Accumulation Of Cystine Research Articles

ERS1 encodes a functional homologue of the human lysosomal cystine transporter

Cystinosis is a lysosomal storage disease caused by an accumulation of insoluble cystine in the lumen of the lysosome. CTNS encodes the lysosomal cystine transporter, mutations in which manifest as a range of disorders and are the most common cause of inherited renal Fanconi syndrome. Cystinosin, the CTNS product, is highly conserved among mammals. Here we show that the yeast Ers1 protein and cystinosin are functional orthologues, despite sharing only limited sequence homology. Ers1 is a vacuolar protein whose loss of function results in growth sensitivity to hygromycin B. This phenotype can be complemented by the human CTNS gene but not by mutant ctns alleles that were previously identified in cystinosis patients. A genetic screen for multicopy suppressors of an ers1Delta yeast strain identified a novel gene, MEH1, which is implicated in regulating Ers1 function. Meh1 localizes to the vacuolar membrane and loss of MEH1 results in a defect in vacuolar acidification, suggesting that the vacuolar environment is critical for normal ERS1 function. This genetic system has also led us to identify Gtr1 as an Meh1 interacting protein. Like Meh1 and Ers1, Gtr1 associates with vacuolar membranes in an Meh1-dependent manner. These results demonstrate the utility of yeast as a model system for the study of CTNS and vacuolar function.

Cysteamine prevents and reverses the inhibition of creatine kinase activity caused by cystine in rat brain cortex

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.

Exfoliated human proximal tubular cells: a model of cystinosis and Fanconi syndrome

The renal Fanconi syndrome (FS) is characterised by generalised proximal tubular dysfunction. Cystinosis is the most common genetic cause of the FS and results from defective function of cystinosin, due to mutations of the CTNS gene leading to intralysosomal cystine accumulation. Despite these advances in our understanding of the molecular basis of cystinosis, the mechanisms of proximal tubular cell (PTC) dysfunction are still unknown. We have further characterised an in vitro model of cultured cells exfoliated in patients' urine. We cultured cells from 9 cystinosis children, 4 children with Lowe syndrome and 8 controls. PTC phenotype and homogeneity were studied by lectin staining, immunocytochemistry (using ZO-1 as an epithelial marker) and enzyme cytochemistry (using gamma-glutamyltransferase as a PTC marker). All cultured cells showed PTC phenotype. Cystinosin was stained using anti-cystinosin antibody and co-localised to the lysosomes with LAMP-2 antibody. Additionally, we have demonstrated significantly elevated intracellular cystine levels in cystinotic cell lines (13.8+/-2.3 nmol 1/2 cystine/mg protein, P <0.001) compared with controls. We believe this in vitro model will allow further investigation of cystinosis and other types of the FS.

A new mutation in two siblings with cystinosis presenting with Bartter syndrome

Nephropathic cystinosis is a severe autosomal recessive inherited metabolic disease characterized by accumulation of free cystine in lysosomes. Cystinosis can lead to renal failure and multiorgan impairment. Only five cases of cystinosis with associated Bartter syndrome are reported in the literature, and no genetic evaluation has been reported. We describe two siblings with nephropathic cystinosis presenting with features of Bartter syndrome and their genetic pattern.

Glutathione precursors replenish decreased glutathione pool in cystinotic cell lines

Cystinosis is an inherited disorder due to mutations in the CTNS gene which encodes cystinosin, a lysosomal transmembrane protein involved in cystine export to the cytosol. Both accumulation of cystine in the lysosome and decreased cystine in the cytosol may participate in the pathogenic mechanism underlying the disease. We observed that cystinotic cell lines have moderate decrease of glutathione content during exponential growth phase. This resulted in increased solicitation of oxidative defences of the cell denoted by concurrent superoxide dismutase induction, although without major oxidative insult under our experimental conditions. Finally, decreased glutathione content in cystinotic cell lines could be counterbalanced by a series of exogenous precursors of cysteine, denoting that lysosomal cystine export is a natural source of cellular cysteine in the studied cell lines.

