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Cystatin C in macular and neuronal degenerations: Implications for mechanism(s) of age-related macular degeneration
Vision Research 50 (2010) 737–742
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Cystatin C in macular and neuronal degenerations: Implications for mechanism(s) of age-related macular degeneration Luminita Paraoan a,*, Paul Hiscott a, Christine Gosden b, Ian Grierson a a b
Unit of Ophthalmology, School of Clinical Sciences, Liverpool L69 3GA, UK Department of Pathology, University of Liverpool, Liverpool L69 3GA, UK
a r t i c l e
i n f o
Article history: Received 11 July 2009 Received in revised form 2 October 2009
Keywords: Cystatin C Age-related macular degeneration Retinal pigment epithelium Secretion Trafficking
a b s t r a c t Cystatin C is a strong inhibitor of cysteine proteinases expressed by diverse cells. Variant B cystatin C, which was associated with increased risk of developing age-related macular degeneration, differs from the wild type protein by a single amino acid (A25T) in the signal sequence responsible for its targeting to the secretory pathway. The same variant conveys susceptibility to Alzheimer disease. Our investigations of the trafficking and processing of variant B cystatin C in living RPE cells highlight impaired secretion of extracellular modulators and inappropriate protein retention in RPE cells as potential molecular mechanisms underpinning macular, and possibly neuronal, degeneration. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Cystatin C is a widely expressed, potent inhibitor of lysosomal and extracellular cysteine proteinases. Its biochemical and structural characteristics, as well as its expression pattern in a large number of human tissues and cells were extensively studied and are presented briefly in Sections 2 and 3, below, respectively. In the retinal pigment epithelium (RPE), cystatin C was firstly recognized as an abundant transcript by analysis of expressed sequence tags in cDNA libraries of human foetal and adult RPE cells (Paraoan, Grierson, & Maden, 2000) – findings which were subsequently confirmed in numerous studies both at transcriptional and protein level (Buraczynska et al., 2002; Ida et al., 2004; Wasselius, Hakansson, Johansson, Abrahamson, & Ehinger, 2001). Later characterization of the intracellular processing of the protein identified the RPE as a major site for secretion of cystatin C in the posterior eye (Paraoan, White, Spiller, Grierson, & Maden, 2001). More recently, epidemiological association of a variant (known as variant B, described in Section 4 below) cystatin C with increased risk of developing exudative age-related macular degeneration (AMD) (Zurdel, Finckh, Menzer, Nitsch, & Richard, 2002) and subsequent characterization of the unexpected trafficking of the variant protein by RPE and other cells (Paraoan et al., 2004) drew attention
* Corresponding author. Address: Unit of Ophthalmology, School of Clinical Sciences, University of Liverpool, UCD Building, Daulby Street, Liverpool L69 3GA, UK. Fax: +44 151 706 5934. E-mail address: [email protected] (L. Paraoan). 0042-6989/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.visres.2009.10.022
to essential molecular events previously not considered in the context of the RPE-driven mechanisms of AMD pathogenesis.
2. Structural and biochemical characteristics of cystatin C Cystatin C, originally called gamma-trace and post-gammaglobulin in early reports (Barrett, Davies, & Grubb, 1984; Brzin, Popovic, Turk, Borchart, & Machleidt, 1984; Cejka & Fleischmann, 1973; Grubb & Lofberg, 1982; Turk et al., 1983), is the main amyloid-forming member of family 2 of the cystatin superfamily of cysteine proteinase inhibitors that present two di-sulphide bridges as defining structural characteristics (for reviews see (Akopyan, 1991; Barrett, 1987; Paraoan & Grierson, 2007; Turk & Bode, 1991)). The active inhibitor is a single polypeptide chain consisting of 120 amino acids residues (molecular weight of 13 kDa) and exhibiting two distinct inhibitory sites conferring its dual inhibitory specificity – as the tightest-binding inhibitor of cysteine peptidases, such as cathepsins B, H, L and S (Abrahamson, Barrett, Salvesen, & Grubb, 1986; Barrett et al., 1984; Turk & Bode, 1991), as well as a strong competitive inhibitor of the lysosomal legumain (Alvarez-Fernandez et al., 1999). Functional studies have identified at least three regions as important for the inhibitory activity of cystatin C: (i) the evolutionary conserved glycine residue in the N-terminal region (Gly-11) together with the three residues preceding it, which afford increased affinity for the target enzymes and optimal interaction with the substrate-binding pockets of cysteine peptidases (Hall, Dalboge, Grubb, & Abrahamson, 1993; Lindahl, Nycander, Ylinenjarvi, Pol,
