Patent Application: US-35415199-A

Abstract:
the present invention concerns a novel cytoskeletal protein , myotilin , which contains ig - like domains homologous to a giant sarcomeric structural protein titin . myotilin is expressed in skeletal and cardiac muscles , it colocalizes with α - actinin in the sarcomeric i - bands and directly interacts with α - actinin . expression of myotilin in mammalian non - muscle cells and in yeast causes reorganization of actin into thick f - actin bundles and inhibits growth of yeast cells .

Description:
a partial cdna encoding myotilin was initially discovered based on a yeast two - hybrid screen for novel cytoskeletal components . using this sequence as a probe , we cloned a 2244 bp cdna from a human skeletal muscle library . the cdna contains a 1494 bp open reading frame ( nucleotides 281 to 1774 ) encoding a 498 amino acid polypeptide ( seq id no : 2 ), which we have termed myotilin ( fig1 a ). the methionine start codon is in partial agreement with the kozak consensus sequence . the nh 2 - terminal sequence is particularly rich in serine residues often arranged in a paired fashion , and contains a 23 amino acid hydrophobic stretch ( residues 57 - 79 ). upon database searches , the nh 2 - terminal sequence is unique and does not contain known structural domains . the cooh - terminus of the protein is predicted to form two ig - like domains with conserved key residues ( fig1 b ) [ 23 ]. several cytoskeletal proteins involved in organization of the muscle sarcomere have recently been shown to contain such structural units . by sequence comparison , the highest homology is detected between myotilin and the region of human striated muscle titin , which contains the z - disk associated ig - domains 7 and 8 ( fig1 c ) ( residues 1406 - 1621 of titin )[ 4 ]. the sequences within the compared regions are 31 % identical and 53 % conserved without any introduced gaps . a similarity comparison using the clustal method indicates a 38 . 3 % similarity between this region of myotilin and titin . the sequence similarity between myotilin and titin is restricted to the ig - domains of myotilin . other characteristic structural features of titin , the fn ( iii )- type domains , the specific sequences of z - disk , i - band and m - band , or the repeating ksp phosphorylation motif [ 4 ] are not present in myotilin . however , the sequence prediction of myotilin reveals several other possible sites for phosphorylation , three of them in the serine - rich region . the organization of myotilin gene was determined by comparing the myotilin cdna with the genomic sequence from chromosome 5 pac clone 9c13 ( genbank accession number ac006084 ). all splice junction sequences are in agreement with the gt - ag consensus ( table 1 ). the exon / intron boundaries were further confirmed by amplification of each exon from a commercial p1 clone with intron specific primers ( not shown ). the gene is composed of ten exons , and the translation initiation signal is in exon ii ( fig2 ). thus the small 69 bp first exon is not translated . the size of the entire gene is under 20 000 bp without the promoter region . the sequences coding for the ig - domains are located in exons vi and vii ( first ig - domain ) and in exons viii and ix ( second ig - domain ). the chromosomal localization of myotilin gene was determined by radiation hybrid mapping . the myotilin gene was mapped to chromosome 5q31 between the markers afm350yb1 and d5s500 ( fig3 ). the gene causing an autosomal , dominantly inherited limb - girdle muscular dystrophy ( lgmd ) 1a has been mapped to chromosome 5q31 between the markers d5s479 and d5s594 [ 16 ]. the myotilin gene is inside this reported area . taken all the data together , myotilin is a candidate gene for lgmd1a . by northern blot analysis we detected two different transcripts ( 2 . 2 and 2 . 