Cystatin C (CST3), the Candidate Gene for Hereditary Cystatin C Amyloid Angiopathy (HCCAA), and Other Members of the Cystatin Gene Family Are Clustered on Chromosome 20p11.2

doi:10.1006/geno.1993.1139

Genomics

Volume 16, Issue 1, April 1993, Pages 50-55

https://doi.org/10.1006/geno.1993.1139 Get rights and content

Abstract

The cystatin C gene (CST3) encodes a low-molecular-weight cysteine proteinase inhibitor belonging to family II of the cystatin super family and is mutated in cases of hereditary cystatin C amyloid angiopathy (HCCAA). CST3, which along with other family II cystatin genes is a member of the cystatin gene family, has been assigned to chromosome 20. To investigate the genomic organization on chromosome 20, the CST3 gene and related sequences were regionally mapped by fluorescence in situ hybridization (FISH), Southern blot, and pulsed-field gel electrophoresis (PFGE) analysis using the cDNA cystatin C probe C6a and three genomic probes, C3E1, C3E2, and C3E2-2. Probe C3E2-2, which like probe C3E2 is specific for CST3, hybridized to only one Hind III and one XbaI fragment on Southern blots and to a 300-kb Bss HII PFGE fragment. FISH with probe C3E2 mapped this locus to chromosome 20p11.2, with an FL-pter value of 0.37 ± 0.07 on the physical map. Probe C3E1 containing the most conserved cystatin gene exon (exon 1) and its flanking sequences hybridized with more fragments, e.g., to eight Xba I and nine HindIII fragments on conventional Southern blots and to eight SmaI, two BssHII (900 and 300 kb), and two Not I fragments after PFGE. FISH with C3E1 revealed only one single site at 20p11.2 with an FL-pter value of 0.37 ± 0.04, identical to that obtained with C3E2. From these results it is concluded that (1) exon 1 and its flanking sequences are preferentially conserved within the cystatin gene family and that (2) CST3 and probably seven other members of the cystatin gene family are clustered within an at maximum 1.2-Mb segment on chromosome 20p11.2.

