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Identification of novel Kirrel3 gene splice variants in adult human skeletal muscle.

Durcan PJ, Conradie JD, Van deVyver M, Myburgh KH - BMC Physiol. (2014)

Bottom Line: Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance.These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, Stellenbosch University, Private Bag X1 Matieland, 7602, Stellenbosch, South Africa. pdurcan@sun.ac.za.

ABSTRACT

Background: Multiple cell types including trophoblasts, osteoclasts and myoblasts require somatic cell fusion events as part of their physiological functions. In Drosophila Melanogaster the paralogus type 1 transmembrane receptors and members of the immunoglobulin superfamily Kin of Irre (Kirre) and roughest (Rst) regulate myoblast fusion during embryonic development. Present within the human genome are three homologs to Kirre termed Kin of Irre like (Kirrel) 1, 2 and 3. Currently it is unknown if Kirrel3 is expressed in adult human skeletal muscle.

Results: We investigated (using PCR and Western blot) Kirrel3 in adult human skeletal muscle samples taken at rest and after mild exercise induced muscle damage. Kirrel3 mRNA expression was verified by sequencing and protein presence via blotting with 2 different anti-Kirrel3 protein antibodies. Evidence for three alternatively spliced Kirrel3 mRNA transcripts in adult human skeletal muscle was obtained. Kirrel3 mRNA in adult human skeletal muscle was detected at low or moderate levels, or not at all. This sporadic expression suggests that Kirrel3 is expressed in a pulsatile manner. Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.

Conclusion: The results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 expressed in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

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Kirrel3 protein detection in adult human skeletal muscle. A – Western Blot results obtained with anti-human Kirrel3 antibody directed toward intracellular region of Kirrel3 on skeletal muscle lysates obtained from adult human males at baseline, days one and two after completion of a DHR exercise protocol. S = subject B – Western blot results from blocking peptide experiment. The antibody in A was incubated with blocking peptide before being added to membrane containing skeletal muscle lysates obtained from adult human males at baseline and one and two days post completion of a DHR exercise protocol. C – A second commercial antibody directed towards the extracellular domain of Kirrel3 was used on a selection of lysates obtained from adult human male skeletal muscle day1 or 2 post completion of the downhill running exercise bout. Ponceau staining was performed on membranes to demonstrate transfer and equal protein loading.
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Fig4: Kirrel3 protein detection in adult human skeletal muscle. A – Western Blot results obtained with anti-human Kirrel3 antibody directed toward intracellular region of Kirrel3 on skeletal muscle lysates obtained from adult human males at baseline, days one and two after completion of a DHR exercise protocol. S = subject B – Western blot results from blocking peptide experiment. The antibody in A was incubated with blocking peptide before being added to membrane containing skeletal muscle lysates obtained from adult human males at baseline and one and two days post completion of a DHR exercise protocol. C – A second commercial antibody directed towards the extracellular domain of Kirrel3 was used on a selection of lysates obtained from adult human male skeletal muscle day1 or 2 post completion of the downhill running exercise bout. Ponceau staining was performed on membranes to demonstrate transfer and equal protein loading.

Mentions: Based on results obtained from our mRNA analysis we wished to investigate Kirrel3 protein presence in human skeletal muscle samples. A commercially available antibody that was raised against the intracellular AA 596-626 of Kirrel3 A was utilised. Lysates obtained from a previous downhill run (DHR) study by our group [32] were utilised for the analysis. Baseline, 1 and 2 days post DHR were the time points assessed. Multiple immunoreactive proteins were detected in all human skeletal muscle samples ranging in size from approximately 45-110 kDa (Figure 4A). To assess for Kirrel3 specificity of the antibody, a blocking peptide was incubated with the antibody prior to addition to the membrane. The immunoreactive proteins observed at approximately 50-100 kDa with antibody alone were eliminated (Figure 4B), thus providing support that these immunoreactive proteins may be isoforms of Kirrel3. To confirm the protein presence of Kirrel3 a second commercially available antibody that was raised against a recombinant human Kirrel3 protein (AA33-535 of NP_001288026) was tested on a selection of lysates from the downhill running study. Immunoreactive proteins were detected at approximately 70-75 kDA while no immunoreactive proteins were detected at approximately 50-55 kDA and 110 (Figure 4C), which is in contrast to results obtained with the intracellular targeting antibody.Figure 4


Identification of novel Kirrel3 gene splice variants in adult human skeletal muscle.

