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Titin (TTN)

( CMH-9, CMD-1G, TMD, LGMD-2J )

(last modified February 10, 2006)


Contents



Summary


Titin (TTN, connectin in chicken) is a giant filamentous protein expressed in cardiac and skeletal muscle. Titin is, after myosin and actin, the third most abundant protein in muscle. Titin spans the entire half of the sarcomere from Z-disk to M-line and is of critical importance for muscle assembly, structural integrity, force transmission at the Z-line and maintenance of resting tension in I-band region. Titin has been shown to interact with a range of sarcomeric proteins, including TCAP (telethonin) and alpha-actinin at the Z-line region, CAPN3 and obscurin at the I-band region, and myosin binding protein C, calmodulin and again CAPN 3 at the M-line region.

Bang et al. (2001) reported that the complete sequence of the human titin gene, spread over 294 kb on the long arm of human chromosome 2 (2q31), contains 363 exons together encoding for 38,138 amino acid residues (4.2 MDa). 

Mutations in the TTN gene have been associated with several genetic diseases; hypertrophic cardiomyopathy (CMH-9, Satoh et al. 1999), autosomal dominant dilated cardiomyopathy (CMD1G, Gerull et al. 2002), autosomal dominant tibial muscular dystrophy (TMD, Hackman et al. 2002) and limb-girdle muscular dystrophy (LGMD-2J, Hackman et al. 2002) . Autoantibodies to titin have been found in patients with  scleroderma, an autoimmune disease.


The titin gene


Links to other databases:
Gene Symbol nomenclature   EntrezGene    OMIM Gene Map   GDB

The titin gene (Gene Symbol TTN) is located on the long arm of human chromosome 2 (2q31). Since part of the gene is duplicated and since it contains a range of small exons, its structure is difficult to determine exactly. Bang et al. (2001) reported the complete sequence of the human titin gene to contain 363 exons, spread over 294 kb. Some exons were predicted using gene prediction software or based on homology, encoding PEVK/Ig repeat consensus sequences. Some of these exons are absent from known cardiac and skeletal muscle transcripts but were present in fetal transcripts.

...N2-A and a cardiac specific N2-B exon.  .... TTN contains 3 unique I-band exons (designated novex-1 to -3, Bang 2001). The novex-3 exon functions as an alternative titin C-terminus. The exon lies 220 kb 5' of the C-terminal M10 exon of normal titins.

In its central I-band region, the TTN gene contains numerous so called PEVK exons. Most PEVK-exons are 78 to 90 bp, encoding conserved 26 to 30 amino acid protien motifs. The central PEVK-region contains a 10.5-kb duplicated segment composed of 3 repeats (A, B, and C, Bang 2001); A and B measure 4,262 bp and are 99.7% similar. Repeat C is 1860 bp and 99.2% similar to the 5' ends of repeats A and B. The two 2.5 kb introns separating A/B and B/C correspond to incomplete 5' LINE-1 repetitive elements. The number of PEVK motifs in the TTN gene seems to have increased during recent evolution. 

Directly 5' (~25 kb to the telomeric side) the TTN gene is flanked by the predicted FLJ39502 gene, further upstream (~300 kb) by the SESTD1 gene (SEC14 and spectrin domains 1). On the centromeric side, ~20 kb downstream, the gene is flanked by the PLEKHA3 gene (pleckstrin homology domain containing, family A, member 3).

Summary table TTN gene (363 exons)


The titin mRNA


Links to other databases:     UniGene: Hs.134602    RefSeq: NM_133378, NM_133437, NM_133432, NM_003319, NM_133379

Transcription of the titin gene has a coding potential of 114,414 bp (4.2 MDa). Transcription can terminate in exon 48 (novex-3 isoform), .... and exon 363. RT-PCR analysis (Bang 2001) showed that isoforms novex-1 to -3 are expressed in human heart and skeletal muscle (most abundant in heart). Splicing of novex-1 and -2 exons is mutually exclusive. The Ig16/N2B isoform is only expressed in heart muscle, while Ig17-Ig27 splicing can be found in both heart and skeletal muscle.

In TTN's I-band region, a range of splice variants have been identified, encoding  titin isoforms ranging in size from 27,000 to 33,000 residues. Effectively, this differential splicing controls the number of spring elements and the size of the titin I-band segments expressed. 

primers for RNA-amplification


The titin protein


Links to other databases:   RefSeq: NP_003310 (N2-B), NP_597681 (novex-2), NP_597676 (novex-1), NP_596869 (N2-A), NP_596870 (novex-3)

The titin protein can be divided into two main regions, an N-terminal I-band and a C-terminal A-band. The I-band is the
elastic part of the molecule and contains two regions of tandem immunoglobulin domains on either side of a so called PEVK region. The PEVK region is rich in amino acids for proline, glutamate, valine and lysine. The A-band is thought to act as a protein-ruler; it contains a large series of both immunoglobulin and fibronectin domains, and it possesses a kinase activity. An N-terminally located Z-disc region and a C-terminal M-line region bind to the Z- and M-line of the sarcomere resp.; consequntly a single titin molecule spans half the length of a sarcomere. In addition titin contains binding sites for several muscle proteins and probably serves as an adhesion site for the assembly of the contractile machinery in muscle cells. 

Considerable variability exists in the I-band, the M-line and the Z-disc regions of titin. Variability in the I-band region contributes to the differences in elasticity of different titin isoforms and consequently to the differences in elasticity of different muscle types. 

 

Together, the 363 exons of the titin gene encode for 38, 138 amino acid residues (4,200 kDa). 