FISH diagnosis of the common 57-kb deletion in CTNS causing cystinosis.

Cystinosis is an autosomal recessive lysosomal storage disease caused by mutations in CTNS. The most prevalent CTNS mutation, a 57-kb deletion, occurs in approximately 60% of patients in the United States and northern Europe and removes exons 1-9, most of exon 10, the CTNS promoter region, and all of an adjacent gene of unknown function called CARKL. CTNS codes for the lysosomal cystine transporter, whose absence leads to intracellular cystine accumulation, widespread cellular destruction, renal Fanconi syndrome in infancy, renal glomerular failure in later childhood, and other systemic complications. Because treatment with oral cysteamine can prevent or delay these complications significantly, early and accurate diagnosis is critical. This study describes the generation of fluorescence in situ hybridization (FISH) probes for the 57-kb deletion in CTNS, enabling cytogenetics laboratories to test for this common mutation. The probes would also be able to detect a less frequent 11.7-kb deletion. A blinded study was performed using multiplex PCR analysis as the gold standard to determine the presence or absence of the 57-kb deletion. The FISH probes, evaluated on 12 lymphoblastoid cell lines from singly deleted, doubly deleted, and nondeleted patients, made the correct diagnosis in every case. This appears to be the first FISH-based diagnostic method described for any lysosomal storage disorder. It can assist in the antenatal and perinatal diagnosis of cystinosis and promote earlier salutary therapy with cysteamine.

Inhibition of pyruvate kinase activity by cystine in brain cortex of rats

Cystinosis is a metabolic disturb associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending of the involved tissues. Accumulation of cystine in the brain may lead to severe neurological symptoms. However, the mechanisms by which cystine is neurotoxic are not fully understood. Considering that pyruvate kinase (PK) is a thiolic enzyme crucial for the glycolytic pathway, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on PK activity in the brain cortex of developing Wistar rats. We also performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol groups protector, and cysteamine (CysN), the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity by two different mechanisms, one through the competition with ADP and phosphoenolpyruvate (PEP), and the other non-competitively, probably through oxidation of the thiol groups of PK. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine. Considering that cysteamine is used in patients with cystinosis because it causes parenkimal organ cystine depletion, the present data provides a possible new beneficial effect for the use of this drug.

Cysteamine prevents and reverses the inhibition of pyruvate kinase activity caused by cystine in rat heart.

Cystinosis is a disorder associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending on the involved tissues. Accumulation of cystine in myocardium may lead to heart failure. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that thiolic enzymes like pyruvate kinase (PK) may be altered by disulfides like cystine, the main objective of the present study was to investigate the effect of cystine on PK activity in the heart of developing rats. We performed kinetic studies and investigated the effects of reduced glutathione (GSH), a biologically occurring thiol groups protector, and cysteamine, the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine, suggesting that cystine inhibits PK activity by oxidation of the sulfhydryl groups of the enzyme. Although there is no definite proof of cystine within cytoplasm, there is indirect proof t it is able to escape lysosomes and come in contact with PK. Considering that cysteamine is used in patients with cystinosis because it causes parenchymal organ cystine depletion, the present data provide a possible new effect for this drug.

New aspects of the pathogenesis of cystinosis.

