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.
Highlights
Cystatin C (Cys C) is a small, 13.3 kDa reversible competitive inhibitor of papain-like cysteine proteases which is ubiquitously expressed throughout the body, including in the testes, liver, pancreas, brain, and retinal pigmented epithelium (RPE) [1, 2]
Untagged A25T Cys C was secreted at 112 ± 9% of untagged WT Cys C; A25T Cys C FLAG was secreted at 100 ± 15% of WT Cys C FLAG levels; and A25T Cys C FLAG green fluorescent protein (GFP) was secreted at 88 ± 17% of WT Cys C FLAG GFP levels (Fig 1D)
We found that WT and A25T Cys C FLAG had overlapping chromatograms with a major species at 14209.8 Da (Fig 6B)
Summary
Cystatin C (Cys C) is a small, 13.3 kDa reversible competitive inhibitor of papain-like cysteine proteases which is ubiquitously expressed throughout the body, including in the testes, liver, pancreas, brain, and retinal pigmented epithelium (RPE) [1, 2]. Cys C inhibits papain as well as cathepsin-family proteases such as cathepsin B, H, L and S [3]. Cys C binds tightly to these proteases, resulting in dissociation constants in the sub-nanomolar to nanomolar range [3]. Appropriate regulation of proteases in general is of fundamental importance for normal development (reviewed in [4]) and for the prevention of a plethora of diseases ranging from retinal degeneration/vascularization [5] to cancer (reviewed in [6]). Cys C has been shown to be involved in a number of anti-bacterial, anti-viral and anti-amyloid processes (reviewed in [7]) as well as other biological events such as inflammation (reviewed in [7]), cancer [8], and cell proliferation [9]. The underlying biological processes that contribute to and regulate Cys C function (and dysfunction) are relatively poorly understood [10]
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