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Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP
Published on: September 6, 2015
Molecular cloning and expression of cDNA encoding a lumenal calcium binding glycoprotein from sarcoplasmic reticulum
E Leberer1, J H Charuk, N M Green
1Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, ON, Canada.
Insights
Researchers discovered a novel calcium-binding glycoprotein in rabbit skeletal muscle sarcoplasmic reticulum, named sarcalumenin. This protein, identified via cDNA screening, plays a role in calcium regulation within the muscle cell.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Sarcoplasmic reticulum (SR) is crucial for calcium (Ca2+) storage and release in muscle cells.
- Glycoproteins within the SR membrane are implicated in Ca2+ handling, but their specific roles are not fully elucidated.
Purpose of the Study:
- To isolate and characterize a novel glycoprotein from rabbit skeletal muscle sarcoplasmic reticulum.
- To investigate the Ca2+ binding properties and cellular localization of this newly identified protein.
Main Methods:
- Antibody screening was employed to isolate the complementary DNA (cDNA) encoding the 160-kDa glycoprotein.
- The isolated cDNA was analyzed for sequence variations, including insertions, compared to known related glycoproteins.
- Calcium binding assays (45Ca2+ gel overlay) and cellular localization studies were performed on the purified and expressed protein.
Main Results:
- A cDNA encoding a 160-kDa glycoprotein was isolated, differing from a 53-kDa glycoprotein by an alternative splicing event.
- The 160-kDa glycoprotein contains a highly acidic, 436-amino acid insert, suggesting Ca2+ binding capabilities.
- The purified and expressed 160-kDa glycoprotein demonstrated Ca2+ binding and was localized to the lumen of the sarcoplasmic reticulum, associated with the membrane via Ca2+.
Conclusions:
- A novel lumenal Ca2+ binding glycoprotein, proposed to be named sarcalumenin, has been identified in the sarcoplasmic reticulum.
- Sarcalumenin likely plays a significant role in intracellular Ca2+ homeostasis within skeletal muscle.
- Alternative splicing is a mechanism generating functional diversity in sarcoplasmic reticulum glycoproteins.
Abstract:
Antibody screening was used to isolate a cDNA encoding the 160-kDa glycoprotein of rabbit skeletal muscle sarcoplasmic reticulum. The cDNA is identical to that encoding the 53-kDa glycoprotein except that it contains an in-frame insertion of 1308 nucleotides near its 5' end, apparently resulting from alternative splicing. The protein encoded by the cDNA would contain a 19-residue NH2-terminal signal sequence and a 453-residue COOH-terminal sequence identical to the 53-kDa glycoprotein. It would also contain a 436-amino acid insert between these sequences. This insert would be highly acidic, suggesting that it might bind Ca2+. The purified 160-kDa glycoprotein and the glycoprotein expressed in COS-1 cells transfected with cDNA encoding the 160-kDa glycoprotein were shown to bind 45Ca2+ in a gel overlay assay. The protein was shown to be located in the lumen of the sarcoplasmic reticulum and to be associated through Ca2+ with the membrane. We propose that this lumenal Ca2+ binding glycoprotein of the sarcoplasmic reticulum be designated "sarcalumenin."
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