Cystinosis is a lysosomal transport disorder characterized by an intra-lysosomal accumulation of cystine, the disulfide of the amino acid cysteine. It is the most common inherited cause of the renal Fanconi syndrome. There are various clinical forms, infantile, juvenile, and ocular, based on age of onset and severity of symptoms. The first clinical description appeared in the early 1900s, but it was not until 1998 that the causative gene, CTNS, was identified. CTNS encodes cystinosin, a novel seven transmembrane domain (TM) protein. Cystinosin is a lysosomal membrane protein that requires two lysosomal targeting signals: a classic GYDQL motif in its C-terminal tail and a novel conformational motif, the core of which is YFPQA, situated in the fifth inter-TM loop. Cystinosin is the lysosomal cystine transporter and its activity is H(+)-driven. A mouse model of cystinosis was recently generated and Ctns(-/-) mice accumulate cystine in all tissues. A high level of cystine accumulates in the kidney, but these mice do not present with proximal tubulopathy or renal dysfunction. The Ctns(-/-) mouse model may provide clues to the cause of the Fanconi syndrome associated with cystinosis, the origin of which remains poorly understood.

Intralysosomal cystine accumulation in mice lacking cystinosin, the protein defective in cystinosis.

Cystinosis is an autosomal recessive disorder characterized by an accumulation of intralysosomal cystine. The causative gene, CTNS, encodes cystinosin, a seven-transmembrane-domain protein, which we recently showed to be a lysosomal cystine transporter. The most severe and frequent form of cystinosis, the infantile form, appears around 6 to 12 months, with a proximal tubulopathy (de Toni-Debré-Fanconi syndrome) and ocular damage. End-stage renal failure is reached by 10 years of age. Accumulation of cystine in all tissues eventually leads to multisystemic disease. Treatment with cysteamine, which reduces the concentration of intracellular cystine, delays disease progression but has undesirable side effects. We report the first Ctns knockout mouse model generated using a promoter trap approach. We replaced the last four Ctns exons by an internal ribosome entry site-betagal-neo cassette and showed that the truncated protein was mislocalized and nonfunctional. Ctns(-/-) mice accumulated cystine in all organs tested, and cystine crystals, pathognomonic of cystinosis, were observed. Ctns(-/-) mice developed ocular changes similar to those observed in affected individuals, bone defects and behavioral anomalies. Interestingly, Ctns(-/-) mice did not develop signs of a proximal tubulopathy, or renal failure. A preliminary therapeutic trial using an oral administration of cysteamine was carried out and demonstrated the efficiency of this treatment for cystine clearance in Ctns(-/-) mice. This animal model will prove an invaluable and unique tool for testing emerging therapeutics for cystinosis.

Follow-up and treatment of adults with cystinosis in the Netherlands.

Cystinosis is a rare autosomal recessive disease, caused by intracellular cystine accumulation due to a defect in the lysosomal cystine carrier. Treatment with cysteamine favours the transport of cystine out of the lysosomes, diminishes organ damage, and postpones the progression of renal failure. The extra-renal deposition of cystine continues after renal transplantation, leading to later complications. The objective of this study was to evaluate the follow-up, the occurrence of late complications, the social status, and the adequacy of cysteamine treatment in adult patients with cystinosis. The medical histories of 10 adult cystinosis patients aged 19-36 years were studied. The impairment of thyroid function, central nervous system, endocrine pancreas, and ocular manifestations, as well as treatment with cysteamine were evaluated. Eight patients received in total 12 renal grafts, one patient was dialysed and one received conservative treatment for chronic renal failure. Extra-renal complications were noted in six patients, loss of visual acuity in four, hypothyroidism in three, diabetes mellitus in one, cerebral atrophy and epilepsy in one, and swallowing difficulties in two patients. Ophthalmic control was not performed in two patients, thyroid function was not controlled in two and glycaemia not controlled in two patients. Seven patients received 2100-4000 mg cysteamine per day in 2 (n=2), 3 (n=1), 4 (n=3), or 6 (n=1) doses. Cystine concentration in leukocytes was measured once or twice a year in eight patients and was within the recommended range only in three patients. A high rate of extra-renal complications in adults with nephropathic cystinosis was found. Optimizing the cysteamine therapy may attenuate these complications. Better communication between paediatric and 'adult's' nephrologists is needed to improve follow-up and treatment of grown-up cystinosis patients.