Using antibodies raised against the novex-3 isoform, Bang et al. (2001) detected  a ~0.7 MDa titin isoform expressed in cardiac and skeletel muscle but not in smooth muscle and other non-muscle tissues. Antibodies against titin's N-terminus (Z1 and Z2 domains), detected bands of ~3 and 0.7 MDa; the 0.7 MDa isoform was one order of magnitude less abundant. Thus, in addition to half-sarcomere spanning full-length titins, the titin gene also encodes shorter isoforms that may constitute a Z-disc-I-band linking filament system. 

As a result of the overlap of titinís N- and C-terminal ends, titins form a continuous filament system along the full length of the myofibril. Titin spans the entire half of the sarcomere from Z-disk to M-line and is of critical importance for muscle assembly, structural integrity, force transmission at the Z-line and maintenance of resting tension in I-band region.

Immunolabeling studies using titin-specific antibodies have shown that titinís 80-kDa N-terminal segment spans the Z-line lattice such that titins from adjacent sarcomeres overlap inside the Z-line in an antiparallel fashion (Gregorio 1998). Titinís I-band region contains multiple spring elements that account for the elastic properties of the titin filament system. Differential splicing in the I-band region yields a range of titin isoforms, varying in size from 27, 000 to 33, 000 amino acid residues (e.g. the 2,970 kDa N2B-isoform in heart muscle and the 3,700 kDa isoform in soleus skeletal muscle). Due to the differential splicing, the number of spring elements and the size of the I-band varies (e.g. ~800 kDa in the heart muscle N2B- isoform and ~1,500 kDa in the soleus muscle isoform). The C-terminal end contains a 2,100 kDa A-band segment. This segment is attached to the thick filaments via multiple myosin binding sites and C-protein ( Trinick & Tskhovrebova 1999). Like the N-terminal ends in the Z-lines, the C-terminal ends from adjacent half-sarcomeres overlap in the M-line (Obermann 1996).

Novex-3 specifically interacts with obscurin, a 721-kDa myofibrillar protein. 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 cardiac myocytes (Bang 2001). Immuno-electron 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 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 (Bang 2001).

Titin has been shown to interact with a range of sarcomeric proteins, including TCAP (telethonin) and alpha-actinin at the Z-line region, CAPN 3 and obscurin at the I-band region, and myosin binding protein C, calmodulin and again CAPN 3 at the M-line region. The titin interaction with alpha-actinin (at the 7 Z-repeats) and alpha-actin at the Z-line of striated muscles consists of a ternary complex.

(15, 16). It is known that titin is
interacting with rod-portion of myosin heavy chain and
myosin binding protein-C in the A band region (15). It
also is known that titin forms a ternary complex with
a-actinin and a-actin at the Z-disk of striated muscles
(17, 18). The Z-disk region of titin has tandem
Z-repeats in the middle of Z-disk (19), and it has been

reported that C-terminal region of a-actinin interacts
with these titin Z-repeats (18, 20). These observations
prompted us to search for a titin mutation in the patients
with HCM, who had no mutation in the known
disease genes, because the titin gene is a good candidate
of the disease gene for HCM.

Titin antibodies


Titin and disease: LGMD-2J


Links to other databases:   OMIM: CMH-9,   CMD-1G,   TMD,   LGMD-2J

Mutations in the TTN gene have been associated with several genetic diseases (see below). Autoantibodies to titin have been found in patients with scleroderma, an autoimmune disease.

CMH-9 (OMIM 192600): mutations in the TTN gene were first reported by Satoh et al. (1999) upon analysis of a Japanese patient with hypertrophic cardiomyopathy (designated CMH-9). CMH is a form of hereditary cardiomyopathy, generally caused by mutations in the genes for sarcomeric proteins in the cardiac muscle. The CMH-related titin mutation in the patient resided in the Z-line region.

CMD-1G (OMIM 604145): Gerull et al. (2002) were first to report the presence of heterozygous TTN mutations in two large Japanese families with autosomal dominant dilated cardiomyopathy (designated CMD1G-OMIM). Both families showed reduced penetrance and, surprisingly since both are expressed in cardiac and noncardiac muscle isoforms, no involvement of noncardiac muscles.

TMD (OMIM 600334): Tibial Muscular Dystrophy is an autosomal dominant late-onset distal myopathy first described in Finnish patients (Udd et al. 1991, Udd et al. 1993). In TMD, weakness and atrophy are usually confined to the anterior compartment of the lower leg, in particular the tibialis anterior muscle. Generally clinical symptoms occur at age 35-45y or later. Cardiomyopathy has not been diagnosed in TMD patients. TMD linkage in the Finnish families (prevalence 16/100,000) was found to a 1 cM region on chromosome 2q31, between markers D2S2173 and D2S2310 (Haravuori et al. 1998). All Finnish patients carry a common haplotype, indicating a single ancestral founder mutation. Linkage to 2q31 was confirmed in a French TMD family showing another haplotype (de Seze et al. 1998). Mutations in the TTN gene for both these families were described recently by Hackman et al. (2002).

LGMD-2J (OMIM 608807): Haravuori et al. 1998 identified three patients with a more severe childhood-onset limb-girdle muscular dystrophy (LGMD-2J) phenotype that were homozygous for the Finish TMD haplotype. Hackman et al. (2002) confirmed the presence of a homozygous Finish mutation, an 11 bp deletion/insertion in the last exon (exon 363) of the TTN gene.

Titin sequence variations (mutations and polymorphisms)


Miscellaneous


DNA-primers

Exons 1-17 (Satoh 1999)

RNA-primers

none reported


Titin sequences




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