The Complete Gene Sequence of Titin, Expression of an Unusual ≈700-kDa Titin Isoform, and Its Interaction With Obscurin Identify a Novel Z-Line to I-Band Linking System
Titin is a giant vertebrate striated muscle protein with critical importance for myofibril elasticity and structural integrity. We show here that the complete sequence of the human titin gene contains 363 exons, which together code for 38 138 residues (4200 kDa). In its central I-band region, 47 novel PEVK exons were found, which contribute to titin’s extensible spring properties. Additionally, 3 unique I-band titin exons were identified (named novex-1 to -3). Novex-3 functions as an alternative titin C-terminus. The novex-3 titin isoform is ≈700 kDa in size and spans from Z1-Z2 (titin’s N-terminus) to novex-3 (C-terminal exon). Novex-3 titin specifically interacts with obscurin, a 721-kDa myofibrillar protein composed of 57 Ig/FN3 domains, followed by one IQ, SH3, DH, and a PH domain at its C-terminus. The obscurin domains Ig48/Ig49 bind to novex-3 titin and target to the Z-line region when expressed as a GFP fusion protein in live cardiac myocytes. Immunoelectron microscopy detected the C-terminal Ig48/Ig49 obscurin epitope near the Z-line edge. The distance from the Z-line varied with sarcomere length, suggesting that the novex-3 titin/obscurin complex forms an elastic Z-disc to I-band linking system. This system could link together calcium-dependent, SH3-, and GTPase-regulated signaling pathways in close proximity to the Z-disc, a structure increasingly implicated in the restructuring of sarcomeres during cardiomyopathies.
Titin is a giant filamentous protein that forms a separate myofilament system in both skeletal and cardiac muscle and is, after myosin and actin, the third most abundant muscle protein.1–3 Immunolabeling studies using epitope-mapped titin-specific antibodies have shown that titin’s 80-kDa N-terminal segment spans the Z-line lattice so that titins from adjacent sarcomeres overlap inside the Z-line in an antiparallel fashion.4 Titin’s I-band region contains multiple, distinct elastic spring elements that account for the elastic properties of the titin filament system. In heart muscle, titin’s elastic spring supports ventricular filling during diastole (for a review, see Linke and Granzier5). Within titin’s I-band segment, a plethora of splice isoform variants have been identified, which explain why titins range in size from 27 000 to 33 000 residues in different striated muscle tissues (eg, heart muscle N2B isoform: 2970 kDa; soleus skeletal muscle isoform: 3700 kDa).6,7 These differential splicing events control the number of spring elements and the size of titin’s I-band segments expressed (eg, ≈800 kDa in heart muscle N2B isoform; ≈1.5 MDa in soleus muscle isoform). The C-terminal 2.1-MDa A-band segment of titin is attached to the thick filaments via its multiple binding sites for myosin and C-protein (for a review, see Trinick and Tskhovrebova8). Similar to the overlap of its N-terminal ends in Z-lines, the C-terminal titin regions from adjacent half-sarcomeres overlap in the M-line.9 As a result of the overlap of titin’s N- and C-termini, titins form a continuous filament system along the full length of the myofibril. This makes titin an ideal candidate for functioning as a template in sarcomere assembly and turnover and for generating and transmitting passive tensions.
On the genomic level, titin is encoded by a single gene located on the long arm of chromosome 2 in both human and mouse.10,11 Recently, we identified 2 bacterial artificial chromosome (BAC) clones that contain the entire coding region of the human titin gene. Analysis of titin’s 294-kb gene sequence reveals 50 novel exons within its 5′ 170-kb genomic portion. Particularly remarkable is a novel 6.5-kb exon located 5′ of the heart-specific N2B exon, referred to as novex-3. Novex-3 is expressed in all striated muscle tissues and functions as an alternative 3′ end, located 220 kb 5′ of the C-terminal M10 exon of conventional titins. Therefore, in addition to half-sarcomere spanning, full-length titins, the titin locus also expresses the truncated novex-3 titin isoform that may constitute a Z-disc-I-band linking filament system. Interestingly, novex-3 titin interacts with the 721-kDa giant protein obscurin, which contains an IQ, SH3, DH, and a PH domain within its C-terminal region.12 PH and DH domains act as guanidine nucleotide exchange factors for Rho/RAC/Cdc42-like GTPAses. IQ domains can interact with calmodulin, whereas SH3 domains generally participate in dynamic multicomponent protein complexes (see http://smart.embl-heidelberg.de/ and Young et al12 and Shaw13) Further studies of the giant novex-3 titin/obscurin protein complex and of obscurin’s potential cellular signaling domains are likely to improve our understanding of Z-line-based signaling pathways and their involvement in cardiac myopathies.