Inhibition of Na(+)-dependent transporters in cystine-loaded human renal cells: electrophysiological studies on the Fanconi syndrome of cystinosis.

Cystinosis is the most common cause of the renal Fanconi syndrome in children, leading to severe electrolyte disturbances and growth failure. A defective lysosomal transporter, cystinosin, results in intralysosomal accumulation of cystine. Loading cells with cystine dimethyl ester (CDME) is the only available model for this disease. This model was used to present electrophysiologic studies on immortalized human kidney epithelial (IHKE-1) cells that had been derived from the proximal tubule with the slow whole-cell patch clamp technique. Basal membrane voltages (V(m)) of IHKE-1 cells were -30.7 +/- 0.4 mV (n = 151). CDME concentration-dependently altered V(m) with an initial depolarization (2.7 +/- 0.2 mV;n = 76; 1 mM CDME) followed by a more pronounced hyperpolarization (-9.9 +/- 1.0 mV;n = 49). Three Na(+)-dependent transporters were examined. Alanine (1 mM) depolarized IHKE-1 cells by 17.6 +/- 0.7 mV (n = 59), and phosphate (1.8 mM) depolarized by 9.7 +/- 1.1 mV (n = 18). Acidification of IHKE-1 cells with propionate (20 mM) resulted in a depolarization of V(m) by 7.1 +/- 0.3 mV (n = 21) followed by a repolarization by 2.9 +/- 0.3 mV/min (n = 17), reflecting Na(+)/H(+)-exchanger activity. Acute addition of 1 mM CDME did not alter the alanine- and propionate-induced changes in V(m), but it reduced the phosphate-induced depolarization by 37 +/- 9% (n = 10). Incubation with 1 mM CDME reduced the activity of all three transporters. Depolarizations by alanine and phosphate and the repolarization after propionate were inhibited by 57 +/- 4% (n =30), 45 +/- 9% (n = 9), and 78 +/- 15% (n = 8), respectively. In conclusion, this study demonstrates that CDME acutely alters V(m) of IHKE-1 cells and that at least three Na(+)-dependent transporters are inhibited, the Na(+)-phosphate cotransporter most sensitively. This might suggest that phosphate depletion and dissipation of the Na(+)-gradient are involved in the development of the Fanconi syndrome of cystinosis.

Cystinosis

Nephropathic cystinosis was first described in the early 1900s in a 21-mo-old boy who died of progressive anorexia; two siblings had previously died in infancy under similar circumstances (1). By meticulous observations and analyses, it became clear that abnormal cystine accumulation was characteristic of this autosomal recessive disease (2-4). Although some considered it to be a severe form of cystinuria, cystinosis was clearly distinguished from cystinuria by Bickel’s excellent clinical and biochemical observations (5). Clinically, untreated cystinosis patients would suffer renal tubular Fanconi syndrome, with hypophosphatemic rickets, hypokalemia, polyuria, polydypsia, dehydration, acidosis, and growth retardation followed by end-stage renal disease (ESRD) and death at approximately 10 yr of age (6,7). Shortly after the distinctive clinical aspects of cystinosis were defined, the intracellular location of cystine storage was determined. In the late 1960s, Schneider et al. (8), Schulman et al. (9), and Patrick and Lake (10) performed elegant biochemical studies to demonstrate that cystinosis cells stored cystine within the lysosome, an organelle discovered only two decades before (11). Subsequent work

Immunolocalization of cystinosin, the protein defective in cystinosis.