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Screening osteoporotic femoral neck without measuring bone mineral density with the use of tartrate resistant acid phosphatase-5b and serum-creatinine-to-cystatin C ratio in Japanese postmenopausal women
2020, Journal of Orthopaedic Science
Screening osteoporosis in Japanese postmenopausal women is an important subject for preventing bone fragility fracture (BFF). Measuring bone mineral density (BMD) is included in the criteria for osteoporosis diagnosis. However, it has not gained popularity.
BMD of lumbar spine (LS) and femoral neck (FN) were measured, the serum creatinine-to-cystatin C ratio (Cr/CysC), tartrate-resistant acid phosphatase-5b (TRACP-5b), body mass index (BMI) were also simultaneously measured. These subjects had no potential secondary osteoporosis factors or concomitant drug administration for osteoporosis (pSubjects; primary subjects). Best subsets regression analysis (BeStR) was evaluated for determining the statistically significant factors, and multivariate regression analysis (MLR) was used to assess the correlation of these factors with BMD at each part. Relationship between osteoporotic criteria whether T score≤−2.5 and each factor was also evaluated at each part with binary logistic regression analysis (BLR). Cut-off index (COI) at each part was determined, and sensitivity and specificity were evaluated in pSubjects and in subjects with whom potential secondary osteoporosis factors were included (sSubjects).
BMI and Cr/CysC had a significant influence on BMD at the LS with BeStR, whereas only BMI demonstrated significant correlation with BMD both with MLR and BLR, with 22.7 of COI and sensitivity and specificity of 48.7% and 94.7%, respectively, in pSubjects with 60 and 49.1% and 87.5%, respectively, in sSubjects with 83. At the FN, Cr/CysC, BMI, and TRACP-5b significantly influenced BMD, whereas all these factors demonstrated significant correlation with BMD. Cr/CysC and TRACP-5b demonstrated significant correlation with T score≤−2.5 in BLR. With COI that was made in combination with these factors and subjects’ age, sensitivity and specificity were 67.7% and 82.4%, respectively, in pSubjects and 55.8% and 82.8%, respectively, in sSubjects.
These results suggested that BMI, TRACP-5b, and Cr/CysC may be realistic surrogate markers for screening osteoporosis in Japanese postmenopausal women.
Journey of cystatins from being mere thiol protease inhibitors to at heart of many pathological conditions
2017, International Journal of Biological Macromolecules
Citation Excerpt :
It possesses the unusual trait of being a glycoprotein, carrying an N-linked carbohydrate chain at position 108 [81]. This protein is only distantly related to the other known family members as reflected by the genomic position of the cystatin E/M gene on chromosome 11q13 [82], whereas all other family II cystatin genes are clustered in a narrow region on chromosome 20p11.2 [83]. Cystatin E/M is synthesized as a pre-protein with a putative 28 residues while mature cystatin E/M contains 121 amino acids [80].
Cystatins are thiol proteinase inhibitors (TPI), present ubiquitously in animals, plants and micro-organisms. These are not merely inhibitors rather they are at heart of many pathological conditions ranging from diabetes to renal failure. These are essential for maintenance of protein balance of the cell; once this balance gets disturbed, it may lead to cell death. Thus, cystatins cannot be merely regarded as TPI’s as these have been found to play a pivotal role in tumorigenesis and neurodegenerative diseases. Many studies have reported the variation in cystatin level in incidences of different types of cancer; highlighting an important role played by these inhibitors in cancer development and progression. Cystatin C is increasingly replacing creatinine as a biomarker of glomerular filtration rate (GFR) thereby highlighting the importance of this important inhibitor. Some recent studies have also reported the interaction pattern of various anti-cancer drugs with cystatins in a bid to find how these drugs affect this important inhibitors and whether these drugs have any side effect on cystatins. Thus, in this growing disease era it can be said that cystatins are no more just inhibitors blocking the activity of thiol proteases rather they play a pivotal role in variety of pathological conditions.
Fertility defects in mice expressing the L68Q variant of human cystatin C: A role for amyloid in male infertility
2014, Journal of Biological Chemistry
Citation Excerpt :
The targeting vector was excised and electroporated into E14.1 129/Sv ES cells using a Bio-Rad Gene Pulser. After selection in G418, the surviving clones were expanded for PCR analysis as well as examined by fluorescence in situ hybridization to confirm the localization of the L68Q construct to the cystatin locus on mouse chromosome 2 as described previously (8). The correctly targeted 129/Sv ES clones were microinjected into C57Bl/6J (B6) mouse blastocysts and implanted into pseudopregnant mice.
