Lysosomal Cystine Transporter Research Articles

Evaluation of the Reproductive and Developmental Safety of Cysteamine • 360

Cystinosis is an autosomal recessive metabolic disease in which the amino acid cystine accumulates in lysosomes due to a defect in lysosomal cystine transport. Cystinosis in infancy is associated with poor growth, muscle wastage and death at about age 10 due to kidney failure. Treatment with cysteamine and kidney transplantation enables cystinotic girls to reach reproductive age and increase the likelihood of a successful pregnancy. It is not known whether exposure to cysteamine will have adverse effects on their offspring exposed as fetuses. It is also possible that some of the complications seen in cystinotic children could be avoided if a pregnant woman carrying a cystinotic fetus were given cysteamine. However, this treatment is not likely to occur until the potential risk for adverse effects on the fetus is known. The goal of this project was to assess the reproductive and developmental safety of cysteamine (as phosphocysteamine) using the rat. The first experiment involved exposure of female rats to cysteamine from pre-mating through day 6.5 post-conception (pc) and assessment of female fertility and early embryonic development. The second experiment involved exposure of pregnant rats to cysteamine from day 6.5 through day 18.5 pc and fetuses were assessed for survival, growth and structural abnormalities. In all cases, cysteamine was administered orally in doses of 0, 37.5, 75, 100 or 150 mg/kg/day. In the first experiment, there were no signs of maternal toxicity during the exposures of 2 to 5 weeks before successful mating. Animals in the 150 mg/kg/day group experienced decreased maternal body weight gain during pregnancy, increased liver and spleen weights, and increased days to coitus suggesting a low level of toxicity. There were no adverse effects on reproductive performance with respect to conception and early development. In experiments involving exposures from day 6.5 through day 18.5 pc, cysteamine produced dose-dependent developmental toxicity with a no observed adverse effect level of 75 mg/kg/day. Specific malformations were associated with this effect (cleft palate, kyphosis, vertebral abnormalities) as well as growth retardation and fetal demise. Investigations continue into the mechanism for the developmental toxicity of cysteamine.

Clinical and molecular aspects of nephropathic cystinosis.

Nephropathic cystinosis, an autosomal recessively inherited lysosomal storage disease, results from impaired transport of the disulfide amino acid cystine out of cellular lysosomes. The consequent accumulation and crystallization of cystine destroys tissues, causing growth retardation in infancy, renal failure at 10 years of age, and a variety of other complications. Early oral therapy with the cystine-depleting agent cysteamine prevents renal deterioration and enhances growth. Although the lysosomal cystine carrier has been extensively studied, its molecular structure remains unknown. The lysosomal cystine transporter gene has been mapped by linkage analysis to human chromosome 17p between polymorphic microsatellite markers D17S1583 and D17S1584. Pertinent recombination events and homozygosity by descent has verified that the cystinosis gene lies in the 3.6 cM genetic interval between these two markers. The cystinosis region has been substantially reduced in size by the observation of recombination events in cystinosis patients between markers D17S1828 and D17S2167. According to radiation hybrid analysis, these two markers are separated by 10.2 cR8000 (centirad using 8000 rad radiation hybrids). Estimates of the physical size of this interval range from 187 to 510 kb. Four yeast artificial chromosomes have been identified which form a contig covering the original cystinosis region. Two P1 clones together may span the new, smaller interval, meaning that the cystinosis gene would lie on one of them. Current efforts are being directed toward using these P1 clones to isolate candidate cDNAs by a variety of methods. The ultimate cloning of the cystinosis gene will reveal how functional lysosomal porters are synthesized, targeted, processed, and integrated into the lysosomal membrane.

Niemann-Pick C Disease: Cystine and Lipids Accumulate in the Murine Model of this Lysosomal Cholesterol Lipidosis

Cystine levels in tissues of the murine BALB/C mouse model of type C Niemann-Pick disease were shown to be greatly elevated. Subcellular fractionation of liver homogenates by differential centrifugation suggested preferential accumulation in a fraction corresponding to lysosomes. Developmentally, a sharp increase in the accumulation of cystine in the mutant mouse liver occurs subsequent to a similar change in the accumulation of cholesterol, sphingomyelin and glucocerebroside. The lysosomal accumulation of cystine in this mutant mouse provides the experimental opportunity to study some aspects of the deficiency of lysosomal cystine transport noted in cystinosis.