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& Bjork, 1992; Machleidt et al., 1989); (ii) the QVVAG region (Gln55-Gly59) involved in formation of a tight beta-hairpin loop with essential role in binding cysteine proteinases (Bode et al., 1988; Engh et al., 1993; Rodziewicz-Motowidlo et al., 2009); and (iii) the Pro105-Trp106 region in the second hairpin loop with role in anchoring the inhibitor to the proteinase once the complex has been formed (Bjork et al., 1996). The high affinity binding of cystatin C to its target peptidases occurs through a competitive and reversible two-step bimolecular mechanism (Barrett et al., 1984; Nycander, Estrada, Mort, Abrahamson, & Bjork, 1998). The propensity of cystatin C to form inactive dimers and oligomers involved in subsequent formation of amyloid protofilaments/ deposits is attributable to a process of three-dimensional domain swapping (Ekiel & Abrahamson, 1996; Janowski et al., 2001; Staniforth et al., 2001). This process occurs in vitro under pre-denaturing conditions while in vivo it is significantly enhanced in the presence of an N-truncated form, which is the main form of cystatin C found in amyloid deposits (Janowski, Abrahamson, Grubb, & Jaskolski, 2004). The oligomerization is dramatically accelerated in the case of a naturally occurring, rare (Icelandic) L68Q mutant variant – the causative factor for an autosomal dominant form of hereditary cystatin C amyloid angiopathy – which retains the inhibitory activity but has decreased stability yielding dimers at physiological temperatures and generates cystatin C amyloid deposits in cerebral blood vessels that lead to fatal amyloidosis in early adult life (Abrahamson, 1996; Cohen, Feiner, Jensson, & Frangione, 1983; Ghiso, Jensson, & Frangione, 1986). Notably, both L68Q mutant variant and the N-truncated form of cystatin C are structurally different from the AMD- and Alzheimer (AD)-associated variant, called variant B cystatin C, whose characteristics are presented in detail in Section 4 below. 3. Overview of expression and localization of cystatin C Presence of cystatin C has been investigated and demonstrated in a variety of tissues, including salivary glands, brain, testis, seminal vesicles, liver, pancreas, lung, placenta and various endocrine glands (Abrahamson et al., 1990; Grubb & Lofberg, 1982; Lofberg, Nilsson, Stromblad, Lasson, & Olsson, 1982; Lofberg et al., 1983). Outside the eye, the highest transcriptional levels of cystatin C, indicated by a semi-quantitative comparative analysis in a wide variety of adult and foetal tissues, appear in salivary glands and
neuronal tissues (Fig. 1A). Although the specific cells responsible for cystatin C synthesis have not been elucidated for all tissues in which the presence of the inhibitor has been documented, some likely contributors are brain cortical neurons and astrocytes (Lofberg, Grubb, & Brun, 1981; Yasuhara et al., 1993), pancreatic islet A cells (Lignelid & Jacobsson, 1992) and various (neuro)endocrine cells including chromaffin cells of the adrenal medulla (Lofberg et al., 1982) and thyroid calcitonin producing cells (Lofberg et al., 1983). Remarkably, cystatin C is present in most biological fluids, including urine, blood serum and cerebrospinal fluid, in concentrations implying a physiological role (Abrahamson et al., 1986; Lofberg & Grubb, 1979; Sickmann et al., 2000). As such, it is thought to be a major local regulator of extracellular proteolytic activity. In the cerebrospinal fluid (CSF) cystatin C is the dominating cysteine proteinase inhibitor (with a 5.5 ratio concentration over that in blood plasma (Lofberg & Grubb, 1979)). Notably, the brain tissue with the highest cystatin C mRNA level is the choroid plexus (Cole et al., 1989), thus explaining the supply of cystatin C to the CSF which is produced mainly by the choroid plexus. In the eye, prior to the identification of the RPE as a major site of cystatin C expression (Paraoan et al., 2000), two partially contradictory studies represented the sum total of the literature on cystatin C expression. Both studies examined the presence of cystatin C mRNA and concluded that in the rat eye the inhibitor is expressed only in the sclera and retina (with a tentative association of the retinal signal with glia and rods) (Barka & van der Noen, 1994), while in the chick eye the expression is localized to the epithelium of the ciliary body and nowhere else (Colella, 1996). The differences were put down to species variation and to a possible shift in the site of expression during evolution of mammalian species. The finding of high incidence of cystatin C precursor expressed sequence tags in cDNA libraries of RPE cells (Paraoan et al., 2000) is further substantiated by the detection of two related transcripts of about 850–900 nucleotides in RPE cells (Fig. 1B). Notably, the presence of two cystatin C mRNAs was previously observed also in non-ocular tissues, pointing towards some tissue specificity of closely related alternative splice variants (Cole et al., 1989; Tsuruta, O’Brien, & Griswold, 1993). However, the mRNA variants detected in RPE cells correspond to alternative splice variants known to differ only in the untranslated regions and to encode same length (146 amino acids) precursor proteins (see CST 3 under AceView genes
Fig. 1. Northerm blot analysis of cystatin C expression. (A) Messenger RNA samples from various tissues in the Human RNA Master Blot (Clontech Laboratories, Inc., Mountain View, USA) were hybridized to a 32P-labelled cystatin C probe derived from a full length cystatin C cDNA clone isolated in-house (Paraoan et al., 2000). Tissues represented are indicated in the diagram alongside the blot. All brain-derived tissues represented in the blot, as well as the spinal cord show a considerable level of expression. Foetal tissues, with the exception of the spleen, present a moderate level of expression compared with adult tissues. Hybridization signals represent the pooled signal of all related transcripts in each tissue that hybridize to the probe. (B) Hybridization of mRNA from human RPE cells, subjected to formaldehyde agarose gel electrophoresis, to the cystatin C-specific probe identifies two closely related transcripts of approximately 850–900 nucleotides (lane 1); ribosomal subunits from yeast total RNA were separated to serve as size markers on lane 2.