5 kb ) strongly expressed in skeletal muscle and weakly in the heart ( fig4 ). the two transcripts in the heart are only seen after a prolonged exposure ( not shown ). smooth muscle and several non - muscular tissues , including brain , placenta , lung , liver , kidney and pancreas , did not contain detectable mrna . in vitro translation of the full - length cdna yielded a 57 kda polypeptide , which is in agreement with the mass of myotilin estimated from the cdna sequence ( fig5 a ). we raised an antibody against myotilin by immunizing rabbits with a synthetic branched 17 amino acid peptide encompassing residues 352 - 368 ( see fig1 a ). in western blotting this antibody revealed a 57 kda protein band and a fainter band near 110 kda from skeletal muscle but not from smooth muscle or non - muscular tissues ( fig5 b ). the reactivity could be blocked by incubating the antibody with 5 - fold molar excess of the corresponding peptide ( fig5 b ). both the mrna and immunoblotting data thus indicate that myotilin is a muscular protein with a clearly restricted expression pattern . the identity of the 110 kda band is unclear . as it migrates at a region twice the size of a myotilin monomer and as myotilin is able to form intermolecular interactions ( see below ), the band possibly represents a myotilin dimer . upon treatment of tissues with 1 % triton x - 100 or with 1m kcl , myotilin was retained in the insoluble fraction suggesting a cytoskeletal association ( data not shown ). to characterize the subcellular localization of myotilin we isolated bundles of striated muscle myofibrils and stained them using the affinity purified peptide antiserum . the immunostaining pattern was compared to several characterized components of the sarcomere ( fig6 ). myotilin staining was detected in the i - bands . the staining pattern was reminiscent of α - actinin , which is known to decorate the z - lines of the i - bands . actin , the major component of thin filaments , gave a more diffuse staining pattern along the entire i - bands . a titin mab , which recognizes an epitope at junctions of thin and thick filaments revealed a staining pattern of a doublet band at each sarcomere . the immunolocalization data demonstrates that myotilin is an integral component of striated muscle sarcomeres . to further study the localization of myotilin in the striated muscle we performed immunohistochemical analyses of frozen tissue sections with the myotilin antibody . in perpendicular sections , where the organization of sarcomeres was visible , we could detect a periodical cross - striated staining of myotilin ( fig7 a ), which was consistent with the pattern in isolated myofibrils . especially in transverse sections ( fig7 b ) the myotilin staining was also localized at the plasma membrane indicating that myotilin is also present at the sarcolemma . in addition to these findings , myotilin antibody stained intramuscular nerve fibers ( fig7 c ). the preimmune serum gave no reactivity ( fig7 d ). we used the yeast two - hybrid method to study protein interactions of myotilin . among the tested partners , the strongest interactions were seen between myotilin and α - actinin ( a construct containing spectrin - like repeats r1 - r4 ) and between myotilin molecules ( fig8 a - c ). α - actinin is known to form dimers via spectrin - like repeats [ 25 , 26 ] and this could be verified also in our two - hybrid analysis ( fig8 b ). however , quantitation of the β - galactosidase values indicate that the intensity of the reaction was weaker than the interaction between α - actinin and myotilin and between two myotilin molecules ( fig8 c ). an nh 2 - terminal deletion construct containing the ig - domains , myotilin 215 - 498 bound full - length myotilin , but did not bind α - actinin ( r1 - r4 ). we were unable to express myotilin 215 - 498 as a bait to test whether the intermolecular interaction of myotilin is mediated by the cooh - terminal ig - domain containing region or by nh 2 - terminal association to the cooh - terminal part . the interaction between myotilin and α - actinin was further tested by an affinity precipitation assay , using in vitro translated 35 s - labelled myotilin and gst - α - actinin constructs bound to glutathione - agarose . myotilin bound the cooh - terminal half of α - actinin ( r3 / r4 / ef - hand ), but not the nh 2 - terminal half ( abd / r1 / r2 ) or gst alone ( fig8 d - e ). based on the two - hybrid and affinity precipitation results , residues important for α - actinin binding reside in the first 215 nh 2 - terminal residues of myotilin and the myotilin binding site in α - actinin apparently locates within spectrin - like repeats 3 and 4 . the cellular localization and function of myotilin was further analyzed by transiently transfecting ha - epitope tagged full length myotilin and a cooh - terminal myotilin construct ( myotilin 215 - 498 ) into cos - 1 cells that do not express endogenous protein . the myotilin 215 - 498 construct was confirmed by in vitro translation to yield a proper size polypeptide ( fig5 a ). β - galactosidase dna was used as a transfection control . after 72 hours the cells were fixed and double stained for myotilin or β - galactosidase and actin . myotilin 215 - 498 showed partly a diffuse cytoplasmic staining pattern but also submembraneous accumulation which colocalized with cortical actin visualized by phalloidin staining ( fig9 b , e ). in contrast , full length myotilin localized within the filament network of the cell body and phalloidin staining revealed a strict colocalization with f - actin in these filaments . remarkably , we could notice formation of thick f - actin containing bundles in cos - 1 cells ( fig9 a , d ), whose actin - containing skeleton is poorly organized under these culture conditions . β - galactosidase showed a diffuse cytoplasmic distribution and did not colocalize with f - actin ( fig9 c , f ). the myotilin - induced actin - containing structures were further characterized by confocal microscopy ( fig1 ). typically , the actin bundles were present in the cell body , although in some cells they were located subcortically in the cell periphery ( fig1 , middle row ). sectioning of the cells revealed that the f - actin and myotilin - containing structures were often haphazardly arranged at different levels in the cytoplasm . this is visualized in the bottom panel of fig1 , in which images on the left and in the middle are composites of f - actin and myotilin staining at two different planes of the same cells and the image on the right is an overlay of the myotilin labelling in the entire cell . importantly , the structures were not present at the ventral surface of the transfected cells indicating that they are not stress fibers . the effect of myotilin on actin - organization is not reminiscent of changes induced by overexpression of previously characterized actin - organizing proteins and suggests a unique mechanism of action . the biological functions of myotilin were also studied by expressing myotilin in saccharomyces cerevisiae . induction of myotilin expression resulted in reorganization of actin into thick filaments not detected in control cells ( fig1 a and c ). the myotilin expressing cells grew in rows and the filaments continued from one cell to another ( fig1 a ). this result indicates that the actin organizing property of myotilin is conserved within different organisms . expression of myotilin or its cooh - terminal part resulted in inhibition of cell growth , whereas expression of nh 2 - terminal constructs ( 1 - 150 and 1 - 250 ) or a shorter cooh - terminal construct ( 270 - 472 ) did not have a similar effect ( fig1 e ). this growth - inhibitory function parallelled with morphogenic changes in yeast cells . when myotilin was expressed simultaneously in two different expression vectors , the growth inhibiting effect was stronger than the effect produced by a single vector indicating that the effect is dependent on the level of protein expression ( fig1 f ). in this experiment , expression of an irrelevant cytoskeletal actin - binding protein , ezrin , did not affect cell growth , further demonstrating the specificity of the effect of myotilin . a partial cdna was used for screening of the full - length myotilin cdna from a skeletal muscle library ( stratagene , la jolla , calif .). positive clones were sequenced with an abi 310 genetic analyzer ( perkin - elmer , foster city , calif .). protein database searches were done with blast program . sequence alignments between ig - domains of myotilin and other cytoskeletal proteins were performed with the megalign software ( dnastar ). the domain predictions were obtained from pfam server . protein motif predictions were done with protein family alignment pfam 2 . 