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The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Gregorio CC, Trombitás K, Centner T, Kolmerer B, Stier G, Kunke K, Suzuki K, Obermayr F, Herrmann B, Granzier H, Sorimachi H, Labeit S - J. Cell Biol. (1998)

Bottom Line: In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region.Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap.Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell Biology and Anatomy, and Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85724, USA. gregorio@u.arizona.edu

ABSTRACT
Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

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Comparison of the T-cap protein sequences from human and mouse predicted by their respective 950-bp full-length  cDNAs. The 19-kD T-cap proteins share 90% sequence identity  and 95% sequence similarity between species. The human T-cap  protein sequence is identical to the recently described telethonin  entry (EMBL data library AJ000491) derived from anonymous  EST entries (Valle et al., 1997); the mouse sequence data from  skeletal muscle are available from GenBank/EMBL/DDBJ under accession number Y14845.
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Figure 4: Comparison of the T-cap protein sequences from human and mouse predicted by their respective 950-bp full-length cDNAs. The 19-kD T-cap proteins share 90% sequence identity and 95% sequence similarity between species. The human T-cap protein sequence is identical to the recently described telethonin entry (EMBL data library AJ000491) derived from anonymous EST entries (Valle et al., 1997); the mouse sequence data from skeletal muscle are available from GenBank/EMBL/DDBJ under accession number Y14845.

Mentions: Titin has a characteristic tissue-specific and developmental expression pattern. In the mouse, titin transcripts first appear in the heart anlage at day 8 pc and remain abundantly expressed in the heart throughout development, whereas expression in the somites occurs about 1 d later (Kolmerer et al., 1996). To determine whether T-cap transcripts have the same characteristics, we performed in situ hybridization of whole-mount embryos using probes from the mouse titin-cap cDNA. The T-cap mouse cDNA sequence was obtained by cross-species PCR (sequence data available from EMBL under accession number Y14845). The comparison of the T-cap sequences from human and mouse predicted by their respective 950-bp full-length cDNAs revealed that the encoded 19-kD T-cap proteins share 90% sequence identity and 95% sequence similarity between species (Fig. 4). At day 9.5 pc, T-cap transcripts are detected in the developing heart and in the somites. At this stage, the transcription pattern of T-cap in the heart and somites is very similar to the transcripts of titin, except that transcripts for T-cap are also detected in the otic vesicle (Fig. 5, A and B). In a survey of adult tissues by RT-PCR, T-cap transcripts were found exclusively in striated muscles, including human fetal heart, adult heart, and skeletal muscles, but were absent in normal and pregnant uterus, fetal brain, liver, and spleen (Fig. 5 C). We conclude that the transcription pattern of titin and T-cap are similar at day 9.5 pc. The significance of the presence of T-cap (but not titin) transcripts in the otic vesicle and the observation that titin transcripts are detectable ∼1 d earlier in somites than T-cap transcripts will require further studies.


The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Gregorio CC, Trombitás K, Centner T, Kolmerer B, Stier G, Kunke K, Suzuki K, Obermayr F, Herrmann B, Granzier H, Sorimachi H, Labeit S - J. Cell Biol. (1998)

Comparison of the T-cap protein sequences from human and mouse predicted by their respective 950-bp full-length  cDNAs. The 19-kD T-cap proteins share 90% sequence identity  and 95% sequence similarity between species. The human T-cap  protein sequence is identical to the recently described telethonin  entry (EMBL data library AJ000491) derived from anonymous  EST entries (Valle et al., 1997); the mouse sequence data from  skeletal muscle are available from GenBank/EMBL/DDBJ under accession number Y14845.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132961&req=5

Figure 4: Comparison of the T-cap protein sequences from human and mouse predicted by their respective 950-bp full-length cDNAs. The 19-kD T-cap proteins share 90% sequence identity and 95% sequence similarity between species. The human T-cap protein sequence is identical to the recently described telethonin entry (EMBL data library AJ000491) derived from anonymous EST entries (Valle et al., 1997); the mouse sequence data from skeletal muscle are available from GenBank/EMBL/DDBJ under accession number Y14845.
Mentions: Titin has a characteristic tissue-specific and developmental expression pattern. In the mouse, titin transcripts first appear in the heart anlage at day 8 pc and remain abundantly expressed in the heart throughout development, whereas expression in the somites occurs about 1 d later (Kolmerer et al., 1996). To determine whether T-cap transcripts have the same characteristics, we performed in situ hybridization of whole-mount embryos using probes from the mouse titin-cap cDNA. The T-cap mouse cDNA sequence was obtained by cross-species PCR (sequence data available from EMBL under accession number Y14845). The comparison of the T-cap sequences from human and mouse predicted by their respective 950-bp full-length cDNAs revealed that the encoded 19-kD T-cap proteins share 90% sequence identity and 95% sequence similarity between species (Fig. 4). At day 9.5 pc, T-cap transcripts are detected in the developing heart and in the somites. At this stage, the transcription pattern of T-cap in the heart and somites is very similar to the transcripts of titin, except that transcripts for T-cap are also detected in the otic vesicle (Fig. 5, A and B). In a survey of adult tissues by RT-PCR, T-cap transcripts were found exclusively in striated muscles, including human fetal heart, adult heart, and skeletal muscles, but were absent in normal and pregnant uterus, fetal brain, liver, and spleen (Fig. 5 C). We conclude that the transcription pattern of titin and T-cap are similar at day 9.5 pc. The significance of the presence of T-cap (but not titin) transcripts in the otic vesicle and the observation that titin transcripts are detectable ∼1 d earlier in somites than T-cap transcripts will require further studies.

Bottom Line: In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region.Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap.Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell Biology and Anatomy, and Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85724, USA. gregorio@u.arizona.edu

ABSTRACT
Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

Show MeSH