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Molecular cloning and characterization of rat and human calpain-5
Anuradhaa Waghray1, Deng-shun Wang, Deborah McKinsey
1Department of Psychiatry, McKnight Brain Institute of the University of Florida, 100 S Newell Drive, P.O. Box 100256, Gainesville, FL 32610-0256, USA. anu@mbi.ufl.edu <anu@mbi.ufl.edu>
Abstract:
Until today, 14 isoforms of mammalian calpains have been identified, including calpain-5. The C. elegans calpain-5 homologue tra-3 is reported to be essential for necrotic neuronal cell death. In this study, we cloned and characterized rat calpain-5, which is highly homologous to human and mouse sequences. The nucleotide sequence is 87% and 93% identical with human and mouse calpain-5, respectively. The protein sequence is well conserved, showing 96% identity in mouse and 92% in human. RT-PCR analysis revealed strong expression of calpain-5 in rat lungs, kidneys, and brain while week expression in heart, whereas in rat brain regions it is ubiquitously expressed. The mRNA expression in different human tissues showed equal expression. However, in human brain regions calpain-5 was strongly expressed in hypothalamus, thalamus, cerebellum, and frontal lobe. Western blot analysis on human neuroblastoma SH-SY5Y cells demonstrated calcium-dependent processing of calpain-5, despite the absence of calmodulin-like domain.
Insights
Researchers characterized rat calpain-5, finding high homology to human and mouse sequences. This study details its expression patterns in various rat and human tissues, including the brain, and its calcium-dependent processing.
Area of Science:
- Molecular Biology
- Neuroscience
- Biochemistry
Background:
- Fourteen mammalian calpain isoforms, including calpain-5, have been identified.
- The C. elegans calpain-5 homologue, tra-3, is crucial for necrotic neuronal cell death.
Purpose of the Study:
- To clone and characterize rat calpain-5.
- To investigate the expression patterns of calpain-5 in rat and human tissues.
- To analyze the calcium-dependent processing of calpain-5.
Main Methods:
- Cloning and sequencing of rat calpain-5.
- Reverse transcription-polymerase chain reaction (RT-PCR) for mRNA expression analysis.
- Western blot analysis for protein processing in human neuroblastoma cells.
Main Results:
- Rat calpain-5 shares high nucleotide (87-93%) and protein (92-96%) sequence identity with human and mouse orthologs.
- Calpain-5 is highly expressed in rat lungs, kidneys, and brain, with ubiquitous expression in rat brain regions.
- Human calpain-5 shows strong expression in the hypothalamus, thalamus, cerebellum, and frontal lobe, and undergoes calcium-dependent processing in SH-SY5Y cells.
Conclusions:
- Rat calpain-5 is a highly conserved protein with significant homology to its human and mouse counterparts.
- Calpain-5 exhibits distinct tissue-specific and regional brain expression patterns in both rats and humans.
- The calcium-dependent processing of calpain-5, even without a calmodulin-like domain, suggests a unique regulatory mechanism.