1 and with motif . the organization of myotilin gene was determined by comparing the myotilin cdna with the genomic sequence from chromosome 5 pac clone 9c13 ( genbank accession number ac006084 ). a commercial p1 clone ( genomesystems inc ., st . louis , mich ., searched by pcr with myotilin primers ) was used for amplification and sequencing of exon - intron boundaries . the chromosomal localization of myotilin gene was determined by radiation hybrid mapping using the genebridge ii panel . pcr assays were performed as duplicates and the resulting data vector was analyzed using whitehead genome center server . a polyclonal antibody was raised in rabbits using a synthetic branched , lysine - cored 17 amino acid peptide of myotilin ( marked in fig1 with a scattered line ) as the antigen . after five immunizations , rabbits were bled . the specific antibody was purified in an affinity column using a corresponding single chain peptide coupled to cnbr - activated sepharose 4b ( pharmacia , uppsala , sweden ) as the ligand . the specificity of the rabbit antibody was verified by reactivity with appropriate gst - fusion protein constructs in western blot analysis ( data not shown ) and by cross - blocking experiments , in which five - fold molar excess of the specific peptide but not an irrelevant myotilin peptide ( residues 199 - 217 ) absorbed the reactivity . northern blot analysis was performed with a multiple tissue mrna filter ( clontech laboratories , inc ., palo alto , calif .) using a 32 p - labelled 320 bp myotilin cdna fragment , which encodes amino acids 369 - 471 , as a probe . in vitro translations were performed with a coupled reticulocyte lysate kit ( promega , madison , wis .) using 35 s - labelled methionine for detection . the templates were full - length myotilin and a construct containing amino acids 215 - 498 ( myotilin 215 - 498 ) in bluescript plasmid vector ( stratagene ). for western blotting , fresh tissues were homogenized in reducing laemmli buffer . equal amounts of protein , as estimated by coomassie blue staining , were separated in 8 % sds - page and transferred to nitrocellulose filters ( schleicher & amp ; schuell gmbh , dassel , germany ). the filters were probed with the myotilin antibody or with a control preimmune serum , followed by peroxidase conjugated goat anti - rabbit igg ( dako a / s , copenhagen , denmark ) and ecl detection ( pierce , rockford , ill .). bundles of bovine and human myofibrils were isolated as described [ 27 ], cytocentrifuged onto objective slides , fixed in − 20 ° c . methanol , and reacted with mab against actin ( ac 40 , sigma chemical co ., st . louis , mo . ), titin ( t11 , sigma ), α - actinin ( 67cb11 ) [ 28 ] and a control mab x63 ( atcc , maryland , usa ), or with affinity purified anti - myotilin antibody or the corresponding preimmune igg . secondary antibodies were fitc - conjugated goat anti - mouse igg ( cappel research products , durham , n . c .) and tritc - conjugated goat anti - rabbit f ( ab ) 2 fragment ( jackson immunoresearch laboratories , inc ., west grove , pa .). staining of bovine and human myofibrils yielded identical results . frozen 2 μm sections of human skeletal muscle were immobilized on poly - l - lysine - coated glass slides , fixed with cold acetone and immediately air - dried . for immunohistochemical staining the sections were reacted with 1 : 100 dilution of affinity - purified myotilin ab or rabbit pre - immune igg at similar concentration . the antibody was detected with elite vectastain abc kit ( vector laboratories , inc ., burlingame , calif .) according to manufacturer &# 39 ; s instructions . the slides were briefly counterstained with hematoxylin - eosin . full length myotilin , myotilin 215 - 498 and spectrin - like repeats r1 - r4 ( residues 267 - 749 ) of chicken smooth muscle α - actinin ( kindly provided by dr . d . critchley , university of leicester , uk ) were subcloned into eg202 and jg4 - 5 plasmids for two - hybrid analysis [ 29 ]. the cooh - terminal construct of myotilin was subcloned from a partial cdna sequence obtained from the skeletal muscle library screen . the authentity of the constructs was verified by sequencing . the genotype of the s . cerevisiae strain boy1 , kindly provided by p . ljungdahl , ludwig institute for cancer research , stockholm , sweden , is mat αhis3 trpl leu2 :: 6lexaop - leu2 ura3 :: 8lexaop - gall - lacz . boyl mating type a was made using the ycpho cut4 plasmid [ 30 ]. yeast strains were grown at 30 ° c . in rich medium or in synthetic minimal medium with appropriate amino acid supplements . bait and prey constructs were transformed into boy1 - yeast of both a and a mating type using the trafo protocol and plated on selection plates . clones were grown to late logarithmic phase in selective medium . for analysis of fusion protein expression , yeast cells from 1 ml of overnight culture were lysed in reducing laemmli sample buffer , the samples were boiled and analyzed by sds - page and immunoblotting . baits and preys were grown on selection plates , replica plated together on rich media plates for mating overnight and replica plated on double ( tryptophane and histidine ) or triple ( tryptophane , histidine and leucine ) selection with or without 5 - bromo - 4 - chloro - 3 - indolyl - β - d - galactopyranoside ( x - gal )( boehringer ) for selection of interactions . for the in vitro binding assay , gst - o - actinin fusion proteins , abd / r1 / r2 , r3 / r4 / ef [ 25 ] or gst alone were produced in e . coli and purified with glutathione - agarose beads ( pharmacia ). 2 μg of fusion proteins on glutathione beads were reacted with 20 μl of in vitro translated , 35 s - labelled myotilin in 10 mm tris - hcl , ph 7 . 5 , 5 mm edta , 130 mm kcl , 0 . 05 % tween 20 . after washes with the same buffer , bound material was eluted by boiling in laemmli buffer , subjected to sds - page and detected by autoradiography . in transfection studies full length myotilin and myotilin 215 - 498 construct in an ha - tagged pahp plasmid ( a derivative of pcdna3 , invitrogen , san diego , calif .) and a control sv - β - galactosidase vector ( clontech ) were used . cos - 1 cells plated on 6 cm tissue culture dishes were transfected with 5 μg of appropriate plasmid cdna using superfect ( qiagen gmbh , hilden , germany ) and grown on glass coverslips . after 72 hours , cells were fixed in 3 . 5 % paraformaldehyde at + 4 ° c . for 10 min . and permeabilized in 0 . 1 % triton x - 100 . transfected protein was immunoreacted with anti - ha mab ( 12ca5 , boehringer gmbh , mannheim , germany ) or anti - β - galactosidase mab ( boehringer ) followed by fitc - conjugated goat anti - mouse igg . f - actin was simultaneously visualized with rhodamine - labelled phalloidin ( molecular probes , eugene , oreg .). the specimens were viewed with a zeiss axiophot ii epifluorescence microscope ( carl zeiss , oberkochen , germany ) or alternatively , with a confocal 410 invert laser scan microscope ( carl zeiss ). the effect of myotilin on yeast actin cytoskeleton and growth rate was studied as follows . for actin staining , cells expressing myotilin or cells transfected with an empty vector were fixed in 4 % paraformaldehyde . f - actin was visualized with rhodamine - labelled phalloidin ( molecular probes , eugene , oreg .) and the cell wall with calcofluor ( 1 mg / ml ) ( sigma ). after washings the cells were resuspended in dabco mounting solution . for analysis of the effect of myotilin in cell growth , haploid cells transfected with myotilin in jg4 - 5 vector were grown in glucose , washed and an equal amount of cells with different myotilin constructs were added into galactose - containing growth medium for induction of protein expression . diploid cells expressing two different proteins from the jg4 - 5 and eg202 vectors were grown in galactose from the initiation of the experiment . at indicated time points a small aliquot of cells was sonicated and od 600 was measured using a spectrophotometer . 1 . fürst , d . o . and gautel , m . 