Cystinosis is an autosomal recessive disorder associated with excessive lysosomal cystine accumulation secondary to defective lysosomal cystine efflux. CTNS, the gene mutated in cystinosis, codes for the lysosomal membrane protein cystinosin. Antisera were raised in rabbits to a carboxy-terminal oligopeptide sequence from cystinosin. Antisera were screened by Western blotting and immunocytochemical analyses of transfected COS-7 cells expressing either human wild-type cystinosin, a wild-type cystinosin-green fluorescent protein (GFP) fusion protein, or a fusion protein of GFP and mutant human cystinosin with a carboxy-terminal deletion. In Western blots, bands corresponding to cystinosin or cystinosin-GFP were observed in transfected cells but no signal was detected in cells expressing the carboxy-terminal mutant; preimmune sera yielded negative results in all three cases. In transfected cells expressing wild-type cystinosin, immunoreactivity appeared in subcellular vesicles. In cells expressing the wild-type cystinosin-GFP fusion protein, immunoreactivity colocalized with GFP fluorescence. Previous studies demonstrated that GFP fluorescence from this construct colocalized with immunostaining for a known lysosomal membrane protein, i.e., lysosome-associated membrane protein 2. In immunohistochemical analyses, cystinosin localized to tubule epithelia in three normal human kidneys, with a pattern similar to that of lysosome-associated membrane protein 2; cystinosin immunoreactivity was absent in kidneys from patients with a CTNS deletion. For the first time, antisera have been raised that localize cystinosin in cells in vitro and in vivo.

Negligible urinary cysteamine loss in cystinosis patients with Fanconi syndrome.

Cystinosis is an inborn error of lysosomal cystine transporter, resulting in cystine accumulation in lysosomes of all cells. Renal Fanconi syndrome is an early sign of kidney involvement in cystinosis patients. Cysteamine, a small amino-thiol, depletes intralysosomal cystine content and reduces organ damage. However, it does not reverse renal Fanconi syndrome and only postpones the progression to renal failure. We examined whether cysteamine could be lost in the urine of cystinosis patients with Fanconi syndrome, which may explain the inefficiency of treatment. Urinary cysteamine loss was studied in 6 cystinosis patients with and without Fanconi syndrome and was less than I% of ingested dose in all patients.

Characterization of a putative founder mutation that accounts for the high incidence of cystinosis in Brittany.

Cystinosis is an autosomal recessive disorder, characterized by an accumulation of intralysosomal cystine, with an incidence of 1 in 100,000 to 200,000 live births. A higher incidence of cystinosis, 1 in 26,000 live births, has been reported in the western French province of Brittany. PCR amplification and sequencing has identified a 27-bp deletion starting 3 bp before the end of exon 8 and continuing into intron 8, 898-900+24del27, which has only been detected in families from this region. Reverse transcription-PCR amplification of RNA from an affected individual has shown that this mutation is indeed a splice-site mutation and results in the production of aberrant transcripts. These transcripts are predicted to either severely truncate cystinosin or alter its topology, thus accounting for the severe phenotype of these individuals. The mutation 898-900+24del27 has been identified in 7 of 18 alleles studied. This mutation is likely to be a founder mutation and would account for the higher incidence of cystinosis in Brittany.(1)

The Targeting of Cystinosin to the Lysosomal Membrane Requires a Tyrosine-based Signal and a Novel Sorting Motif

Cystinosis is a lysosomal transport disorder characterized by an accumulation of intra-lysosomal cystine. Biochemical studies showed that the lysosomal cystine transporter was distinct from the plasma membrane cystine transporters and that it exclusively transported cystine. The gene underlying cystinosis, CTNS, encodes a predicted seven-transmembrane domain protein called cystinosin, which is highly glycosylated at the N-terminal end and carries a GY-XX-Phi (where Phi is a hydrophobic residue) lysosomal-targeting motif in its carboxyl tail. We constructed cystinosin-green fluorescent protein fusion proteins to determine the subcellular localization of cystinosin in transfected cell lines and showed that cystinosin-green fluorescent protein colocalizes with lysosomal-associated membrane protein 2 (LAMP-2) to lysosomes. Deletion of the GY-XX-Phi motif resulted in a partial redirection to the plasma membrane as well as sorting to lysosomes, demonstrating that this motif is only partially responsible for the lysosomal targeting of cystinosin and suggesting the existence of a second sorting signal. A complete relocalization of cystinosin to the plasma membrane was obtained after deletion of half of the third cytoplasmic loop (amino acids 280-288) coupled with the deletion of the GY-DQ-L motif, demonstrating the presence of the second signal within this loop. Using site-directed mutagenesis studies we identified a novel conformational lysosomal-sorting motif, the core of which was delineated to YFPQA (amino acids 281-285).

