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Molecular cloning and characterization of a novel tissue-specific calpain predominantly expressed in the digestive
H J Lee1, H Sorimachi, S Y Jeong
1Department of Molecular Biology, Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Abstract:
In the course of the genomic cloning of nCL-2, a stomach-specific calpain, we identified a genomic clone encoding a novel member of the calpain large subunit family and designated it 'nCL-4'. First, using exon sequences, we cloned the cDNA for mouse nCL-4. Based on this sequence, we also cloned the cDNAs for rat and human nCL-4. In the case of human nCL-4, the longest open reading frame encodes 690 amino acid residues (Mr 79095) with equal sequence similarities (50-55%) to both ubiquitous and organ-specific calpain large subunits from mammals. The deduced amino acid sequence revealed that nCL-4 is highly conserved among mammals. nCL-4 can be aligned without significant deletions or insertions, and, thus, like other calpains, can be divided into four domains (I-IV). The significant similarity of domains II and IV to those in conventional calpain large subunits suggests the potential protease activity and Ca2+-binding ability of nCL-4. Northern blot analysis revealed that the mRNA for nCL-4 is expressed predominantly in stomach and small intestine but not in uterus, suggesting specialized functions of nCL-4 in the digestive tract. When overexpressed in COS-7 cells, a specific band for nCL-4 was detected. In addition, the gene coding for nCL-4 was localized on human chromosome 1.
Insights
Researchers identified a novel calpain large subunit, nCL-4, highly conserved across mammals. This protein is primarily expressed in the digestive tract, suggesting specialized roles in stomach and intestinal functions.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Calpains are calcium-dependent proteases involved in various cellular processes.
- The calpain large subunit family includes ubiquitous and organ-specific members.
- nCL-2 is a known stomach-specific calpain.
Purpose of the Study:
- To identify and characterize novel calpain large subunit members.
- To investigate the sequence, conservation, and tissue expression of a newly identified calpain, designated nCL-4.
- To explore the potential function and localization of nCL-4.
Main Methods:
- Genomic cloning and cDNA sequencing of nCL-4 from mouse, rat, and human.
- Sequence analysis to determine protein domains and similarities.
- Northern blot analysis for mRNA expression profiling.
- Gene localization using human chromosome mapping.
Main Results:
- A novel calpain large subunit, nCL-4, was identified and cloned across multiple mammalian species.
- Human nCL-4 (690 amino acids) shows 50-55% similarity to known calpain subunits and is highly conserved.
- nCL-4 possesses four domains, with domains II and IV suggesting protease and Ca2+-binding activity.
- nCL-4 mRNA is predominantly expressed in the stomach and small intestine.
- The gene for nCL-4 was localized to human chromosome 1.
Conclusions:
- nCL-4 represents a novel mammalian calpain large subunit.
- Its sequence and domain structure suggest functional similarities to conventional calpains.
- Predominant expression in the digestive tract indicates specialized roles in gastrointestinal functions.
- nCL-4 is a conserved protein with its gene located on human chromosome 1.