Cloning, Sequencing, and the Expression of the Elusive Sarcomeric TPM4α Isoform in Humans.

Dipak K Dube,Syamalima Dube,Bernard J Poiesz,Lynn Abbott,Charles Mitschow,Ruham Alshiekh-Nasany

doi:10.1155/2016/3105478

Dipak K Dube, Syamalima Dube + Show 4 more

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https://doi.org/10.1155/2016/3105478

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Abstract

In mammals, tropomyosin is encoded by four known TPM genes (TPM1, TPM2, TPM3, and TPM4) each of which can generate a number of TPM isoforms via alternative splicing and/or using alternate promoters. In humans, the sarcomeric isoform(s) of each of the TPM genes, except for the TPM4, have been known for a long time. Recently, on the basis of computational analyses of the human genome sequence, the predicted sequence of TPM4α has been posted in GenBank. We designed primer-pairs for RT-PCR and showed the expression of the transcripts of TPM4α and a novel isoform TPM4δ in human heart and skeletal muscle. qRT-PCR shows that the relative expression of TPM4α and TPM4δ is higher in human cardiac muscle. Western blot analyses using CH1 monoclonal antibodies show the absence of the expression of TPM4δ protein (~28 kDa) in human heart muscle. 2D western blot analyses with the same antibody show the expression of at least nine distinct tropomyosin molecules with a mass ~32 kD and above in adult heart. By Mass spectrometry, we determined the amino acid sequences of the extracted proteins from these spots. Spot “G” reveals the putative expression of TPM4α along with TPM1α protein in human adult heart.

Highlights

Tropomyosin (TPM) is a coiled-coil dimer protein that plays important role(s) in regulating muscle contraction in conjunction with other sarcomeric proteins like actin, troponins, tropomodulin, and so forth
In order to detect the expression of TPM4α and TPM4δ in human striated muscles, we used conventional RT-PCR with the RNA from adult human heart and skeletal muscles
Conventional RT-PCR results presented in Figure 2(a) show that TPM4δ is expressed in both fetal and adult human heart

Summary

Introduction

Tropomyosin (TPM) is a coiled-coil dimer protein that plays important role(s) in regulating muscle contraction in conjunction with other sarcomeric proteins like actin, troponins, tropomodulin, and so forth. The rodent TPM4 gene has lost the use of exons 1a and 2; the high molecular weight isoforms encoding 284 amino acid residues such as TPM4α and TPM4β are not expressed as they are in nonmammalian species. It was speculated for a long time that the TPM4 gene in humans as in rodents does not encode sarcomeric isoform with exon 9a/b [2, 3]. The first report on the expression of a high molecular weight TPM4 protein was reported in human ovary tumor tissues in 2004 (accession number AK023385) This isoform, defined as TPM4β, contains exons 1a, 2, 3, 4, 5, 6, 7, 8, and 9d (Figure 1), which differs from the sarcomeric TPM4α isoform containing exon 9a/b instead of 9d.

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