Durcan PJ, Conradie JD, Van deVyver M, Myburgh KH - BMC Physiol. (2014)

Kirrel3 protein detection in adult human skeletal muscle. A – Western Blot results obtained with anti-human Kirrel3 antibody directed toward intracellular region of Kirrel3 on skeletal muscle lysates obtained from adult human males at baseline, days one and two after completion of a DHR exercise protocol. S = subject B – Western blot results from blocking peptide experiment. The antibody in A was incubated with blocking peptide before being added to membrane containing skeletal muscle lysates obtained from adult human males at baseline and one and two days post completion of a DHR exercise protocol. C – A second commercial antibody directed towards the extracellular domain of Kirrel3 was used on a selection of lysates obtained from adult human male skeletal muscle day1 or 2 post completion of the downhill running exercise bout. Ponceau staining was performed on membranes to demonstrate transfer and equal protein loading.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4269076&req=5

Fig4: Kirrel3 protein detection in adult human skeletal muscle. A – Western Blot results obtained with anti-human Kirrel3 antibody directed toward intracellular region of Kirrel3 on skeletal muscle lysates obtained from adult human males at baseline, days one and two after completion of a DHR exercise protocol. S = subject B – Western blot results from blocking peptide experiment. The antibody in A was incubated with blocking peptide before being added to membrane containing skeletal muscle lysates obtained from adult human males at baseline and one and two days post completion of a DHR exercise protocol. C – A second commercial antibody directed towards the extracellular domain of Kirrel3 was used on a selection of lysates obtained from adult human male skeletal muscle day1 or 2 post completion of the downhill running exercise bout. Ponceau staining was performed on membranes to demonstrate transfer and equal protein loading.
Mentions: Based on results obtained from our mRNA analysis we wished to investigate Kirrel3 protein presence in human skeletal muscle samples. A commercially available antibody that was raised against the intracellular AA 596-626 of Kirrel3 A was utilised. Lysates obtained from a previous downhill run (DHR) study by our group [32] were utilised for the analysis. Baseline, 1 and 2 days post DHR were the time points assessed. Multiple immunoreactive proteins were detected in all human skeletal muscle samples ranging in size from approximately 45-110 kDa (Figure 4A). To assess for Kirrel3 specificity of the antibody, a blocking peptide was incubated with the antibody prior to addition to the membrane. The immunoreactive proteins observed at approximately 50-100 kDa with antibody alone were eliminated (Figure 4B), thus providing support that these immunoreactive proteins may be isoforms of Kirrel3. To confirm the protein presence of Kirrel3 a second commercially available antibody that was raised against a recombinant human Kirrel3 protein (AA33-535 of NP_001288026) was tested on a selection of lysates from the downhill running study. Immunoreactive proteins were detected at approximately 70-75 kDA while no immunoreactive proteins were detected at approximately 50-55 kDA and 110 (Figure 4C), which is in contrast to results obtained with the intracellular targeting antibody.Figure 4

Bottom Line: Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance.These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiological Science, Stellenbosch University, Private Bag X1 Matieland, 7602, Stellenbosch, South Africa. pdurcan@sun.ac.za.

ABSTRACT

Background: Multiple cell types including trophoblasts, osteoclasts and myoblasts require somatic cell fusion events as part of their physiological functions. In Drosophila Melanogaster the paralogus type 1 transmembrane receptors and members of the immunoglobulin superfamily Kin of Irre (Kirre) and roughest (Rst) regulate myoblast fusion during embryonic development. Present within the human genome are three homologs to Kirre termed Kin of Irre like (Kirrel) 1, 2 and 3. Currently it is unknown if Kirrel3 is expressed in adult human skeletal muscle.

Results: We investigated (using PCR and Western blot) Kirrel3 in adult human skeletal muscle samples taken at rest and after mild exercise induced muscle damage. Kirrel3 mRNA expression was verified by sequencing and protein presence via blotting with 2 different anti-Kirrel3 protein antibodies. Evidence for three alternatively spliced Kirrel3 mRNA transcripts in adult human skeletal muscle was obtained. Kirrel3 mRNA in adult human skeletal muscle was detected at low or moderate levels, or not at all. This sporadic expression suggests that Kirrel3 is expressed in a pulsatile manner. Several anti Kirrel3 immunoreactive proteins were detected in all adult human skeletal muscle samples analysed and results suggest the presence of different isoforms or posttranslational modification, or both.

Conclusion: The results presented here demonstrate for the first time that there are at least 3 splice variants of Kirrel3 expressed in adult human skeletal muscle, two of which have never previously been identified in human muscle. Importantly, mRNA of all splice variants was not always present, a finding with potential physiological relevance. These initial discoveries highlight the need for more molecular and functional studies to understand the role of Kirrel3 in human skeletal muscle.

Show MeSH
Related in: MedlinePlus