Effects of Cycloheximide and Tunicamycin on Lysosomal Cystine Transport in Rat FRTL-5 Cells

Rat thyroid FRTL-5 cells were employed to study the synthesis and degradation of a functional integral lysosomal membrane protein, the lysosomal cystine transporter. This carrier exhibited countertransport and closely resembled the cystine transport system of human leucocytes, fibroblasts, and lymphoblasts shown to be defective in the lysosomal storage disease, nephropathic cystinosis. Using cycloheximide to prevent new protein synthesis, the half-life of the FRTL-5 cell lysosomal cystine carrier was determined to approximate 21 h. Carrier function was not influenced by the N-glycosylation inhibitor tunicamycin, nor by the oligosaccharide processing inhibitors castanospermine and deoxymannojirimycin. The data suggest that the lysosomal cystine carrier is a protein without strict functional requirements for N-linked oligosaccharides, and that rat FRTL-5 cells can be employed in future investigations into the structure and function of other integral lysosomal membrane proteins as well.

Description of a selection method highly cytotoxic for cystinotic fibroblasts but not normal human fibroblasts.

Nephropathic cystinosis is an inherited disorder characterized by a high intralysosomal accumulation of cystine due to a defect in lysosomal cystine transport. Cystine can be specifically loaded into the lysosomal compartment of intact cells by incubating cells with cystine dimethyl ester (CDME). We have applied this methyl ester loading technique to develop a selection method that is highly cytotoxic for cystinotic fibroblasts but not normal human fibroblasts and that is based on the inherent differences in lysosomal cystine transport activity of normal and cystinotic fibroblasts. Thus, only 0-0.03% of fetal cystinotic fibroblasts survive exposure to 2 mM CDME for 20 min whereas 70-80% of normal fetal fibroblasts survive these same conditions. Following transfection of cystinotic fibroblasts with normal human genomic DNA or cDNA, this CDME selection method can be used to select for those cells that have been transformed to the normal phenotype and thus aid in the identification of the gene coding for the lysosomal cystine transport protein.

Characterization of the lysosomal cystine transport system in mouse L-929 fibroblasts.

We characterize here a lysosomal cystine transporter in mouse L-929 fibroblasts. Granular fractions from cells preloaded with cystine demonstrated countertransport that showed no dependence on Na+ or K+. The Michaelis constant for infinite-trans influx was 0.27 +/- 0.06 mM (n = 3), and a nonsaturable component of cystine entry was observed with Kd = 0.8-1.8 nmol of cystine.min-1.unit of hexosaminidase-1.mM-1. We found no evidence that cystine was also carried on any of the other known lysosomal amino acid transporters. Over 50 analogs were tested for their ability to inhibit countertransport. The inhibition constants are reported for selenocystine, cystathionine, selenomethionine, and leucine. Significant requirements for recognition by the transporter were the presence of amino groups, L configuration, and a chain length not greater than eight atoms. A net positive or negative charge was not required. Some di- as well as tetrapolar amino acids were recognized. We have surmised that the binding site has polar and apolar domains, the latter being large enough to accommodate branching on C-3 and the substitution of selenium or carbon in place of sulfur.

Update on nephropathic cystinosis

The cystine that accumulates within cystinotic lysosomes comes primarily from proteins which have been degraded within this organelle. The individual amino acids have specific transport mechanisms to exit the lysosome. The lysosomal cystine transporter is defective in all types of cystinosis. When cells from patients with nephropathic and benign cystinosis were fused, the defect was not corrected and the cystine level remained elevated. This strongly indicates that the genetic defects are allelic (i.e., on the same chromosome). Cysteamine is a weak base which enters the cystinotic lysosome and reacts with cysteamine. forming a mixed disulfide of half-cystine and cysteamine. This mixed disulfide rapidly exits the lysosome via the transport system for cationic amino acids which is normal in cystinosis. Because of the success of renal transplantation, many cystinosis patients are alive in their twenties and even early thirties. Unfortunately, these patients have developed damage to other organs including thyroid, eye, central nervous system, pancreas, and muscle. Cysteamine and its analog, phosphocysteamine, are very beneficial to cystinosis patients, especially when started early in life. These drugs may prevent the need for transplantation. It is too early to know if they will prevent damage to other organs.