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Fig. 2. Immunohistochemical analysis of cystatin C in human eyes. Sections were exposed to a polyclonal rabbit anti-Cystatin C antibody (Upstate/Millipore). Immunoreactive sites were revealed with red chromogen AEC (Dako Envision); haematoxylin counterstain. Strongest immunoreactivity is detected in the RPE cell layer (A). Cystatin C is also seen in the multistratified epithelium of the ciliary body – CBE (C), lens epithelium – LE (D), cells in the ganglion cell layer of the retina (arrow) (E) and in the vitreous (F). Panel B shows a negative control in which the primary antibody was substituted with normal serum. Scale bars represent 50 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
www.ncbi.nlm.nih.gov/IEB/Research/Acembly (Thierry-Mieg & Thierry-Mieg, 2006)). Immunohistochemical analysis of whole human eyes sections (Fig. 2) reveals immunoreactivity for cystatin C protein in the neurorepithelium of the iris, ciliary body, lens epithelium and RPE, with the strongest and most confluent staining present in the RPE. Inflammatory cells in the uveal tract and some cells in the
neuroretina, mostly in the ganglion cell layer, are positive. The cornea, trabecular meshwork, iris stroma, uveal stroma, optic nerve and sclera are negative. The vitreous (especially base) also contains cystatin C (Fig. 2F). Staining of the ciliary body epithelium (Fig. 2C) is consistent with reports of cystatin C in the aqueous (Russell & Epstein, 1992; Wasselius, Hakansson, Abrahamson, & Ehinger, 2004). Similar distribution of cystatin C was reported in mouse
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and rat eye, with the main difference being the presence of cystatin C in the cornea and iris stromal cells in rodents (Wasselius et al., 2001, 2004). 4. Variant B cystatin C The human precursor cystatin C gene, CST 3, (accession number NM_000099) is located alongside the majority of type 2 cystatin genes and pseudogenes in the cystatin locus on chromosome 20 (gene map locus 20p11.21; (Abrahamson, Islam, Szpirer, Szpirer, & Levan, 1989; Schnittger, Rao, Abrahamson, & Hansmann, 1993)). The gene encodes a 146 amino acids precursor whose highly hydrophobic N-terminal sequence of 26 amino acids residues was demonstrated experimentally to function as a signal sequence directing the precursor to the secretory pathway in RPE and other cells (Paraoan, Grierson, & Maden, 2003; Paraoan et al., 2001). A variation in the CST 3 gene was recently designated ARMD11 (http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=611953) and is characterized by three KspI (SstII) polymorphisms at positions 157 and 72 in the 50 untranslated region and at position +73 of the coding sequence of the transcript (Balbin & Abrahamson, 1991). However, as a result of strong linkage disequilibrium, all three polymorphisms appear linked and define a single variant allele named variant B (with a frequency in population of 0.29) in addition to the wild type one, also referred to as variant A (whose frequency is 0.71) (Balbin & Abrahamson, 1991; Finckh et al., 2000). One of the polymorphisms mentioned above translates into an amino acid substitution in the penultimate position of the signal sequence of precursor cystatin C, namely A25T in the variant B. The substitution does not affect any of the protein’s domains involved in its biochemical/inhibitory function. However, the A25T substitution has striking consequences on the trafficking and intracellular processing of the variant precursor (Paraoan et al., 2004). Thus, processing of the variant B cystatin C through the secretory pathway in RPE cells is significantly impaired through a mechanism dependent on the reduced hydrophobicity of the signal sequence which is affected by the substitution (Ratnayaka et al., 2007) and results in a reduction by approximately half of the secreted, mature cystatin C (Paraoan et al., 2004). Furthermore, the unprocessed or partially processed variant B precursor cystatin C associates surprisingly with the mitochondria (Fig. 3A) in a manner
not dependent on the mitochondrial membrane potential and suggestive of a relative anchorage in the mitochondrial membrane system (Paraoan et al., 2004). A small but distinct amount of variant B molecules also appear diffusely throughout the cytoplasm and nucleus, but not nucleoli in RPE cells. The implications for the biology of RPE cells of both reduced secretion and intracellular mislocalization of the variant B cystatin C were discussed at large previously (Paraoan et al., 2004). Reduced secretion of cystatin C is likely to affect the proteolytic balance in the extracellular space of the RPE cells with possible consequences on attachment of cells, extracellular transport mechanisms and/or neovascularization. Conversely, the inappropriate association of the variant inhibitor with the mitochondria, alongside a gradual increase of intracellular misfolded protein may lead to further, progressive functional impairment of the RPE. 5. Involvement in disease of the variant B cystatin C Homozygosity for variant B cystatin C allele was shown to correlate with increased risk of developing exudative AMD and with a relative early onset of the disease (Zurdel et al., 2002). The data pointed to the CST 3 haplotype B acting as a recessive allele contributing to AMD risk in up to 6.6% of the patients investigated in the above study, with an odds ratio for disease association with the CST 3 B/B genotype of 2.97 (95% confidence interval [CI]: 1.28–6.86). There are no studies to date investigating the effect of CST 3 haplotype B in the presence of other contributory factors to the multifactorial etiology of exudative AMD. A series of studies over the last decade addressed the association of cystatin C and neurological disorders, with particular emphasis on neuronal degenerative pathology. These studies were in part triggered by the detection of cystatin C co-deposited with b-amyloid protein in amyloid deposits, in senile plaques and in arteriolar walls in the brain of AD patients (Levy et al., 2001), suggesting involvement of the protein in the pathogenic processes leading to amyloid deposition. A significant genetic association was unveiled between CST 3 B/B genotype and late-onset AD with an overall 3.8 odds ratio (95% CI: 1.56–9.25) for AD in association with the respective genotype (Finckh et al., 2000) and a doubled risk to develop the disease for homozygous patients over 80 years old (Crawford et al., 2000). Although CST 3 appears to act independently of the apolipoprotein E gene (APOE), the finding that
