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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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Interaction of T-cap and the  titin Z1-Z2 domains. (a) Titin repeats Z1  and Z2 specifically interact with the 19-kD  titin-cap protein by yeast two-hybrid analysis. Numbers above the schematic layout  of the NH2-terminal titin region indicate  the nucleotide residue numbers corresponding to the human entry (EMBL data  library X90568) (top) and residue numbers of the deduced amino acid sequence  (bottom). Two immunoglobulin domains  at the NH2 terminus of titin are indicated  as Z1 and Z2, respectively. Bars indicate  positions of the cDNAs used for the bait plasmid (pAS2-Z1-Z2-is1), and the truncation constructs, pAS2-Z1-Z2 (containing the  Z1 and Z2 domains), pAS2-Z1 (containing Z1 domain only), and  pAS2Z2-is1 (containing the Z2 domain fused to residues 200– 257), used for the binding assay to T-cap. +/− signs refer to  growth on minus (Leu, Trp, and His) plates supplemented with  3-AT, and numbers in parentheses refer to β-galactosidase activities (arbitrary units). (b) Interaction of the NH2-terminal 16 kD  of T-cap with the titin Z1-Z2 Ig domains in the yeast two-hybrid  system. Schematic of T-cap cDNA clone. Numbers above the  titin-cap cDNA indicate the nucleotide residues of the human  full-length cDNA sequence (sequence data available from  EMBL under accession number AJ000491; from Valle et al.,  1997) (top) and residue numbers of the deduced amino acid sequence (bottom). AA...A, a poly(A+) tail at the 3′ terminus of the  corresponding cDNA clone. Bars indicate positions of cDNA inserts derived from the two-hybrid screening using pAS2-Z1-Z2-is1 (see a) as bait (T-cap-1, -3, -5, -6, and -7), and the truncation  constructs of T-cap (T-cap-Δ1 and -Δ2) described in Materials  and Methods. +/− signs refer to growth on minus (Leu, Trp, and  His) plates supplemented with 3-AT, and numbers in parentheses  refer to β-galactosidase activities (arbitrary units). (c) Interaction  of expressed T-cap and titin Z1-Z2 peptides. A Coomassie blue– stained 18% Laemmli SDS gel of a whole-cell lysate of BL21 cells  expressing tagless titin Z1-Z2 (lane C). These cells were mixed  with a His6-tagged 16-kD fragment of T-cap and lysed. Lysates  were passed over Ni-NTA agarose columns. Interaction of a stable complex of titin Z1-Z2 and T-cap is indicated by retaining  also the nontagged titin Z1-Z2 on the column (arrow marks Z1-Z2 and T-cap in third lane). Lane M, molecular mass markers as  in legend for Fig. 2.
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Figure 3: Interaction of T-cap and the titin Z1-Z2 domains. (a) Titin repeats Z1 and Z2 specifically interact with the 19-kD titin-cap protein by yeast two-hybrid analysis. Numbers above the schematic layout of the NH2-terminal titin region indicate the nucleotide residue numbers corresponding to the human entry (EMBL data library X90568) (top) and residue numbers of the deduced amino acid sequence (bottom). Two immunoglobulin domains at the NH2 terminus of titin are indicated as Z1 and Z2, respectively. Bars indicate positions of the cDNAs used for the bait plasmid (pAS2-Z1-Z2-is1), and the truncation constructs, pAS2-Z1-Z2 (containing the Z1 and Z2 domains), pAS2-Z1 (containing Z1 domain only), and pAS2Z2-is1 (containing the Z2 domain fused to residues 200– 257), used for the binding assay to T-cap. +/− signs refer to growth on minus (Leu, Trp, and His) plates supplemented with 3-AT, and numbers in parentheses refer to β-galactosidase activities (arbitrary units). (b) Interaction of the NH2-terminal 16 kD of T-cap with the titin Z1-Z2 Ig domains in the yeast two-hybrid system. Schematic of T-cap cDNA clone. Numbers above the titin-cap cDNA indicate the nucleotide residues of the human full-length cDNA sequence (sequence data available from EMBL under accession number AJ000491; from Valle et al., 1997) (top) and residue numbers of the deduced amino acid sequence (bottom). AA...A, a poly(A+) tail at the 3′ terminus of the corresponding cDNA clone. Bars indicate positions of cDNA inserts derived from the two-hybrid screening using pAS2-Z1-Z2-is1 (see a) as bait (T-cap-1, -3, -5, -6, and -7), and the truncation constructs of T-cap (T-cap-Δ1 and -Δ2) described in Materials and Methods. +/− signs refer to growth on minus (Leu, Trp, and His) plates supplemented with 3-AT, and numbers in parentheses refer to β-galactosidase activities (arbitrary units). (c) Interaction of expressed T-cap and titin Z1-Z2 peptides. A Coomassie blue– stained 18% Laemmli SDS gel of a whole-cell lysate of BL21 cells expressing tagless titin Z1-Z2 (lane C). These cells were mixed with a His6-tagged 16-kD fragment of T-cap and lysed. Lysates were passed over Ni-NTA agarose columns. Interaction of a stable complex of titin Z1-Z2 and T-cap is indicated by retaining also the nontagged titin Z1-Z2 on the column (arrow marks Z1-Z2 and T-cap in third lane). Lane M, molecular mass markers as in legend for Fig. 2.

