [3-51. channel gating [B], and [fH]ryanodine binding activity [5,9]. To date none of the various ligand binding sites (e.g. Ca’+, Mg?“. ATP. calmodulin [I]) which regulate this channel have been identified. Further study of the MHS porcine ryunodine receptor mutation should help define structure/function relationships within this Ca” channel protein. .I~~k/,trll~l~~~/~c~rrrc,r/.~: The authors wish to hunk Dr. Clivc Sl;myhtcr 01 the Howard Huphss Mcdicul loslitutc. University or TL’XIS Southwcstcrn Medical Center, Ibr protein-suqucnciii~ cxpcrtisc. This work was supported by pr;lnts rrom 111~’Muscular Dystrophy Association ol’Amcric;i ;II~ the Niltiunill Pork Producers COUIIC~I (lo J.R.M. und C.F.L.). NIH GM 31382 (to C.F.L.). ;mrl by the University or.Minncsotil Agricultural Experiment Stiltion (C.F,L. und W.E,R,), K.P.C. is UII Invcstiyutor ol’ The Ho\vurd Hughes Mcdicul Institute.
REFERENCES [I] Lai. F.A.. Erickson. H.P.. Roussc;n~. E.. Liu. Q.-Y. and Mcissncr. G. (19Y8) NilLure 331. 315-319. [?I T;lkcshimil. H,. Nishinnux. S.. bkllSl~lllUlO. T.. Ishkh. l-l.. KillI~;Iw;I. K.. Miniunino. N.. Miltsuoa Ii.. Ucdti. M.. H:III;IO~;I. M.. Hirosc. T. iInd Numa. S. (1989) Nmurc 339. 439-445, 1.11Ndson. T.E. (1983) J. Clin. Invest. 72. 862-870. [A] Kim. D.H,, Srekr. F.A.. Ohnishi. ST.. Ryan. J.F.. Roberts. J., Allen. P.D.. Mcszlros. LG.. Antoniu. 13.and Ikcmolo. N, (19S4) Biochim. Biophys. Actu 775. 320-327, [5] Mickclson. J.11.. Gullilnt. E.M.. Liticrcr, L.A., !ohson, K.M., Rcmpcl, W.E, and Louis, C.F. (19%) J. Biol. Chcni. 263. 93lO9315, [O] Fill. M.. Coronudo. R.. Mickclson. J.R.. Vilvcn. J.. Mu. J.. J;icobson. B./z, and Louis, CF. i 1990) Biophys. J, 50. 471--175. F.. DcLcdn. S,. Khannu. V.K.. 171 Fujii. J.. Otsu. K.. Zorznto. Wcilcr. J,E.. O’Brien. P,J. and MucLcnnan. D.H. ( 1991) Science 253. 448-45 I. 181 Rcik, T.R.. Rcmpcl. W.E.. McCir;~th. C.J. und Addis. P.B. ( 1983) J. An. Sci. 57, X26-831, W.E.. Johnson. K.M.. [91 Mickclso~~.J.R.. GulliLnt. E.M.. Run@. Littcrcr. L,A,, Jitcobson. B.A. illId Louis. C,F. (1989) At11. J. Physiul. 257. C7X7-C794. J-R.. Louis. C.F. and Cumpbsll, [lOI Ktmdson, C %I.. Mirkelson, K.P. (1990) J. Biol. Chcm. 265. 2421-2414. D. ( 1998). in: Antibodies, A I.uhor;lt~ry [l II Hurlow, E. and Lax, M;mual. Cold Spring Hurbor Luborutory. p. 498. Matsuduira. P. (1987) J. Biol. Chum. 262. 10035-10038. ii:; Tubor. S. i\nd RIchardsol:, CC. (1989) J. Biol. Chem. X4,6447M58.