Fig. 3. Intracellular localization and trafficking of variant B (A25T) precursor cystatin C (A) is dramatically altered compared with that of wild-type precursor cystatin C (B). Confocal micrographs of living human RPE cells transiently transfected with (A) variant B cystatin C fused to enhanced green fluorescent protein (EGFP), (B) wild-type cystatin C-EGFP and (C) EGFP only, 24 h post-transfection. Green fluorescence image is merged in (A) with the red fluorescence of the oxidated Mitotracker Red CM-H2Xros dye (Molecular Probes™, Invitrogen, Paisley, UK) concentrated in the mitochondria of actively respiring cells. The majority of variant B precursor cystatin C molecules colocalize with mitochondria (indicated by yellow colour); some diffuse green fluorescence is also present in the cytoplasm and nucleus. In contrast, targeting of wild-type precursor cystatin C is to the ER/golgi apparatus (B), as previously described (Paraoan et al., 2001). Bright field image is overlaid with the image of RPE cells transfected with EGFP only (C) to illustrate cell morphology. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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patients with both risk factors had a significantly earlier onset of disease (Finckh et al., 2000) raises the possibility that the combination of adverse alleles of CST 3 and APOE leads to an accelerated upsetting of the intracellular processing of cellular fractions involved in AD pathology. This hypothesis is further supported by the finding of a synergistic association between the variant B CST 3 haplotype, APOE4 and AD in patients between 60 and 74 years old (Beyer et al., 2001; Cathcart, Huang, Lanham, Corder, & Poduslo, 2005). While variant B cystatin C allele has become firmly recognized as a potential AD susceptibility gene (Bertram, McQueen, Mullin, Blacker, & Tanzi, 2007) and the protective role of the wild type cystatin C in AD pathogenesis has been established (Kaeser et al., 2007; Mi et al., 2007), the functional effects of the variant protein in neurons remain to be clarified. Conversely, in relation to the eye pathology there is an obvious need for study of possible interactions of the CST 3 haplotype B with other known genetic determinants, including APOE4, of the AMD. Our investigation into the processing and trafficking of variant B cystatin C in living RPE cells has highlighted two molecular mechanisms previously not considered in relation to AMD, and possibly AD, pathology: (i) reduced secretion of modulators of extracellular environment; and (ii) inappropriate intracellular retention and targeting of proteins, other than in drusen or amyloid format. The specific consequences upon RPE cells of the reduction of extracellular function of cystatin C and of its intracellular accumulation around mitochondria are currently addressed in our laboratory. It is likely that at least some of the functional effects of, on one hand, the impaired secretion of a major regulator of proteolysis and, on the other, of the errant intracellular accumulation of the variant protein are shared by RPE and neuronal cells on the progressive path to respective pathologies. Acknowledgments The authors thank Dr. Dave Spiller for help with cell imaging, Daniel Brotchie for help with preparation and editing of figures and manuscript and Donna Gray for technical support. Confocal imaging was performed in the Centre for Cell Imaging, School of Biological Sciences, University of Liverpool. Dr Paraoan’s research on cystatin C was supported by The Wellcome Trust and The Guide Dogs for the Blind Association, UK. References Abrahamson, M. (1996). Molecular basis for amyloidosis related to hereditary brain hemorrhage. Scandinavian Journal of Clinical and Laboratory Investigation Supplement, 226, 47–56. Abrahamson, M., Barrett, A. J., Salvesen, G., & Grubb, A. (1986). Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. Journal of Biological Chemistry, 261, 11282–11289. Abrahamson, M., Islam, M. Q., Szpirer, J., Szpirer, C., & Levan, G. (1989). The human cystatin C gene (CST 3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20. Human Genetics, 82, 223–226. Abrahamson, M., Olafsson, I., Palsdottir, A., Ulvsback, M., Lundwall, A., Jensson, O., et al. (1990). Structure and expression of the human cystatin C gene. Biochemical Journal, 268, 287–294. Akopyan, T. (1991). Protein inhibitors of proteinases from brain. Neurochemical Research, 16, 513–517. Alvarez-Fernandez, M., Barrett, A. J., Gerhartz, B., Dando, P. M., Ni, J., & Abrahamson, M. (1999). Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site. Journal of Biological Chemistry, 274, 19195–19203. Balbin, M., & Abrahamson, M. (1991). SstII polymorphic sites in the promoter region of the human cystatin C gene. Human Genetics, 87, 751–752. Barka, T., & van der Noen, H. (1994). Expression of the cysteine proteinase inhibitor cystatin C mRNA in rat eye. Anatomical Record, 239, 343–348. Barrett, A. J. (1987). The cystatins: A new class of peptidase inhibitors. Trends in Biochemical Sciences, 12, 193–196. Barrett, A. J., Davies, M. E., & Grubb, A. (1984). The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors. Biochemical and Biophysical Research Communications, 120, 631–636.
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Russell, P., & Epstein, D. L. (1992). Protein analysis of monkey aqueous humor. Current Eye Research, 11, 1239–1243. Schnittger, S., Rao, V. V., Abrahamson, M., & Hansmann, I. (1993). Cystatin C (CST 3), the candidate gene for hereditary cystatin C amyloid angiopathy (HCCAA), and other members of the cystatin gene family are clustered on chromosome 20p11.2. Genomics, 16, 50–55. Sickmann, A., Dormeyer, W., Wortelkamp, S., Woitalla, D., Kuhn, W., & Meyer, H. E. (2000). Identification of proteins from human cerebrospinal fluid, separated by two-dimensional polyacrylamide gel electrophoresis. Electrophoresis, 21, 2721–2728. Staniforth, R. A., Giannini, S., Higgins, L. D., Conroy, M. J., Hounslow, A. M., Jerala, R., et al. (2001). Three-dimensional domain swapping in the folded and moltenglobule states of cystatins, an amyloid-forming structural superfamily. EMBO Journal, 20, 4774–4781. Thierry-Mieg, D., & Thierry-Mieg, J. (2006). AceView: A comprehensive cDNAsupported gene and transcripts annotation. Genome Biology, 7(Suppl. 1), S12–S14. Tsuruta, J. K., O’Brien, D. A., & Griswold, M. D. (1993). Sertoli cell and germ cell cystatin C: Stage-dependent expression of two distinct messenger ribonucleic acid transcripts in rat testes. Biology of Reproduction, 49, 1045–1054. Turk, V., & Bode, W. (1991). The cystatins: Protein inhibitors of cysteine proteinases. FEBS Letters, 285, 213–219. Turk, V., Brzin, J., Longer, M., Ritonja, A., Eropkin, M., Borchart, U., et al. (1983). Protein inhibitors of cysteine proteinases. III. Amino-acid sequence of cystatin from chicken egg white. Hoppe-Seylers Zeitschrift Fur Physiologische Chemie, 364, 1487–1496. Wasselius, J., Hakansson, K., Abrahamson, M., & Ehinger, B. (2004). Cystatin C in the anterior segment of rat and mouse eyes. Acta Ophthalmologica Scandinavica, 82, 68–75. Wasselius, J., Hakansson, K., Johansson, K., Abrahamson, M., & Ehinger, B. (2001). Identification and localization of retinal cystatin C. Investigative Ophthalmology and Visual Science, 42, 1901–1906. Yasuhara, O., Hanai, K., Ohkubo, I., Sasaki, M., McGeer, P. L., & Kimura, H. (1993). Expression of cystatin C in rat, monkey and human brains. Brain Research, 628, 85–92. Zurdel, J., Finckh, U., Menzer, G., Nitsch, R. M., & Richard, G. (2002). CST 3 genotype associated with exudative age related macular degeneration. British Journal of Ophthalmology, 86, 214–219.
