Abstract
Here, we identify the rat brain (rb) CLCA1 metalloprotease motif and its role in rbCLCA1 processing. GFP tagging or c-myc tagging adjacent to the rbCLCA1 signal sequence was used to detect rbCLCA1 expression and localization patterns if they matched those of other CLCA family members. Immunoblot analysis revealed that massive deletion of the metalloprotease motif affects the protein cleavage process by restricting two cleavage products to only one product. rbCLCA1 as well as the mutant proteins H155A, E156Q, H159A, D166A, E167A, E170A, and D171A overexpressed in HEK293T cells showed plasma membrane localization; and intracellular localizations of H159A and E167A were observed in permeabilized and non-permeabilized conditions. C-terminally GFP-tagged rbCLCA1 showed either ER localization or overall signal within the cells rather than on the cell surface. Cell surface biotinylation analysis was used to show that rbCLCA1, H155A, E156Q, D166A, E170A, and D171A reach the cell surface while little H159A and E167A reach the cell surface. Taken together, our findings indicate that the amino acids H159 and E167 in the rbCLCA1 metalloprotease motif are important in rbCLCA1 processing for localization to the cell surface. Our data demonstrate that rbCLCA1 localization is dependent on the H159 and E167, suggesting either the metalloprotease motif including H159 and E167 may be the key site for rbCLCA1 cellular processing or that a novel rbCLCA1 regulation mechanism exists with a metalloprotease activity.
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Acknowledgments
This study was supported by the Veterinary Science Research Institute of the Konkuk University. This work was investigated and revised in the memory of Dr. Sang Min Jeong who initially supported and influenced this investigation. We appreciate his insights into the work and hope that he rests in peace.
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Lee, R.M., Han, K.H. & Han, J.S. rbCLCA1 is a putative metalloprotease family member: localization and catalytic domain identification. Amino Acids 48, 707–720 (2016). https://doi.org/10.1007/s00726-015-2119-6
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DOI: https://doi.org/10.1007/s00726-015-2119-6