Materials and Methods
A human BAC library was probed with pooled partial titin cDNAs, representing both the 5′ and 3′ regions of titin, respectively (Incyte Genomics, St Louis, Mo). Four titin BAC clones were identified, and their polymerase chain reaction (PCR)-typing showed that the BAC clone No. 14104 spanned from titin’s 5′ end region to I86, whereas the BAC clone No. 23155 extended from I84 to titin’s 3′ end (Figure 1). The complete sequence of BAC No. 14104 was determined by Medigenomix (Munich, Germany), whereas ≈96% of the 3′ BAC No. 23155 was found in the public databases. Three 1 to 2 kb segments not found in the public databanks were amplified and sequenced. The 294 kb annotated titin sequence assembled from BAC No. 14104 and BAC No. 23155 is available from the EMBL data library under accession AJ277892 and from http://www.embl-heidelberg.de/ExternalInfo/Titin/. The obscurin gene sequence spanning from I4 to its putative 3′ end12 was identified from Celera’s database (Celera contig GA_x2HTBKLELYV).
Isoform Transcript Studies
RNAs from human tissues (Stratagene, Heidelberg, Germany) were analyzed by reverse transcriptase (RT)-PCR with combinations of I9S to I29R primers essentially as described.7 Rapid amplification of cDNA ends (RACE) was performed with human heart marathon-ready cDNAs (Clontech, La Jolla, Calif) using the Advantage 2 Polymerase (Clontech). Novex-3 polyadenylated transcripts were amplified with a reverse primer including 12 nucleotides from the 3′ end of novex-3 followed by T18 (T18: TTTTTTTTTTTTTTTTTTAGGTAGCCAAGG; see Figure 2E).
Antibodies, SDS Page, and Western Blot Analysis of Titin
A novex-3 fragment (N-terminal of I21, see Figure 5A) and obscurin fragments (F6/I7 and I48/I49/F50, respectively; see Figure 5C) were expressed as His6-fusion proteins in E. coli and purified on Ni-NTA columns essentially as described.4,14 Rabbit polyclonal antibodies were raised and affinity-purified by Biogenes (Berlin, Germany). Gel electrophoresis and Western blot conditions were as described previously.7,15–17 The molecular mass of novex-3 titin and obscurin were estimated by comparing their mobility on SDS-gels with that of titin T2 (2.1 MDa), nebulin (Mr 780 kDa), and myosin heavy chain (205 kDa).