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Vision Research journal homepage: www.elsevier.com/locate/visres
Cystatin C in macular and neuronal degenerations: Implications for mechanism(s) of age-related macular degeneration Luminita Paraoan a,*, Paul Hiscott a, Christine Gosden b, Ian Grierson a a b
Unit of Ophthalmology, School of Clinical Sciences, Liverpool L69 3GA, UK Department of Pathology, University of Liverpool, Liverpool L69 3GA, UK
a r t i c l e
i n f o
Article history: Received 11 July 2009 Received in revised form 2 October 2009
Keywords: Cystatin C Age-related macular degeneration Retinal pigment epithelium Secretion Trafficking
a b s t r a c t Cystatin C is a strong inhibitor of cysteine proteinases expressed by diverse cells. Variant B cystatin C, which was associated with increased risk of developing age-related macular degeneration, differs from the wild type protein by a single amino acid (A25T) in the signal sequence responsible for its targeting to the secretory pathway. The same variant conveys susceptibility to Alzheimer disease. Our investigations of the trafficking and processing of variant B cystatin C in living RPE cells highlight impaired secretion of extracellular modulators and inappropriate protein retention in RPE cells as potential molecular mechanisms underpinning macular, and possibly neuronal, degeneration. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Cystatin C is a widely expressed, potent inhibitor of lysosomal and extracellular cysteine proteinases. Its biochemical and structural characteristics, as well as its expression pattern in a large number of human tissues and cells were extensively studied and are presented briefly in Sections 2 and 3, below, respectively. In the retinal pigment epithelium (RPE), cystatin C was firstly recognized as an abundant transcript by analysis of expressed sequence tags in cDNA libraries of human foetal and adult RPE cells (Paraoan, Grierson, & Maden, 2000) – findings which were subsequently confirmed in numerous studies both at transcriptional and protein level (Buraczynska et al., 2002; Ida et al., 2004; Wasselius, Hakansson, Johansson, Abrahamson, & Ehinger, 2001). Later characterization of the intracellular processing of the protein identified the RPE as a major site for secretion of cystatin C in the posterior eye (Paraoan, White, Spiller, Grierson, & Maden, 2001). More recently, epidemiological association of a variant (known as variant B, described in Section 4 below) cystatin C with increased risk of developing exudative age-related macular degeneration (AMD) (Zurdel, Finckh, Menzer, Nitsch, & Richard, 2002) and subsequent characterization of the unexpected trafficking of the variant protein by RPE and other cells (Paraoan et al., 2004) drew attention
* Corresponding author. Address: Unit of Ophthalmology, School of Clinical Sciences, University of Liverpool, UCD Building, Daulby Street, Liverpool L69 3GA, UK. Fax: +44 151 706 5934. E-mail address: [email protected] (L. Paraoan). 0042-6989/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.visres.2009.10.022
to essential molecular events previously not considered in the context of the RPE-driven mechanisms of AMD pathogenesis.
