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Molecular cloning, functional expression and tissue distribution of the cDNA encoding frog skeletal muscle calsequestrin.

ABSTRACT: We have cloned, sequenced and expressed the cDNA encoding frog skeletal muscle calsequestrin. The processed frog calsequestrin is 398 residues long, with an Mr of 45941 (unglycosylated form), and exhibits 77% sequence similarity with its rabbit counterpart. Consensus sequences for glycosylation and phosphorylation of the protein were conserved. Compared with rabbit calsequestrin, the mature amphibian protein has peculiar structural properties, which include (i) a higher content of negatively charged residues (142 versus 109), and (ii) a striking repeat sequence at the C-terminal region of 44 aspartic acid residues. Furthermore, this is the first report on the expression of calsequestrin cDNA in COS-1 cells; the expressed protein exhibited an Mr and antigenic properties which were indistinguishable from those of the native protein. In addition, it was capable of binding 45Ca in a ligand overlay. Northern blot analysis of frog skeletal muscle, liver, heart and brain RNA showed that the protein is mainly expressed in skeletal muscle. The high density of negative charges at the C-terminus might constitute high-capacity low-affinity Ca(2+)-binding sites, which may account for the higher Ca(2+)-binding capacity of frog calsequestrin compared with other members of the calsequestrin family (56 mol/mol versus 40-44 mol/mol of protein).

SUBMITTER: Treves S

PROVIDER: S-EPMC1130952 | biostudies-other | 1992 May

REPOSITORIES: biostudies-other

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Project description:We describe a new method for determining the concentration of total Ca in whole skeletal muscle samples ([CaT]WM in units of mmoles/kg wet weight) using the Ca-dependent UV absorbance spectra of the Ca chelator BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid). Muscle tissue was homogenized in a solution containing 0.15 mM BAPTA and 0.5% sodium dodecyl sulfate (to permeabilize membranes and denature proteins) and then centrifuged. The solution volume was adjusted so that BAPTA captured essentially all of the Ca. [CaT]WM was obtained with Beer's law from the absorbance change produced by adding 1 mM EGTA to capture Ca from BAPTA. Results from mouse, rat, and frog muscles were reasonably consistent with results obtained using other methods for estimating total [Ca] in whole muscles and in single muscle fibers. Results with external Ca removed before determining [CaT]WM indicate that most of the Ca was intracellular, indicative of a lack of bound Ca in the extracellular space. In both fast-twitch (extensor digitorum longus, EDL) and slow-twitch (soleus) muscles from mice, [CaT]WM increased approximately linearly with decreasing muscle weight, increasing approximately twofold with a twofold decrease in muscle weight. This suggests that the Ca concentration of smaller muscles might be increased relative to that in larger muscles, thereby increasing the specific force to compensate for the smaller mass. Knocking out the high capacity Ca-binding protein calsequestrin (CSQ) did not significantly reduce [CaT]WM in mouse EDL or soleus muscle. However, in EDL muscles lacking CSQ, muscle weights were significantly lower than in wild-type (WT) muscles and the values of [CaT]WM were, on average, about half the expected WT values, taking into account the above [CaT]WM versus muscle weight relationship. Because greater reductions in [CaT]WM would be predicted in both muscle types, we hypothesize that there is a substantial increase in Ca bound to other sites in the CSQ knockout muscles.

Project description:BackgroundMany human diseases and phenotypes are related to RNA expression, levels of which are influenced by a wide spectrum of genetic and exposure-related factors. In a large genome-wide study of muscle tissue expression, we found that some genes exhibited a bimodal distribution of RNA expression, in contrast to what is usually assumed in studies of a single healthy tissue. As bimodality has classically been considered a hallmark of genetic control, we assessed the genome-wide prevalence, cause, and association of this phenomenon with diabetes-related phenotypes in skeletal muscle tissue from 225 healthy Pima Indians using exon array expression chips.ResultsTwo independent batches of microarrays were used for bimodal assessment and comparison. Of the 17,881 genes analyzed, eight (GSTM1, HLA-DRB1, ERAP2, HLA-DRB5, MAOA, ACTN3, NR4A2, and THNSL2) were found to have bimodal expression replicated in the separate batch groups, while 24 other genes had evidence of bimodality in only one group. Some bimodally expressed genes had modest associations with pre-diabetic phenotypes, of note ACTN3 with insulin resistance. Most of the other bimodal genes have been reported to be involved with various other diseases and characteristics. Association of expression with cis genetic variation in a subset of 149 individuals found all but one of the confirmed bimodal genes and nearly half of all potential ones to be highly significant expression quantitative trait loci (eQTL). The rare prevalence of these bimodally expressed genes found after controlling for batch effects was much lower than the prevalence reported in other studies. Additional validation in data from separate muscle expression studies confirmed the low prevalence of bimodality we observed.ConclusionsWe conclude that the prevalence of bimodal gene expression is quite rare in healthy muscle tissue (<0.2%), and is much lower than limited reports from other studies. The major cause of these clearly bimodal expression patterns in homogeneous tissue appears to be cis-polymorphisms, indicating that such bimodal genes are, for the most part, eQTL. The high frequency of disease associations reported with these genes gives hope that this unique feature may identify or actually be an underlying factor responsible for disease development.

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Molecular cloning, functional expression and tissue distribution of the cDNA encoding frog skeletal muscle calsequestrin.

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