Yeast 2-Hybrid (Y2H) Screens and GST Pulldown Assays
Novex-3 exon fragments were amplified from the BAC clone No. 14104 by PCR.18 Amplified novex-3 fragments were inserted into the BTM117c vectors19 to obtain LexA-BD fusions. Screening of human skeletal and cardiac muscle cDNA libraries (HL4010AB and HL4042AH, Clontech, Palo Alto, Calif) by the Y2H method, identification of β-galactosidase expressing prey clones, in vitro transcription and translation of titin novex-3 and obscurin in the presence of [35S] Methionine (Amersham Pharmacia Biotech) and glutathione S-transferase (GST) pulldown assays were performed as described.20
Immunoelectron and Immunofluorescence Microscopy
Rat cardiac muscles were stretched, fixed, immuno-labeled, embedded, and processed for immunoelectron microscopy as described.16,21 For immunofluorescence microscopy, isolated rat skeletal myofibrils20 were incubated with anti-F6/I7 obscurin rabbit antiserum (1:100), followed by Texas Red conjugated donkey anti-rabbit IgG (1:600), antimyomesin monoclonal antibodies (generously provided by Drs Perriard and Ehler, Institute for Cell Biology, Zurich, Switzerland),22 and Cy-2 conjugated goat anti-mouse IgG (1:600). All coverslips were analyzed and prepared for presentation as described.20
Cell Culture and Transfection Procedures
For expression studies, an obscurin fragment (coding for I48/I49/F50) was amplified by PCR and cloned into pEGFP-C1 (Clontech, Palo Alto, Calif). The recombinant pEGFP-C1 construct was purified using QIAGEN columns and verified by sequencing. Primary cultures of rat cardiac myocytes were isolated and maintained as described.23 Cells were plated and transfected as described.20 The transfected cells were incubated with antimyomesin B4 antibodies, followed by Texas red-conjugated goat anti-mouse IgG+IgM (1:600; Jackson ImmunoResearch Laboratories). Over 200 transfected cells were analyzed as described.20
Titin’s Complete Gene Sequence Reveals 363 Exons in a 283-kb Segment and LINE-1 Associated Duplication Events
Alignment of the titin cardiac and skeletal cDNA sequences6 to the gene sequence revealed that the titin N-terminus encoding the Z1 exon and the titin C-terminus encoding M10 exon are 283 kb apart. The known 101 kb of titin coding sequences can be assigned to 313 exons (Figure 1). Fifty additional exons in the gene sequence were discovered by Artemis, Genefinder, SMART,24 and by homology to PEVK/Ig repeat consensus sequences (see annotations to AJ277892).
In the PEVK region, 47 exons were identified that are absent in the known cardiac and skeletal cDNA sequences. A subset of these exons have recently been noted in human fetal transcripts25 (in Figure 1 marked in orange), raising the possibility of their fetal-specific expression. About 75% of the PEVK exons correspond to phased 78 to 90 nucleotide units, which on the protein level code for conserved 26- to 30-residue motifs.7
Remarkably, the central PEVK region contains a 10.5-kb segment that is composed of 3 nucleotide repeats, A, B, and C (Figure 1). A and B are both 4262 bp in length and are 99.7% conserved, whereas C is 1860 bp in length and is 99.2% conserved to the 5′ regions of A and B. The two 2.5 kb introns separating A/B and B/C, respectively, correspond to incomplete 5′ truncated LINE-1 repeats, mobile elements known to have randomly moved around in mammalian genomes during evolution. The 99.7% conservation of the LINE-1 repeats suggests that the duplication of A and B occurred about 1 million years ago, whereas the 99.2% conservation of B and C would be consistent with a Line-1 insertion about 3 million years ago.26 The >99% conserved and >4 kb long nucleotide repeats A, B, C are a technical challenge for correct sequence fragment assembly. This, in part, may explain why some titin exons were missed in previous titin cDNA sequencing projects6 and in the analysis of the human genome based on shot-gun approaches.27,28
Three novel exons, 375, 576, and 6398 bp in length were identified 5′ of titin’s heart-specific N2B exon and are referred to as titin novex-1 to novex-3, respectively (Figure 1). Novex-1, -2, and -3 together contain 8 Ig repeats, which correspond to Ig repeats I16 to I24, when using a revised Ig nomenclature based on the titin gene sequence (Figure 1; see also7).
Striated Muscle-Specific Expression of Novex-1, -2, and -3 and Polyadenylation of Novex-3
RT-PCR studies using I9-sense to I27-reverse primer pairs showed that novex-1, -2, and -3 are expressed in the human heart and skeletal muscles. Semiquantitative PCR experiments suggested that novex-1, -2, and -3 transcripts are less abundant in skeletal than in heart muscle (Figures 2A through 2C). Splicing together of the I16 and N2B exons corresponds to a heart-specific isoform, whereas the splicing of I17 to I27 was found in both heart and skeletal muscles. The I16/I17 exons appeared to be mutually exclusive because they were never amplified in the same fragments (Figure 3). I23-sense primers in combination with downstream reverse primers did not amplify fragments extending to the 3′ end. Therefore, we performed cDNA extensions to the 3′ end from novex-3 by anchored PCR. This identified novex-3 transcripts with poly-A-tails (Figures 2D and 2E). Consistent with this, the intron downstream of novex-3 contains polyadenylation signals (Figure 2E).