2. Structural and biochemical characteristics of cystatin C Cystatin C, originally called gamma-trace and post-gammaglobulin in early reports (Barrett, Davies, & Grubb, 1984; Brzin, Popovic, Turk, Borchart, & Machleidt, 1984; Cejka & Fleischmann, 1973; Grubb & Lofberg, 1982; Turk et al., 1983), is the main amyloid-forming member of family 2 of the cystatin superfamily of cysteine proteinase inhibitors that present two di-sulphide bridges as defining structural characteristics (for reviews see (Akopyan, 1991; Barrett, 1987; Paraoan & Grierson, 2007; Turk & Bode, 1991)). The active inhibitor is a single polypeptide chain consisting of 120 amino acids residues (molecular weight of 13 kDa) and exhibiting two distinct inhibitory sites conferring its dual inhibitory specificity – as the tightest-binding inhibitor of cysteine peptidases, such as cathepsins B, H, L and S (Abrahamson, Barrett, Salvesen, & Grubb, 1986; Barrett et al., 1984; Turk & Bode, 1991), as well as a strong competitive inhibitor of the lysosomal legumain (Alvarez-Fernandez et al., 1999). Functional studies have identified at least three regions as important for the inhibitory activity of cystatin C: (i) the evolutionary conserved glycine residue in the N-terminal region (Gly-11) together with the three residues preceding it, which afford increased affinity for the target enzymes and optimal interaction with the substrate-binding pockets of cysteine peptidases (Hall, Dalboge, Grubb, & Abrahamson, 1993; Lindahl, Nycander, Ylinenjarvi, Pol,
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& Bjork, 1992; Machleidt et al., 1989); (ii) the QVVAG region (Gln55-Gly59) involved in formation of a tight beta-hairpin loop with essential role in binding cysteine proteinases (Bode et al., 1988; Engh et al., 1993; Rodziewicz-Motowidlo et al., 2009); and (iii) the Pro105-Trp106 region in the second hairpin loop with role in anchoring the inhibitor to the proteinase once the complex has been formed (Bjork et al., 1996). The high affinity binding of cystatin C to its target peptidases occurs through a competitive and reversible two-step bimolecular mechanism (Barrett et al., 1984; Nycander, Estrada, Mort, Abrahamson, & Bjork, 1998). The propensity of cystatin C to form inactive dimers and oligomers involved in subsequent formation of amyloid protofilaments/ deposits is attributable to a process of three-dimensional domain swapping (Ekiel & Abrahamson, 1996; Janowski et al., 2001; Staniforth et al., 2001). This process occurs in vitro under pre-denaturing conditions while in vivo it is significantly enhanced in the presence of an N-truncated form, which is the main form of cystatin C found in amyloid deposits (Janowski, Abrahamson, Grubb, & Jaskolski, 2004). The oligomerization is dramatically accelerated in the case of a naturally occurring, rare (Icelandic) L68Q mutant variant – the causative factor for an autosomal dominant form of hereditary cystatin C amyloid angiopathy – which retains the inhibitory activity but has decreased stability yielding dimers at physiological temperatures and generates cystatin C amyloid deposits in cerebral blood vessels that lead to fatal amyloidosis in early adult life (Abrahamson, 1996; Cohen, Feiner, Jensson, & Frangione, 1983; Ghiso, Jensson, & Frangione, 1986). Notably, both L68Q mutant variant and the N-truncated form of cystatin C are structurally different from the AMD- and Alzheimer (AD)-associated variant, called variant B cystatin C, whose characteristics are presented in detail in Section 4 below. 3. Overview of expression and localization of cystatin C Presence of cystatin C has been investigated and demonstrated in a variety of tissues, including salivary glands, brain, testis, seminal vesicles, liver, pancreas, lung, placenta and various endocrine glands (Abrahamson et al., 1990; Grubb & Lofberg, 1982; Lofberg, Nilsson, Stromblad, Lasson, & Olsson, 1982; Lofberg et al., 1983). Outside the eye, the highest transcriptional levels of cystatin C, indicated by a semi-quantitative comparative analysis in a wide variety of adult and foetal tissues, appear in salivary glands and
neuronal tissues (Fig. 1A). Although the specific cells responsible for cystatin C synthesis have not been elucidated for all tissues in which the presence of the inhibitor has been documented, some likely contributors are brain cortical neurons and astrocytes (Lofberg, Grubb, & Brun, 1981; Yasuhara et al., 1993), pancreatic islet A cells (Lignelid & Jacobsson, 1992) and various (neuro)endocrine cells including chromaffin cells of the adrenal medulla (Lofberg et al., 1982) and thyroid calcitonin producing cells (Lofberg et al., 1983). Remarkably, cystatin C is present in most biological fluids, including urine, blood serum and cerebrospinal fluid, in concentrations implying a physiological role (Abrahamson et al., 1986; Lofberg & Grubb, 1979; Sickmann et al., 2000). As such, it is thought to be a major local regulator of extracellular proteolytic activity. In the cerebrospinal fluid (CSF) cystatin C is the dominating cysteine proteinase inhibitor (with a 5.5 ratio concentration over that in blood plasma (Lofberg & Grubb, 1979)). Notably, the brain tissue with the highest cystatin C mRNA level is the choroid plexus (Cole et al., 1989), thus explaining the supply of cystatin C to the CSF which is produced mainly by the choroid plexus. In the eye, prior to the identification of the RPE as a major site of cystatin C expression (Paraoan et al., 2000), two partially contradictory studies represented the sum total of the literature on cystatin C expression. Both studies examined the presence of cystatin C mRNA and concluded that in the rat eye the inhibitor is expressed only in the sclera and retina (with a tentative association of the retinal signal with glia and rods) (Barka & van der Noen, 1994), while in the chick eye the expression is localized to the epithelium of the ciliary body and nowhere else (Colella, 1996). The differences were put down to species variation and to a possible shift in the site of expression during evolution of mammalian species. The finding of high incidence of cystatin C precursor expressed sequence tags in cDNA libraries of RPE cells (Paraoan et al., 2000) is further substantiated by the detection of two related transcripts of about 850–900 nucleotides in RPE cells (Fig. 1B). Notably, the presence of two cystatin C mRNAs was previously observed also in non-ocular tissues, pointing towards some tissue specificity of closely related alternative splice variants (Cole et al., 1989; Tsuruta, O’Brien, & Griswold, 1993). However, the mRNA variants detected in RPE cells correspond to alternative splice variants known to differ only in the untranslated regions and to encode same length (146 amino acids) precursor proteins (see CST 3 under AceView genes
Fig. 1. Northerm blot analysis of cystatin C expression. (A) Messenger RNA samples from various tissues in the Human RNA Master Blot (Clontech Laboratories, Inc., Mountain View, USA) were hybridized to a 32P-labelled cystatin C probe derived from a full length cystatin C cDNA clone isolated in-house (Paraoan et al., 2000). Tissues represented are indicated in the diagram alongside the blot. All brain-derived tissues represented in the blot, as well as the spinal cord show a considerable level of expression. Foetal tissues, with the exception of the spleen, present a moderate level of expression compared with adult tissues. Hybridization signals represent the pooled signal of all related transcripts in each tissue that hybridize to the probe. (B) Hybridization of mRNA from human RPE cells, subjected to formaldehyde agarose gel electrophoresis, to the cystatin C-specific probe identifies two closely related transcripts of approximately 850–900 nucleotides (lane 1); ribosomal subunits from yeast total RNA were separated to serve as size markers on lane 2.