Identification and characterisation of the murine homologue of the gene responsible for cystinosis, Ctns.

BackgroundCystinosis is an autosomal recessive disorder characterised by an intralysosomal accumulation of cystine, and affected individuals progress to end-stage renal failure before the age of ten. The causative gene, CTNS, was cloned in 1998 and the encoded protein, cystinosin, was predicted to be a lysosomal membrane protein.ResultsWe have cloned the murine homologue of CTNS, Ctns, and the encoded amino acid sequence is 92.6% similar to cystinosin. We localised Ctns to mouse chromosome 11 in a region syntenic to human chromosome 17 containing CTNS. Ctns is widely expressed in all tissues tested with the exception of skeletal muscle, in contrast to CTNS.ConclusionsWe have isolated, characterised and localised Ctns, the murine homologue of CTNS underlying cystinosis. Furthermore, our work has brought to light the existence of a differential pattern of expression between the human and murine homologues, providing critical information for the generation of a mouse model for cystinosis.

New Method for Determining Cystine in Leukocytes and Fibroblasts

Cystinosis is a rare inborn error of cystine transport, leading to accumulation of cystine in the lysosomes. To diagnose cystinosis and monitor treatment with cysteamine, adequate measurements of cystine concentrations in leukocytes and cultured fibroblasts are required. Cells were sonicated in the presence of excess N-ethylmaleimide to prevent oxidation of cysteine to cystine and disulfide exchange reactions of cystine with available sulfhydryl moieties. Cystine was measured as cysteine after reduction with sodium borohydride and derivatization with monobromobimane, followed by separation with automated HPLC and fluorescence detection. The assay was linear to 200 micromol/L cysteine. Within-run and day-to-day (total) imprecision (CV) was <5%, and the detection limit was 0.3 micromol/L. Added cysteine, up to 200 micromol/L, was completely removed, and recovery of added cystine was 69-86%. Cystine was stable for at least 2 months in leukocytes frozen in liquid nitrogen and stored at -80 degrees C CONCLUSIONS: Oxidation of cysteine to cystine and disulfide exchange reactions of cystine with sulfhydryl moieties are prevented by N-ethylmaleimide. The detection limit for the determination of cystine is adequate to measure cystine in leukocytes and cultured fibroblasts for diagnosis of cystinosis and monitoring treatment with cysteamine.

Molecular analysis of cystinosis: probable Irish origin of the most common French Canadian mutation.

Infantile nephropathic cystinosis, an autosomal recessive disease characterized by a lysosomal accumulation of cystine, presents as failure to thrive, rickets and proximal renal tubular acidosis. The cystinosis gene, CTNS, which maps to chromosome 17p13, encodes a predicted 55 kDa protein with characteristics of a lysosomal membrane protein. We have conducted extensive linkage analysis in a French Canadian cystinosis cohort identifying a founding haplotype present in approximately half (21/40) of the chromosomes studied. Subsequent mutational analysis, in addition to identifying two novel mutations, has unexpectedly revealed a mutation which has been previously found in Irish (but not French) cystinotic families on these 21 French Canadian chromosomes. Haplotype analysis of two Irish families with this mutation supports the hypothesis that Celtic chromosomes represent an extensive portion of cystinosis chromosomes in French Canada. Our analysis underlines the genetic heterogeneity of the French Canadian population, reflecting a frequently unrecognized contribution from non-Gallic sources including the Irish.