If novex-3 is an alternative C-terminus, novex-3 titin should represent an unusually small titin isoform. To test this, we raised antibodies to the novex-3 exon. These antibodies recognized, on Western blots of cardiac and skeletal muscle tissues, a protein of ≈700 kDa. In skeletal muscles, novex-3 titin migrated just below nebulin (Figure 4A). Novex-3 titin was not detected in smooth muscle and other nonmuscle tissues (Figure 4A).
The novex-3 titin molecular mass of ≈700 kDa suggests that most or all exons 5′ of novex-3 are included in this isoform. To test if the most N-terminal region of the conventional titins is included in novex-3 titin, we performed Western blot studies using our anti-Z1-Z2 antibodies.4 When using the highly sensitive enhanced chemiluminescence (ECL) method, we detected reactivity to both a ≈3000-kDa and ≈700-kDa species (Figure 4B). Comparison of the intensities suggested that novex-3 titin is more than one order of magnitude less abundant than conventional titins.
Novex-3 Titin Interacts With Obscurin, a Novel 721-kDa Myofibrillar Protein
To search for potential ligands of the novex-3 titin isoform, Y2H studies were performed with 5 baits covering the novex-3 exon (novex-3.1 to novex-3.5; see Figure 5A). Confirmed prey clones were only obtained with one bait (novex-3.5). Novex-3.5 includes the C-terminal 564 amino acids of novex-3, corresponding to a nonrepetitive segment and the 3 Ig repeats I21/I22/I23. From 650 000 skeletal muscle and 450 000 cardiac muscle cDNA prey clones screened, 14 potentially interacting prey clones were identified. Five of these, 3 from the skeletal and 2 from the cardiac library screen, were positive for β-galactosidase expression; their inserts corresponded to partial cDNAs of the giant obscurin protein.12 To narrow down the binding sites of novex-3 titin and obscurin, deletion analysis was performed. This showed that a 198 residue unique sequence N-terminal to the novex-3 Ig repeat I21 is sufficient for the interaction with obscurin (corresponding to bps 73 011 to 73 599 of accession AJ277892, see Figure 5A). Interestingly, this sequence contains 6 repeats of the amino acid sequence RYSTPPGETLE (Figure 5A). In obscurin, the Ig-repeats I48/I49 interact with novex-3 as shown by GST pulldown assays (Figure 5B) and Y2H deletion constructs (Figure 5C). A search of the Celera database assigned the obscurin cDNA to the contig GA_x2HTBKLELYV on human chromosome 1q41.2. Analysis of this contig by Genefinder (http://dot.imgen.bcm.tmc.edu:9331/gene-finder/gf.html) and SMART24 revealed 55 Ig repeats, 2 FN3 repeats, and also IQ, SH3, DH, and PH domains within obscurin’s C-terminal region (Figure 5C). This is consistent with the obscurin cDNA sequencing studies of Gautel and colleagues.12
On Northern multiple tissue dot blots, both obscurin and novex-3 titin mRNA expression appeared to be specific to striated muscle tissues (Figure 5D). On Western blots, the obscurin-specific antisera recognized a ≈750-kDa species from rat heart muscle (Figure 4C).
Localization of the Novex-3/Obscurin Complex
To investigate the localization of the obscurin/novex-3 titin complex in the sarcomere, antibodies were raised to obscurin’s F6/I7 domains (close to its N-terminus), and to the I48/I49/F50 domains (located C-terminal of obscurin’s tandem Ig segment; see Figure 5C). The antiserum directed to the N-terminal F6/I7 epitopes stained both the Z- and M-line region by immunoelectron microscopy (Figure 6A). The C-terminally directed I48/I49/F50 antiserum localized these epitopes within the I-band region close to the Z-line (Figure 6B). When comparing different sarcomere lengths, the distance of this epitope to the periphery of the Z-line ranged from ≈60 to ≈120 nm at sarcomere lengths from 1.8 to 2.3 μm (Figures 6C and 2D). Similarly, in immunofluorescence microscopy studies using our anti-obscurin F6/I7 antibodies, in most preparations of heart and skeletal rat muscle isolated myofibrils or frozen sections, Z-line-associated staining was detected; however, in some preparations staining at the M-line was detected (obscurin Z-line staining on isolated rat skeletal myofibrils is shown: Figure 7B). Although we could not correlate the different staining patterns with the conditions of tissue isolation, antibodies used, or developmental stage, this staining paradox is consistent with that reported recently by others.12
Additionally, in immunofluorescence studies, our anti-novex-3 antibodies stained M-lines, whereas immunoelectron microscopy using the same antibodies did not show any specific label (data not shown). Whether potential novex-3 epitopes were not accessible under the conditions used, or if our novex-3 antibodies cross-react nonspecifically to M-line proteins, remains to be established.