Corneal thickness in nephropathic cystinosis.

Cystinosis is a rare autosomal recessive metabolic disorder in which non-protein cystine accumulates within cellular lysosomes owing to a defect in lysosomal cystine transport. The pathognomonic ocular manifestation of cystinosis is the deposition of distinctive iridescent crystals in the cornea, not associated with any inflammatory response or recognised change in corneal function. We measured corneal thickness in nine patients with infantile nephropathic cystinosis. We also studied a corneal button from one of these patients who underwent corneal transplantation. All nine patients had increased corneal thickness in comparison with an age matched control population. Electron microscopy analysis of the cystinotic button revealed structural changes of both epithelium and endothelium layers. Increased corneal thickness in patients with nephropathic cystinosis may reflect subclinical corneal oedema.

Contrast sensitivity function in nephropathic cystinosis.

Cystinosis is a rare autosomal recessive metabolic disorder in which nonprotein cystine accumulates within most body organs due to a defect in lysosomal cystine transport. The pathognomonic ocular manifestations of cystinosis are the presence of distinctive iridescent crystals within ocular tissue and a pigmentary retinopathy. We measured spatial contrast sensitivity in seven patients with infantile-onset nephropathic cystinosis and compared their contrast sensitivity function with that measured in ten age-matched controls. Spatial contrast sensitivities in the patient group were significantly lower than those in the normal group. Loss of contrast sensitivity in the patients with nephropathic cystinosis was more pronounced at higher spatial frequencies. We speculate that this loss of contrast function is primarily a manifestation of corneal disease, with secondary contributions from retinal changes and central nervous system dysfunction.

Polyamines stimulate lysosomal cystine transport.

Lysosomal cystine transport is a carrier-dependent process that, in isolated lysosomes, is stimulated by proton gradients, membrane potential, and millimolar concentrations of divalent cations. The importance of these regulatory factors in vivo is not well established. Polyamines were found to stimulate cystine transport in Percoll gradient purified rat liver lysosomes with spermidine greater than putrescine = cadaverine greater than spermine in order of effectiveness. Maximal stimulation was achieved with 500 microM spermidine. The effects of optimal concentrations of polyamines and divalent cations on cystine transport were not additive. Spermidine stimulated cystine efflux from lysosomes of cultured human diploid fibroblasts, but had no effect on lysosomes of cystinotic fibroblasts which have defective cystine transport. Spermidine did not accumulate within lysosomes in exchange for cystine, had no effect on lysosomal pH, had only slight effects on the lysosomal membrane potential, and had little effect on either methionine or tyrosine efflux. Polyamines are cellular cytoplasmic components that, in physiologic concentrations, stimulate lysosomal cystine transport.

Glare disability in nephropathic cystinosis.

Cystinosis is a rare metabolic disorder in which nonprotein cystine accumulates within lysosomes due to a defect in lysosomal cystine transport. Although cystine accumulates within most ocular tissues, patients with cystinosis generally complain only of photophobia and glare. We measured glare sensitivity in 12 patients with infantile cystinosis and compared their results with an age-matched control population. Ten of the 12 patients with cystinosis had demonstrable glare disability when compared with the control group. Glare disability scores in the patients with cystinosis ranged from 5% to 50%. Dazzle glare resulting from the accumulation of cystine crystals in ocular tissue may account for glare disability seen in these patients and contribute to their complaints of photophobia.

Lysosomal cystine transport. Effect of intralysosomal pH and membrane potential.

