1. Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism
- Author
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John D. Hulleman and Annie Nguyen
- Subjects
0301 basic medicine ,Physiology ,Intracellular Space ,lcsh:Medicine ,Protein Sequencing ,Endoplasmic Reticulum ,Biochemistry ,Mass Spectrometry ,0302 clinical medicine ,Cell Signaling ,Medicine and Health Sciences ,lcsh:Science ,chemistry.chemical_classification ,Multidisciplinary ,Secretory Pathway ,Proteases ,Cysteine protease ,Amino acid ,Enzymes ,Protein Transport ,Cell Processes ,Cellular Structures and Organelles ,Sequence Analysis ,Research Article ,Signal Transduction ,Signal peptide ,Signal Inhibition ,Biology ,Protein Sorting Signals ,Cleavage (embryo) ,Transfection ,Research and Analysis Methods ,Polymorphism, Single Nucleotide ,Cell Line ,03 medical and health sciences ,FLAG-tag ,Humans ,Cystatin C ,Molecular Biology Techniques ,Sequencing Techniques ,Molecular Biology ,Secretion ,Endoplasmic reticulum ,lcsh:R ,Biology and Life Sciences ,Protein Secretion ,Proteins ,Cell Biology ,Molecular biology ,030104 developmental biology ,Secretory protein ,chemistry ,Mutation ,Proteolysis ,Unfolded Protein Response ,Enzymology ,lcsh:Q ,Physiological Processes ,030217 neurology & neurosurgery - Abstract
Cystatin C (Cys C) is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T) polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer's disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP), were all secreted as efficiently from immortalized human cells as their wild-type (WT) counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively). Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER)-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as 'site 2' cleavage). However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K) mutation), A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 ('site 1') or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease inhibition function of Cys C.
- Published
- 2016