As an independent approach to study the sarcomeric localization of obscurin, we expressed the obscurin fragment I48/I49/F50 (see Figure 5C) as a GFP fusion protein in live rat cardiac myocytes. Based on costaining with anti-myomesin antibodies that stain the M-line, we observed that the obscurin I48/I49/F50 fragment specifically targeted to the Z-line region in more than 80% of transfected cells (the remaining <20% demonstrated diffuse staining). This is consistent with our immunoelectron microscopy data (because it is difficult to resolve a distance of ≈60 nm from the Z-line by light microscopy) (Figure 7A).
We determined titin’s human gene sequence and found that a 283 kb genomic segment contains 363 titin exons, including 50 novel exons. Together, titin’s 363 exons have a coding capacity of 114 414 bp (4200 kDa), significantly higher than the 80 780-bp coding sequences found in 178 exons as part of the public human genome project27 and the 234 exons identified by Celera (hCT34523-Celera databank).28
Of the 50 novel exons, 47 exons code for PEVK elements. All novel PEVK exons and 70% of the previously identified PEVK exons are 84 to 99 nucleotides in length and code for conserved 28- to 33-residue motifs. These motifs may represent structural units of the titin PEVK spring.7 Interestingly, the number of PEVK motifs in the titin gene appears to have increased during evolution. Three highly conserved nucleotide repeats (boxes A through C in Figure 1) each contain 9 PEVK exons and share 99.7% (A and B) to 99.2% sequence identity (A and C). The two 2.5 kb introns separating A/B and B/C correspond to truncated Line-1 repeats. Line repeats are mobile elements that constitute about 16% to 21% of the human genome.26,27 Their expansion may have contributed to the development and extinction of mammalian species during evolution.27,29 In titin, LINE-1 mediated duplication events apparently modified the genomic region responsible for titin’s spring properties. Some of the duplicated PEVK exons have been identified in human cDNA transcripts from fetal tissues.25 This further supports the functional relevance of the duplication events, which presumably increased myofibril elasticity and/or muscle tissue diversity by expanding the differentially spliced PEVK region.7
The nucleotide divergence of A, B, and C (A and B: 0.3%; B and C: 0.8%) predicts that the two LINE-1 elements inserted roughly about 3 million (B) and 1 million (A) years ago, and therefore, after the divergence of humans and humanoids such as chimpanzee and orangutans.26 Further studies of the PEVK region in different mammals with respect to how many LINE-1 repeats are present should provide clues to explain how the PEVK titin region was restructured during evolution, eventually linking together speciation to species-specific adaptations of myofibril mechanics.
5′ of the PEVK and N2B spring elements, 3 novel exons novex-1, -2, and -3 were identified that are expressed in heart and skeletal muscles (Figure 2). Inclusion of the C-terminal novex-3 is linked to the expression of a ≈700-kDa titin isoform, which spans to the N-terminal Z1-Z2 Ig repeats, located at the periphery of the Z-line of the opposite sarcomere.4 Why are 2 very differently sized classes of titin expressed from the single titin gene locus? The >3000 kDa titin polypeptides are integral components of both thick and thin filament structures. To date, it is unclear how the presumed 6 titin molecules per half thick filament can participate in the assembly of structures, which have both a 3-fold (thick filaments) and 2-fold symmetry (thin filaments), a problem that has been referred to as the titin symmetry paradox.30 Because the truncated ≈700 kDa titin isoform can integrate into the tetragonal Z-line lattice but is too short to reach into the A-band, the coexpression of half-sarcomere spanning titins together with shorter titins could be involved in adjusting the titin filament to both 3- and 2-fold symmetries.