The regulation of lysosomal cystine transport was studied using cystine dimethyl ester-loaded lysosomes isolated from human diploid fibroblasts. Net efflux from normal fibroblast lysosomes was compared to that from lysosomes of cystinotic fibroblasts, which contain an inherited mutation decreasing lysosomal cystine transport. This exodus of cystine from normal fibroblast lysosomes was greater than from cystinotic fibroblast lysosomes. When lysosomes were incubated with both 5 mM MgCl2 and 2 mM ATP (Mg/ATP), the amount of lysosomal cystine lost from normal lysosomes doubled, but the amount of cystine lost from cystinotic lysosomes remained small. This effect of Mg/ATP on cystine loss from lysosomes isolated from normal fibroblasts was abolished when either carbonyl cyanide m-chlorophenylhydrazone or N-ethylmaleimide was present, suggesting that the effect of Mg/ATP was mediated by the action of a lysosomal proton-translocating ATPase. Addition of KCl, RbCl, or NaCl to normal lysosomes caused smaller increases in cystine exodus. A variety of experimental conditions altered lysosomal pH, membrane potential, and the cystine lost from normal fibroblast lysosomes. These same experimental conditions produced similar alterations in the lysosomal pH and membrane potential of cystinotic fibroblast lysosomes without a comparable alteration in cystine loss. These results have led us to propose a model in which the transport of cystine out of the normal lysosome is regulated by both the lysosomal membrane potential gradient and the transmembrane pH gradient.

Impaired clearance of free cystine from lysosome-enriched granular fractions of I-cell-disease fibroblasts.

Cultured fibroblasts from patients with I-cell disease (mucolipidosis II) accumulate excessive amounts of free cystine, similarly to cells from patients with nephropathic cystinosis, a disorder of lysosomal cystine transport. To clarify whether the intralysosomal accumulation of cystine in I-cell-disease fibroblasts was due to a defective disposal mechanism, we measured the rates of clearance of free [35S]cystine from intact normal, cystinotic and I-cell-disease fibroblasts. Loss of radioactivity from the two mutant cell types occurred slowly (t 1/2 = 500 min) compared with the rapid loss from normal cells (t 1/2 = 40 min). Lysosome-rich granular fractions isolated from three different cystine-loaded normal, cystinotic and I-cell-disease fibroblast strains were similarly examined for non-radioactive cystine egress. Normal granular fractions lost cystine rapidly (mean t 1/2 = 43 min), whereas cystinotic granular fractions did not lose any cystine (mean t 1/2 = infinity). I-cell-disease granular fractions displayed prolonged half-times for cystine disposal (mean = 108 min), suggesting that I-cell-disease fibroblasts, like cystinotic cells, possess a defective carrier mechanism for cystine transport.

860 CYSTINOSIS: DEFECTIVE FIBROBLAST LYSOSOMAL CYSTINE TRANSPORT

Defective cystine efflux has been demonstrated in lysosomes from both peripheral leukocytes and cultured EBV-lymphocytes from cystinotic patients. However, this abnormality has been difficult to demonstrate in fibroblast lysosomes, raising the question of whether lysosomal transport is the only defect in cystinosis. We have substantiated that some previous methods of loading fibroblast lysosomes with cystine, using cystine dimethyl ester (CDME), lead to cystine accumulation in a non-lysosomal as well as the lysosomal compartment (E. Harms, personal communication). If fibroblasts are removed from their anchorage substrate and incubated with 0.25 mM CDME for 15 min., only lysosomes are loaded with cystine as shown on Percoll gradients. In cystine-loaded lysosomes from two normal control fibroblast cultures the 20 min. cystine efflux was 19% and 20% without ATP and 56% and 74% with 2 mM MgATP. Lysosomes from two cystinotic fibroblast cultures had no cystine efflux over 20 min. both with and without ATP. We have used the dye, dipropylthiodicarbocyanine iodide to measure lysosomal membrane potential. MgATP causes a positive shift in membrane potential, in both cystinotic and normal lysosomes. In both cases, inhibitor and anion effects are consistent with an electrogenic proton pump. All of these findings support the concept of an abnormal lysosomal cystine transport protein as the primary defect in this disease.