Mentions: Next, we attempted to identify more precisely the binding sites for the titin/T-cap interaction. Truncation analysis of the bait plasmid, pAS2-Z1-Z2-is1, using the constructs pAS2-Z1-Z2 (containing the Z1 and Z2 domains), pAS2-Z1 (containing only the Z1 domain), and pAS2-Z2-is1 (containing the Z2 domain fused to residues 200-257) indicated that, individually, Z1 and Z2 are not capable of binding T-cap, but that both Z1 and Z2 Ig motifs are required for its binding activity to T-cap (Fig. 3 a). Truncation analyses of T-cap indicated that the COOH-terminal SP-rich domain is not involved in the binding of the Z1-Z2 domains (Fig. 3 b), whereas the deletion mutant T-cap-Δ2 has lost its interaction potential (representing nucleotide residues 19–246).


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)

Interaction of T-cap and the  titin Z1-Z2 domains. (a) Titin repeats Z1  and Z2 specifically interact with the 19-kD  titin-cap protein by yeast two-hybrid analysis. Numbers above the schematic layout  of the NH2-terminal titin region indicate  the nucleotide residue numbers corresponding to the human entry (EMBL data  library X90568) (top) and residue numbers of the deduced amino acid sequence  (bottom). Two immunoglobulin domains  at the NH2 terminus of titin are indicated  as Z1 and Z2, respectively. Bars indicate  positions of the cDNAs used for the bait plasmid (pAS2-Z1-Z2-is1), and the truncation constructs, pAS2-Z1-Z2 (containing the  Z1 and Z2 domains), pAS2-Z1 (containing Z1 domain only), and  pAS2Z2-is1 (containing the Z2 domain fused to residues 200– 257), used for the binding assay to T-cap. +/− signs refer to  growth on minus (Leu, Trp, and His) plates supplemented with  3-AT, and numbers in parentheses refer to β-galactosidase activities (arbitrary units). (b) Interaction of the NH2-terminal 16 kD  of T-cap with the titin Z1-Z2 Ig domains in the yeast two-hybrid  system. Schematic of T-cap cDNA clone. Numbers above the  titin-cap cDNA indicate the nucleotide residues of the human  full-length cDNA sequence (sequence data available from  EMBL under accession number AJ000491; from Valle et al.,  1997) (top) and residue numbers of the deduced amino acid sequence (bottom). AA...A, a poly(A+) tail at the 3′ terminus of the  corresponding cDNA clone. Bars indicate positions of cDNA inserts derived from the two-hybrid screening using pAS2-Z1-Z2-is1 (see a) as bait (T-cap-1, -3, -5, -6, and -7), and the truncation  constructs of T-cap (T-cap-Δ1 and -Δ2) described in Materials  and Methods. +/− signs refer to growth on minus (Leu, Trp, and  His) plates supplemented with 3-AT, and numbers in parentheses  refer to β-galactosidase activities (arbitrary units). (c) Interaction  of expressed T-cap and titin Z1-Z2 peptides. A Coomassie blue– stained 18% Laemmli SDS gel of a whole-cell lysate of BL21 cells  expressing tagless titin Z1-Z2 (lane C). These cells were mixed  with a His6-tagged 16-kD fragment of T-cap and lysed. Lysates  were passed over Ni-NTA agarose columns. Interaction of a stable complex of titin Z1-Z2 and T-cap is indicated by retaining  also the nontagged titin Z1-Z2 on the column (arrow marks Z1-Z2 and T-cap in third lane). Lane M, molecular mass markers as  in legend for Fig. 2.