As a first attempt to study the functional role(s) of novex-3 titin, we performed Y2H studies using novex-3 baits. We identified obscurin, another giant myofibrillar protein,12 as a potential binding partner of novex-3. Expression of both novex-3 titin and obscurin transcripts are restricted to striated muscle tissues (Figure 5D).
Interestingly, the antiserum directed to obscurin’s F6/I7 epitopes stained both Z-lines and M-lines in adult rat heart and skeletal muscle (Figure 6A). Although we did not correlate obscurin staining patterns with the developmental stage of the striated muscle, this observation may be related to a recent report that also described an M-line localization of obscurin.12 In this study, a model was presented in which a fetal form/isoform of obscurin is present in Z-lines (≤10-somite stage in chick and E9.5 in mouse) whereas in later, fully-mature myofibrils, obscurin is redistributed and is localized to the M-line region.12 In contrast, the obscurin fragment I48/I49/F50 (which binds to novex-3 titin) localized exclusively to the Z-line region of the sarcomere when expressed in live neonatal rat cardiac myocytes containing mature myofibrils (Figure 7). Consistent with this, immunoelectron microscopy localized the endogenous C-terminal I48/I49/F50 obscurin epitope close to the Z-line in adult rat cardiac muscle (Figure 6C). The distance of this epitope to the Z-line increased with stretch up to 120 nm (Figures 6C and 6D). Future studies such as the expression of N- and C-terminal obscurin GFP fusion proteins are required to determine if the N- and C-terminal regions of obscurin are indeed differentially targeted during development.
The titin Z1-Z2 N-terminal modules, which are included in the novex-3-titin isoform (Figure 4B), have been shown previously to localize to Z-lines, presumably by their interaction with T-cap4 (also called telethonin31). This would be consistent with a model that the novex-3 titin N-terminus (Z1-Z2) integrates novex-3 titin within the Z-line, whereas its tandem Ig segment (I1 to I15) and its C-terminal novex-3 segment project ≈100 nm into the I-band (Figure 6). The extension of the obscurin I48/I49/F50 epitope to Z-line distance during stretch raises the possibility that both novex-3 titin and obscurin form filamentous structures that have elastic properties similar to conventional titins. These elastic properties may be conferred by the tandem Ig segments contained in both novex-3 titin and obscurin.
The novex-3 titin/obscurin complex appears to be another example of Z-line-associated proteins that is likely to participate in myofibrillar signaling. Other members of this group include muscle LIM protein (MLP),32 cardiac-restricted ankyrin repeat protein (CARP),33 T-cap/telethonin,4,31 palladin,34 and myopalladin.20 Because the novex-3 titin/obscurin complex extends when sarcomeres are stretched (Figure 6), the complex may have signaling properties that respond to the strain imposed on the sarcomere. We speculate that this complex may be involved in strain/stress-initiated sarcomeric restructuring that is known to occur during muscle development and cardiac disease.35–37
The discovery of novel titin exons and of the coexpression of ≈700-kDa and >3000-kDa titins will be relevant for future studies on genetic diseases linked to titin. Familial cardiac myopathies with dominant inheritance and linkage to titin gene markers have been identified.38 Similarly, a skeletal muscular dystrophy was identified in the Finnish population which affects distal leg muscles (TMD; tibialis anterior muscular dystrophy). TMD is recessive and is closely linked to titin markers.39 For both 2q-linked familiar cardiac myopathies and for TMD, no disease-causing mutations have been identified thus far. Mutational searches of the 363 titin exons are likely to address whether mutations are present in the titin gene and whether particular cardiomyopathies and muscular dystrophies are indeed caused by dysfunctions of the titin filament system.
We gratefully acknowledge the support of the Deutsche Forschungsgemeinschaft (La 668/6-1 to S.L.), the American Heart Association (No. 0120009Y to M.L.B.), and the National Institutes of Health (HL61497 and HL62881 to H.G.; HL03985 and HL63926 to C.C.G.). We thank Mamata Pochampalli for initiating the GFP-targeting experiments, Joseph Bahl for rat cardiac myocytes, and Brenda Gerull for genome database analysis.
↵*Both authors contributed equally to this work.
Original received June 15, 2001; revision received October 17, 2001; accepted October 18, 2001.
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