title: Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism.
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.
authors: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX, United States of America.
authors: Annie
authors: A
authors: Nguyen
authors: Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX, United States of America.|Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX, United States of America.
authors: John D
authors: JD
authors: Hulleman
mesh_terms: D002460:Cell Line; D055316:Cystatin C / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space; D013058:Mass Spectrometry; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D021382:Protein Sorting Signals; D021381:Protein Transport; D059748:Proteolysis; D014162:Transfection; D056811:Unfolded Protein Response
chemical_list: D055316:Cystatin C; D021382:Protein Sorting Signals