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Fig. 2. Immunohistochemical analysis of cystatin C in human eyes. Sections were exposed to a polyclonal rabbit anti-Cystatin C antibody (Upstate/Millipore). Immunoreactive sites were revealed with red chromogen AEC (Dako Envision); haematoxylin counterstain. Strongest immunoreactivity is detected in the RPE cell layer (A). Cystatin C is also seen in the multistratified epithelium of the ciliary body – CBE (C), lens epithelium – LE (D), cells in the ganglion cell layer of the retina (arrow) (E) and in the vitreous (F). Panel B shows a negative control in which the primary antibody was substituted with normal serum. Scale bars represent 50 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
www.ncbi.nlm.nih.gov/IEB/Research/Acembly (Thierry-Mieg & Thierry-Mieg, 2006)). Immunohistochemical analysis of whole human eyes sections (Fig. 2) reveals immunoreactivity for cystatin C protein in the neurorepithelium of the iris, ciliary body, lens epithelium and RPE, with the strongest and most confluent staining present in the RPE. Inflammatory cells in the uveal tract and some cells in the
neuroretina, mostly in the ganglion cell layer, are positive. The cornea, trabecular meshwork, iris stroma, uveal stroma, optic nerve and sclera are negative. The vitreous (especially base) also contains cystatin C (Fig. 2F). Staining of the ciliary body epithelium (Fig. 2C) is consistent with reports of cystatin C in the aqueous (Russell & Epstein, 1992; Wasselius, Hakansson, Abrahamson, & Ehinger, 2004). Similar distribution of cystatin C was reported in mouse
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and rat eye, with the main difference being the presence of cystatin C in the cornea and iris stromal cells in rodents (Wasselius et al., 2001, 2004). 4. Variant B cystatin C The human precursor cystatin C gene, CST 3, (accession number NM_000099) is located alongside the majority of type 2 cystatin genes and pseudogenes in the cystatin locus on chromosome 20 (gene map locus 20p11.21; (Abrahamson, Islam, Szpirer, Szpirer, & Levan, 1989; Schnittger, Rao, Abrahamson, & Hansmann, 1993)). The gene encodes a 146 amino acids precursor whose highly hydrophobic N-terminal sequence of 26 amino acids residues was demonstrated experimentally to function as a signal sequence directing the precursor to the secretory pathway in RPE and other cells (Paraoan, Grierson, & Maden, 2003; Paraoan et al., 2001). A variation in the CST 3 gene was recently designated ARMD11 (http://www.ncbi.nlm.nih.gov/entrez/dispomim.cgi?id=611953) and is characterized by three KspI (SstII) polymorphisms at positions 157 and 72 in the 50 untranslated region and at position +73 of the coding sequence of the transcript (Balbin & Abrahamson, 1991). However, as a result of strong linkage disequilibrium, all three polymorphisms appear linked and define a single variant allele named variant B (with a frequency in population of 0.29) in addition to the wild type one, also referred to as variant A (whose frequency is 0.71) (Balbin & Abrahamson, 1991; Finckh et al., 2000). One of the polymorphisms mentioned above translates into an amino acid substitution in the penultimate position of the signal sequence of precursor cystatin C, namely A25T in the variant B. The substitution does not affect any of the protein’s domains involved in its biochemical/inhibitory function. However, the A25T substitution has striking consequences on the trafficking and intracellular processing of the variant precursor (Paraoan et al., 2004). Thus, processing of the variant B cystatin C through the secretory pathway in RPE cells is significantly impaired through a mechanism dependent on the reduced hydrophobicity of the signal sequence which is affected by the substitution (Ratnayaka et al., 2007) and results in a reduction by approximately half of the secreted, mature cystatin C (Paraoan et al., 2004). Furthermore, the unprocessed or partially processed variant B precursor cystatin C associates surprisingly with the mitochondria (Fig. 3A) in a manner
not dependent on the mitochondrial membrane potential and suggestive of a relative anchorage in the mitochondrial membrane system (Paraoan et al., 2004). A small but distinct amount of variant B molecules also appear diffusely throughout the cytoplasm and nucleus, but not nucleoli in RPE cells. The implications for the biology of RPE cells of both reduced secretion and intracellular mislocalization of the variant B cystatin C were discussed at large previously (Paraoan et al., 2004). Reduced secretion of cystatin C is likely to affect the proteolytic balance in the extracellular space of the RPE cells with possible consequences on attachment of cells, extracellular transport mechanisms and/or neovascularization. Conversely, the inappropriate association of the variant inhibitor with the mitochondria, alongside a gradual increase of intracellular misfolded protein may lead to further, progressive functional impairment of the RPE. 5. Involvement in disease of the variant B cystatin C Homozygosity for variant B cystatin C allele was shown to correlate with increased risk of developing exudative AMD and with a relative early onset of the disease (Zurdel et al., 2002). The data pointed to the CST 3 haplotype B acting as a recessive allele contributing to AMD risk in up to 6.6% of the patients investigated in the above study, with an odds ratio for disease association with the CST 3 B/B genotype of 2.97 (95% confidence interval [CI]: 1.28–6.86). There are no studies to date investigating the effect of CST 3 haplotype B in the presence of other contributory factors to the multifactorial etiology of exudative AMD. A series of studies over the last decade addressed the association of cystatin C and neurological disorders, with particular emphasis on neuronal degenerative pathology. These studies were in part triggered by the detection of cystatin C co-deposited with b-amyloid protein in amyloid deposits, in senile plaques and in arteriolar walls in the brain of AD patients (Levy et al., 2001), suggesting involvement of the protein in the pathogenic processes leading to amyloid deposition. A significant genetic association was unveiled between CST 3 B/B genotype and late-onset AD with an overall 3.8 odds ratio (95% CI: 1.56–9.25) for AD in association with the respective genotype (Finckh et al., 2000) and a doubled risk to develop the disease for homozygous patients over 80 years old (Crawford et al., 2000). Although CST 3 appears to act independently of the apolipoprotein E gene (APOE), the finding that