Hereditary cystatin C amyloid angiopathy is an autosomal dominant disorder in which a variant form of cystatin C (L68Q) readily forms amyloid deposits in cerebral arteries in affected individuals resulting in early death. L68Q protein deposits in human cystatin C amyloid angiopathy patients have also been found in tissues outside of the brain including the testis, suggesting possible effects on fertility. Heterozygous transgenic mice (L68Q) that express the human L68Q variant of cystatin C under the control of the mouse cystatin C promoter were unable to generate offspring, suggesting the presence of L68Q cystatin C amyloid affected sperm function. In vitro studies showed that epididymal spermatozoa from L68Q mice were unable to fertilize oocytes and exhibited poor sperm motility. Furthermore, spermatozoa from L68Q mice exhibited reduced cell viability compared with wild type (WT) spermatozoa and often were detected in large agglutinated clumps. Examination of the epididymal fluid and spermatozoa from L68Q mice showed increased levels and distinct forms of cystatin C amyloid that were not present in WT mice. The addition of epididymal fluid from L68Q mice to WT spermatozoa resulted in a recapitulation of the L68Q phenotype in that WT spermatozoa showed reduced cell viability and motility compared with WT spermatozoa incubated in epididymal fluid from WT mice. L68Q epididymal fluid that was depleted of cystatin C amyloids, however, did not impair the motility of WT spermatozoa. Taken together these studies suggest that amyloids in the epididymal fluid can be cytotoxic to the maturing spermatozoa resulting in male infertility.
Cystatin C as a Biomarker in Kidney Disease
2011, Biomarkers of Kidney Disease
Presently, 12 human proteins are known to contain polypeptide chains with sequences homologous to that of cystatin C and form the human cystatin protein superfamily of cysteine protease inhibitors. Cystatin C is the strongest human inhibitor of important cysteine proteases such as cathepsin K and H and is, in contrast to the other cystatins, present in significant concentrations in all investigated human body fluids. The structures of the cystatin C gene, comprising three exons and two introns, and of its promoter, indicate that the gene is a so-called housekeeping gene suggesting a stable rate of production of the protein in all nucleated cell types. As a low molecular mass protein (13.4 kDa) cystatin C is mainly catabolised by free filtration in the glomeruli followed by virtually complete tubular reabsorption. Although GFR is a good general indicator of renal disease, it will not detect renal diseases in which the composition of the glomerular filtrate is deranged in the presence of a normal GFR. Studies using gold standard determinations of GFR are still required to unequivocally demonstrate that cystatin C has an advantage over measured GFR as a predictor of cardiovascular events. Although a raised level of cystatin C might indicate a decline in GFR due to significant atherosclerosis in the renal arteries and thus an increased probability of simultaneous significant atherosclerosis in other vital arteries, e.g. the coronary and brain arteries, other mechanisms explaining the usefulness of cystatin C as a predictor of aging success might be involved.
Cystatins - Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells
2010, Biochimie
Citation Excerpt :
Like the other type 2 cystatins, it is synthesised as a preprotein with a signal peptide (26 residues long in the case of cystatin C [12]). As for the other type 2 cystatins (with one exception), its gene is located in the cystatin multigene locus at chromosome 20 [13]. Cystatin C is produced in a wide variety of human tissues and cell lines [14,15] and it is the type 2 cystatin showing the most wide-spread body distribution.
Cystatins are present in mammals, birds, fish, insects, plants, fungi and protozoa and constitute a large protein family, with most members sharing a cysteine protease inhibitory function. In humans 12 functional cystatins exist, forming three groups based on molecular organisation and distribution in the organism. The type 1 cystatins (A and B) are known as intracellular, type 2 cystatins (C, D, E/M, F, G, S, SN and SA) extracellular and type 3 cystatins (L- and H-kininogen) intravascular proteins. The present paper is focused on the human cystatins and especially those of type 2, which are directed (with signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels, but also contain high amounts of cystatin C are presented. Culturing of these cells in medium containing cystatin C at concentrations found in body fluids resulted in increased intracellular cystatin C, as a result of an uptake process. At immunofluorescence cytochemistry a pronounced vesicular cystatin C staining was observed. The simplistic denotation of the type 2 cystatins as extracellular inhibitors is thus challenged, and possible biological functions of the internalised cystatins are discussed. To illustrate the special case of high cellular cystatin content seen in cells of patients with hereditary cystatin C amyloid angiopathy, expression vectors for wild-type and L68Q mutated cystatin C were used to transfect SK-N-BE(2) cells. Clones overexpressing the two variants showed increased secreted levels of cystatin C. Within the cells the L68Q variant appeared to mainly localise to the endoplasmic reticulum rather than to acidic vesicular organelles, indicating limitations in the transport out from the cell rather than increased uptake as explanation for the elevated cellular cystatin levels seen in hereditary cystatin C amyloid angiopathy.
Cystatin C in macular and neuronal degenerations: Implications for mechanism(s) of age-related macular degeneration
2010, Vision Research
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.

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Regular ArticleCystatin C (CST3), the Candidate Gene for Hereditary Cystatin C Amyloid Angiopathy (HCCAA), and Other Members of the Cystatin Gene Family Are Clustered on Chromosome 20p11.2

Abstract

Regular Article
Cystatin C (CST3), the Candidate Gene for Hereditary Cystatin C Amyloid Angiopathy (HCCAA), and Other Members of the Cystatin Gene Family Are Clustered on Chromosome 20p11.2