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Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Young P, Ehler E, Gautel M - J. Cell Biol. (2001)

Bottom Line: It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle.Both proteins coassemble during myofibrillogenesis.The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Structural Biology Division, 69117 Heidelberg, Germany.

ABSTRACT
Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

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Detection of obscurin by Western blotting. (A) The polyclonal antibody α-Ob48–49 against obscurin detects a large protein of approximately the same size as nebulin in striated muscle. Lanes 1, 2, and 3: human Vastus lateralis muscle sample run on 3% polyacrylamide gel. Lane 1, Coomassie stain of a lane from the gel; lane 2, titin detected with S53 monoclonal antibody on a Western blot of one lane from the same gel; lane 3, obscurin detected with α-Ob48–49 polyclonal antibody on a Western blot of an adjacent lane. Note, titin and nebulin were well blotted and their positions are marked on the blot. Titin is resolved as a single band. Obscurin is detected slightly above the position of nebulin as marked on the blot. Lanes 4–7, human Vastus lateralis muscle sample run on 4% polyacrylamide gel. Lane 4, Coomassie stain of a lane from the gel; lane 5; obscurin detected with α-Ob48–49 on a lane cut from of a Western blot of the same gel; lane 6, the same blot as lane 5 stripped and reprobed with antinebulin NSH3-ra. Note, blots have been accurately aligned and obscurin can be distinguished from nebulin. The α-Ob48–49 antibody has not been completely stripped from the blot, so, in lane 6 there is some carry over of the obscurin signal appearing just above the darker nebulin band. Lane 7, obscurin detected with α-ObDH; lane 8, human cardiac muscle sample run on a 4% polyacrylamide gel. Obscurin detected with α-Ob48–49 on a Western blot. Note, a protein of a similar size is detected in cardiac and skeletal muscle. M, myosin; T, titin; N, nebulin; O, obscurin.
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fig3: Detection of obscurin by Western blotting. (A) The polyclonal antibody α-Ob48–49 against obscurin detects a large protein of approximately the same size as nebulin in striated muscle. Lanes 1, 2, and 3: human Vastus lateralis muscle sample run on 3% polyacrylamide gel. Lane 1, Coomassie stain of a lane from the gel; lane 2, titin detected with S53 monoclonal antibody on a Western blot of one lane from the same gel; lane 3, obscurin detected with α-Ob48–49 polyclonal antibody on a Western blot of an adjacent lane. Note, titin and nebulin were well blotted and their positions are marked on the blot. Titin is resolved as a single band. Obscurin is detected slightly above the position of nebulin as marked on the blot. Lanes 4–7, human Vastus lateralis muscle sample run on 4% polyacrylamide gel. Lane 4, Coomassie stain of a lane from the gel; lane 5; obscurin detected with α-Ob48–49 on a lane cut from of a Western blot of the same gel; lane 6, the same blot as lane 5 stripped and reprobed with antinebulin NSH3-ra. Note, blots have been accurately aligned and obscurin can be distinguished from nebulin. The α-Ob48–49 antibody has not been completely stripped from the blot, so, in lane 6 there is some carry over of the obscurin signal appearing just above the darker nebulin band. Lane 7, obscurin detected with α-ObDH; lane 8, human cardiac muscle sample run on a 4% polyacrylamide gel. Obscurin detected with α-Ob48–49 on a Western blot. Note, a protein of a similar size is detected in cardiac and skeletal muscle. M, myosin; T, titin; N, nebulin; O, obscurin.

Mentions: To monitor the expression of obscurin protein and gain estimates of the size distributions of the polypeptide, the rabbit polyclonal antisera α-Ob19–20, α-Ob48–49, α-Ob-DH and α-Ob51–52 were raised (Fig. 1). α-Ob48–49, α-Ob51–52 and α-Ob-DH were affinity purified and used to detect obscurin on Western blots from low porosity SDS polyacrylamide gels. Using α-Ob48–49 and α-Ob-DH to probe blots of human Vastus lateralis muscle, a very high molecular weight protein was detected (Fig. 3) . This protein was seen to migrate slightly slower than the visible nebulin band. A band of similar molecular weight was detected on blots of cardiac tissue (Fig. 3). The blots were also probed with anti-titin antibodies. Although titin can be detected, the obscurin band does not react with several anti-titin antibodies (S54-4 and CH11, Whiting et al., 1989; Gautel et al., 1996b). α-Ob48–49 and α-Ob-DH react with neither nebulin nor titin. Nebulin has a molecular weight of ∼700–900 kD (Labeit and Kolmerer, 1995a; Wang et al., 1996) and thus obscurin is expected to be of a similar or slightly larger size. This is in agreement with the molecular weight of at least 720 kD predicted for obscurin from the cDNA sequence. A band of similar size is also detected using the α-Ob-DH antibody (Fig. 3). On Coomassie-stained low porosity gels with normal loading (20–40 μg total protein) of adult muscle, there is no appreciable protein in the region between nebulin and titin (Fig. 3), suggesting that obscurin is expressed at much lower levels than either of these proteins. Estimations by densitometric analysis of double-probed Western blots of adult skeletal muscle suggest that the ratio of nebulin to obscurin is at least 10:1.


Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Young P, Ehler E, Gautel M - J. Cell Biol. (2001)

Detection of obscurin by Western blotting. (A) The polyclonal antibody α-Ob48–49 against obscurin detects a large protein of approximately the same size as nebulin in striated muscle. Lanes 1, 2, and 3: human Vastus lateralis muscle sample run on 3% polyacrylamide gel. Lane 1, Coomassie stain of a lane from the gel; lane 2, titin detected with S53 monoclonal antibody on a Western blot of one lane from the same gel; lane 3, obscurin detected with α-Ob48–49 polyclonal antibody on a Western blot of an adjacent lane. Note, titin and nebulin were well blotted and their positions are marked on the blot. Titin is resolved as a single band. Obscurin is detected slightly above the position of nebulin as marked on the blot. Lanes 4–7, human Vastus lateralis muscle sample run on 4% polyacrylamide gel. Lane 4, Coomassie stain of a lane from the gel; lane 5; obscurin detected with α-Ob48–49 on a lane cut from of a Western blot of the same gel; lane 6, the same blot as lane 5 stripped and reprobed with antinebulin NSH3-ra. Note, blots have been accurately aligned and obscurin can be distinguished from nebulin. The α-Ob48–49 antibody has not been completely stripped from the blot, so, in lane 6 there is some carry over of the obscurin signal appearing just above the darker nebulin band. Lane 7, obscurin detected with α-ObDH; lane 8, human cardiac muscle sample run on a 4% polyacrylamide gel. Obscurin detected with α-Ob48–49 on a Western blot. Note, a protein of a similar size is detected in cardiac and skeletal muscle. M, myosin; T, titin; N, nebulin; O, obscurin.
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fig3: Detection of obscurin by Western blotting. (A) The polyclonal antibody α-Ob48–49 against obscurin detects a large protein of approximately the same size as nebulin in striated muscle. Lanes 1, 2, and 3: human Vastus lateralis muscle sample run on 3% polyacrylamide gel. Lane 1, Coomassie stain of a lane from the gel; lane 2, titin detected with S53 monoclonal antibody on a Western blot of one lane from the same gel; lane 3, obscurin detected with α-Ob48–49 polyclonal antibody on a Western blot of an adjacent lane. Note, titin and nebulin were well blotted and their positions are marked on the blot. Titin is resolved as a single band. Obscurin is detected slightly above the position of nebulin as marked on the blot. Lanes 4–7, human Vastus lateralis muscle sample run on 4% polyacrylamide gel. Lane 4, Coomassie stain of a lane from the gel; lane 5; obscurin detected with α-Ob48–49 on a lane cut from of a Western blot of the same gel; lane 6, the same blot as lane 5 stripped and reprobed with antinebulin NSH3-ra. Note, blots have been accurately aligned and obscurin can be distinguished from nebulin. The α-Ob48–49 antibody has not been completely stripped from the blot, so, in lane 6 there is some carry over of the obscurin signal appearing just above the darker nebulin band. Lane 7, obscurin detected with α-ObDH; lane 8, human cardiac muscle sample run on a 4% polyacrylamide gel. Obscurin detected with α-Ob48–49 on a Western blot. Note, a protein of a similar size is detected in cardiac and skeletal muscle. M, myosin; T, titin; N, nebulin; O, obscurin.
Mentions: To monitor the expression of obscurin protein and gain estimates of the size distributions of the polypeptide, the rabbit polyclonal antisera α-Ob19–20, α-Ob48–49, α-Ob-DH and α-Ob51–52 were raised (Fig. 1). α-Ob48–49, α-Ob51–52 and α-Ob-DH were affinity purified and used to detect obscurin on Western blots from low porosity SDS polyacrylamide gels. Using α-Ob48–49 and α-Ob-DH to probe blots of human Vastus lateralis muscle, a very high molecular weight protein was detected (Fig. 3) . This protein was seen to migrate slightly slower than the visible nebulin band. A band of similar molecular weight was detected on blots of cardiac tissue (Fig. 3). The blots were also probed with anti-titin antibodies. Although titin can be detected, the obscurin band does not react with several anti-titin antibodies (S54-4 and CH11, Whiting et al., 1989; Gautel et al., 1996b). α-Ob48–49 and α-Ob-DH react with neither nebulin nor titin. Nebulin has a molecular weight of ∼700–900 kD (Labeit and Kolmerer, 1995a; Wang et al., 1996) and thus obscurin is expected to be of a similar or slightly larger size. This is in agreement with the molecular weight of at least 720 kD predicted for obscurin from the cDNA sequence. A band of similar size is also detected using the α-Ob-DH antibody (Fig. 3). On Coomassie-stained low porosity gels with normal loading (20–40 μg total protein) of adult muscle, there is no appreciable protein in the region between nebulin and titin (Fig. 3), suggesting that obscurin is expressed at much lower levels than either of these proteins. Estimations by densitometric analysis of double-probed Western blots of adult skeletal muscle suggest that the ratio of nebulin to obscurin is at least 10:1.

Bottom Line: It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle.Both proteins coassemble during myofibrillogenesis.The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Structural Biology Division, 69117 Heidelberg, Germany.

ABSTRACT
Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

Show MeSH
Related in: MedlinePlus