Fig. 3. Intracellular localization and trafficking of variant B (A25T) precursor cystatin C (A) is dramatically altered compared with that of wild-type precursor cystatin C (B). Confocal micrographs of living human RPE cells transiently transfected with (A) variant B cystatin C fused to enhanced green fluorescent protein (EGFP), (B) wild-type cystatin C-EGFP and (C) EGFP only, 24 h post-transfection. Green fluorescence image is merged in (A) with the red fluorescence of the oxidated Mitotracker Red CM-H2Xros dye (Molecular Probes™, Invitrogen, Paisley, UK) concentrated in the mitochondria of actively respiring cells. The majority of variant B precursor cystatin C molecules colocalize with mitochondria (indicated by yellow colour); some diffuse green fluorescence is also present in the cytoplasm and nucleus. In contrast, targeting of wild-type precursor cystatin C is to the ER/golgi apparatus (B), as previously described (Paraoan et al., 2001). Bright field image is overlaid with the image of RPE cells transfected with EGFP only (C) to illustrate cell morphology. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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patients with both risk factors had a significantly earlier onset of disease (Finckh et al., 2000) raises the possibility that the combination of adverse alleles of CST 3 and APOE leads to an accelerated upsetting of the intracellular processing of cellular fractions involved in AD pathology. This hypothesis is further supported by the finding of a synergistic association between the variant B CST 3 haplotype, APOE4 and AD in patients between 60 and 74 years old (Beyer et al., 2001; Cathcart, Huang, Lanham, Corder, & Poduslo, 2005). While variant B cystatin C allele has become firmly recognized as a potential AD susceptibility gene (Bertram, McQueen, Mullin, Blacker, & Tanzi, 2007) and the protective role of the wild type cystatin C in AD pathogenesis has been established (Kaeser et al., 2007; Mi et al., 2007), the functional effects of the variant protein in neurons remain to be clarified. Conversely, in relation to the eye pathology there is an obvious need for study of possible interactions of the CST 3 haplotype B with other known genetic determinants, including APOE4, of the AMD. Our investigation into the processing and trafficking of variant B cystatin C in living RPE cells has highlighted two molecular mechanisms previously not considered in relation to AMD, and possibly AD, pathology: (i) reduced secretion of modulators of extracellular environment; and (ii) inappropriate intracellular retention and targeting of proteins, other than in drusen or amyloid format. The specific consequences upon RPE cells of the reduction of extracellular function of cystatin C and of its intracellular accumulation around mitochondria are currently addressed in our laboratory. It is likely that at least some of the functional effects of, on one hand, the impaired secretion of a major regulator of proteolysis and, on the other, of the errant intracellular accumulation of the variant protein are shared by RPE and neuronal cells on the progressive path to respective pathologies. Acknowledgments The authors thank Dr. Dave Spiller for help with cell imaging, Daniel Brotchie for help with preparation and editing of figures and manuscript and Donna Gray for technical support. Confocal imaging was performed in the Centre for Cell Imaging, School of Biological Sciences, University of Liverpool. Dr Paraoan’s research on cystatin C was supported by The Wellcome Trust and The Guide Dogs for the Blind Association, UK. References Abrahamson, M. (1996). Molecular basis for amyloidosis related to hereditary brain hemorrhage. Scandinavian Journal of Clinical and Laboratory Investigation Supplement, 226, 47–56. Abrahamson, M., Barrett, A. J., Salvesen, G., & Grubb, A. (1986). Isolation of six cysteine proteinase inhibitors from human urine. Their physicochemical and enzyme kinetic properties and concentrations in biological fluids. Journal of Biological Chemistry, 261, 11282–11289. Abrahamson, M., Islam, M. Q., Szpirer, J., Szpirer, C., & Levan, G. (1989). The human cystatin C gene (CST 3), mutated in hereditary cystatin C amyloid angiopathy, is located on chromosome 20. Human Genetics, 82, 223–226. Abrahamson, M., Olafsson, I., Palsdottir, A., Ulvsback, M., Lundwall, A., Jensson, O., et al. (1990). Structure and expression of the human cystatin C gene. Biochemical Journal, 268, 287–294. Akopyan, T. (1991). Protein inhibitors of proteinases from brain. Neurochemical Research, 16, 513–517. Alvarez-Fernandez, M., Barrett, A. J., Gerhartz, B., Dando, P. M., Ni, J., & Abrahamson, M. (1999). Inhibition of mammalian legumain by some cystatins is due to a novel second reactive site. Journal of Biological Chemistry, 274, 19195–19203. Balbin, M., & Abrahamson, M. (1991). SstII polymorphic sites in the promoter region of the human cystatin C gene. Human Genetics, 87, 751–752. Barka, T., & van der Noen, H. (1994). Expression of the cysteine proteinase inhibitor cystatin C mRNA in rat eye. Anatomical Record, 239, 343–348. Barrett, A. J. (1987). The cystatins: A new class of peptidase inhibitors. Trends in Biochemical Sciences, 12, 193–196. Barrett, A. J., Davies, M. E., & Grubb, A. (1984). The place of human gamma-trace (cystatin C) amongst the cysteine proteinase inhibitors. Biochemical and Biophysical Research Communications, 120, 631–636.
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