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Figure 3: Interaction of T-cap and the titin Z1-Z2 domains. (a) Titin repeats Z1 and Z2 specifically interact with the 19-kD titin-cap protein by yeast two-hybrid analysis. Numbers above the schematic layout of the NH2-terminal titin region indicate the nucleotide residue numbers corresponding to the human entry (EMBL data library X90568) (top) and residue numbers of the deduced amino acid sequence (bottom). Two immunoglobulin domains at the NH2 terminus of titin are indicated as Z1 and Z2, respectively. Bars indicate positions of the cDNAs used for the bait plasmid (pAS2-Z1-Z2-is1), and the truncation constructs, pAS2-Z1-Z2 (containing the Z1 and Z2 domains), pAS2-Z1 (containing Z1 domain only), and pAS2Z2-is1 (containing the Z2 domain fused to residues 200– 257), used for the binding assay to T-cap. +/− signs refer to growth on minus (Leu, Trp, and His) plates supplemented with 3-AT, and numbers in parentheses refer to β-galactosidase activities (arbitrary units). (b) Interaction of the NH2-terminal 16 kD of T-cap with the titin Z1-Z2 Ig domains in the yeast two-hybrid system. Schematic of T-cap cDNA clone. Numbers above the titin-cap cDNA indicate the nucleotide residues of the human full-length cDNA sequence (sequence data available from EMBL under accession number AJ000491; from Valle et al., 1997) (top) and residue numbers of the deduced amino acid sequence (bottom). AA...A, a poly(A+) tail at the 3′ terminus of the corresponding cDNA clone. Bars indicate positions of cDNA inserts derived from the two-hybrid screening using pAS2-Z1-Z2-is1 (see a) as bait (T-cap-1, -3, -5, -6, and -7), and the truncation constructs of T-cap (T-cap-Δ1 and -Δ2) described in Materials and Methods. +/− signs refer to growth on minus (Leu, Trp, and His) plates supplemented with 3-AT, and numbers in parentheses refer to β-galactosidase activities (arbitrary units). (c) Interaction of expressed T-cap and titin Z1-Z2 peptides. A Coomassie blue– stained 18% Laemmli SDS gel of a whole-cell lysate of BL21 cells expressing tagless titin Z1-Z2 (lane C). These cells were mixed with a His6-tagged 16-kD fragment of T-cap and lysed. Lysates were passed over Ni-NTA agarose columns. Interaction of a stable complex of titin Z1-Z2 and T-cap is indicated by retaining also the nontagged titin Z1-Z2 on the column (arrow marks Z1-Z2 and T-cap in third lane). Lane M, molecular mass markers as in legend for Fig. 2.
Mentions: Next, we attempted to identify more precisely the binding sites for the titin/T-cap interaction. Truncation analysis of the bait plasmid, pAS2-Z1-Z2-is1, using the constructs pAS2-Z1-Z2 (containing the Z1 and Z2 domains), pAS2-Z1 (containing only the Z1 domain), and pAS2-Z2-is1 (containing the Z2 domain fused to residues 200-257) indicated that, individually, Z1 and Z2 are not capable of binding T-cap, but that both Z1 and Z2 Ig motifs are required for its binding activity to T-cap (Fig. 3 a). Truncation analyses of T-cap indicated that the COOH-terminal SP-rich domain is not involved in the binding of the Z1-Z2 domains (Fig. 3 b), whereas the deletion mutant T-cap-Δ2 has lost its interaction potential (representing nucleotide residues 19–246).

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