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( 1994 ) activation of a yeast replication origin near a double - stranded dna break . genes & amp ; development 8 : 554 - 562 . tagtaataat ttgccttcat cttccatata ccaactaagc atg ttt aac tac gaa 295 cgt cca aaa cac ttc atc cag tcc caa aac cca tgt ggc tcc aga ttg 343 arg pro lys his phe ile gln ser gln asn pro cys gly ser arg leu cag cct cct gga cca gaa acc tcc agc ttc tct agc cag acc aaa cag 391 tct tcc att atc atc cag ccc cgc cag tgt aca gag caa aga ttt tct 439 gcc tcc tca aca ctg agc tct cac atc acc atg tcc tcc tct gct ttc 487 cct gct tct ccc cag cag cat gct ggc tcc aac cca ggc caa agg gtt 535 aca acc acc tat aac cag tcc cca gcc agc ttc ctc agc tcc ata tta 583 cca tca cag cct gat tac aat agc agt aaa atc cct tcc gct atg gat 631 tcc aac tat caa cag tcc tca gct ggc caa cct ata aat gca aag cca 679 tcc caa act gca aat gct aag ccc ata cca aga act cct gat cat gaa 727 ser gln thr ala asn ala lys pro ile pro arg thr pro asp his glu ata caa gga tca aaa gaa gct ttg att caa gat ttg gaa aga aag ctg 775 aaa tgc aag gac acc ctt ctt cat aat gga aat caa cgt cta aca tat 823 lys cys lys asp thr leu leu his asn gly asn gln arg leu thr tyr gaa gag aag atg gct cgc aga ttg cta gga cca cag aat gca gct gct 871 gtg ttt caa gct cag gat gac agt ggt gca caa gac tcg cag caa cac 919 aac tca gaa cat gcg cga ctg caa gtt cct aca tca caa gta aga agt 967 asn ser glu his ala arg leu gln val pro thr ser gln val arg ser aga tca acc tca agg gga gat gtg aat gat cag gat gca atc cag gag 1015 arg ser thr ser arg gly asp val asn asp gln asp ala ile gln glu aaa ttt tac cca cca cgt ttc att caa gtg cca gag aac atg tcg att 1063 lys phe tyr pro pro arg phe ile gln val pro glu asn met ser ile gat gaa gga aga ttc tgc aga atg gac ttc aaa gtg agt gga ctg cca 1111 asp glu gly arg phe cys arg met asp phe lys val ser gly leu pro gct cct gat gtg tca tgg tat cta aat gga aga aca gtt caa tca gat 1159 ala pro asp val ser trp tyr leu asn gly arg thr val gln ser asp gat ttg cac aaa atg ata gtg tct gag aag ggt ctt cat tca ctc atc 1207 ttt gaa gta gtc aga gct tca gat gca ggg gct tat gca tgt gtt gcc 1255 aag aat aga gca gga gaa gcc acc ttc act gtg cag ctg gat gtc ctt 1303 lys asn arg ala gly glu ala thr phe thr val gln leu asp val leu gca aaa gaa cat aaa aga gca cca atg ttt atc tac aaa cca cag agc 1351 ala lys glu his lys arg ala pro met phe ile tyr lys pro gln ser aaa aaa gtt tta gag gga gat tca gtg aaa cta gaa tgc cag atc tcg 1399 gct ata cct cca cca aag ctt ttc tgg aaa aga aat aat gaa atg gta 1447 ala ile pro pro pro lys leu phe trp lys arg asn asn glu met val caa ttc aac act gac cga ata agc tta tat caa gat aac act gga aga 1495 gln phe asn thr asp arg ile ser leu tyr gln asp asn thr gly arg gtt act tta ctg ata aaa gat gta aac aag aaa gat gct ggg tgg tat 1543 val thr leu leu ile lys asp val asn lys lys asp ala gly trp tyr act gtg tca gca gtt aat gaa gct gga gtg act aca tgt aac aca aga 1591 tta gac gtt acg gca cgt cca aac caa act ctt cca gct cct aag cag 1639 tta cgg gtt cga cca aca ttc agc aaa tat tta gca ctt aat ggg aaa 1687 leu arg val arg pro thr phe ser lys tyr leu ala leu asn gly lys ggt ttg aat gta aaa caa gct ttt aac cca gaa gga gaa ttt cag cgt 1735 gly leu asn val lys gln ala phe asn pro glu gly glu phe gln arg ttg gca gct caa tct gga ctc tat gaa agt gaa gaa ctt taataacttt 1784 met phe asn tyr glu arg pro lys his phe ile gln ser gln asn pro glu gln arg phe ser ala ser ser thr leu ser ser his ile thr met pro gly gln arg val thr thr thr tyr asn gln ser pro ala ser phe thr pro asp his glu ile gln gly ser lys glu ala leu ile gln asp gln arg leu thr tyr glu glu lys met ala arg arg leu leu gly pro asp ser gln gln his asn ser glu his ala arg leu gln val pro thr asp ala ile gln glu lys phe tyr pro pro arg phe ile gln val pro glu asn met ser ile asp glu gly arg phe cys arg met asp phe lys val ser gly leu pro ala pro asp val ser trp tyr leu asn gly arg thr val gln ser asp asp leu his lys met ile val ser glu lys gly leu his ser leu ile phe glu val val arg ala ser asp ala gly ala tyr ala cys val ala lys asn arg ala gly glu ala thr phe thr val gln leu asp val leu ala lys glu his lys arg ala pro met phe ile glu cys gln ile ser ala ile pro pro pro lys leu phe trp lys arg asn asn glu met val gln phe asn thr asp arg ile ser leu tyr gln pro ala pro lys gln leu arg val arg pro thr phe ser lys tyr leu cys leu glu gly ala asn cys arg phe asp leu lys val val gly arg pro met pro glu thr phe trp phe his asp gly gln gln ile val asn asp tyr thr his lys val val ile lys glu asp gly thr gln ser leu asn val asn ile lys glu gly ser arg leu glu met lys val arg ala thr gly asn pro asn pro asp ile val trp leu lys asn ser asp ile glu ala ala leu lys ile asp ser thr val ser gln asp ser ala trp lys leu thr ser leu arg leu lys arg phe gly pro ala his phe glu