J Am Soc Nephrol 13: 2189–2191, 2002 Cystinosis: Antibodies and Healthy Bodies

ROBERT KLETA AND WILLIAM A. GAHL Section on Human Biochemical Genetics, Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland.

Nephropathic cystinosis was first described in the early 1900s transplant cystinosis patients. At the same time, eyedrops con- in a 21-mo-old boy who died of progressive anorexia; two taining cysteamine (0.5%) were shown to dissolve the corneal siblings had previously died in infancy under similar circum- crystals, which cause a painful photophobia and occasional stances (1). By meticulous observations and analyses, it be- epithelial erosions (21-23). The crystals are pathognomic for came clear that abnormal cystine accumulation was character- cystinosis and can be identified by an experienced ophthalmol- istic of this autosomal recessive disease (2-4). Although some ogist as early as 1 yr of age (24). considered it to be a severe form of cystinuria, cystinosis was The era of molecular biology has brought with it an under- clearly distinguished from cystinuria by Bickel’s excellent standing of the genetic basis of cystinosis. In the mid 1990s, clinical and biochemical observations (5). Clinically, untreated the cystinosis gene was mapped to chromosome 17p (25); in cystinosis patients would suffer renal tubular Fanconi syn- 1998, the gene CTNS, coding for a lysosomal transport protein drome, with hypophosphatemic rickets, hypokalemia, polyuria, named cystinosin, was isolated (26). A 57,257-bp deletion (27) polydypsia, dehydration, acidosis, and growth retardation fol- was found to be responsible for approximately half of Northern lowed by end-stage renal disease (ESRD) and death at approx- European and North American cystinosis patients (26,28); this imately 10 yr of age (6,7). Shortly after the distinctive clinical deletion is easily detected by a multiplex PCR amplification aspects of cystinosis were defined, the intracellular location of assay (29). Cystinosis occurs in all ethnic groups, and 56 cystine storage was determined. In the late 1960s, Schneider et different mutations have been described to date, including al. (8), Schulman et al. (9), and Patrick and Lake (10) per- promoter, missense, nonsense, deletion, insertion, and splice- formed elegant biochemical studies to demonstrate that cysti- site mutations (26,28,30–36). The allelic disorders, intermedi- nosis cells stored cystine within the lysosome, an organelle ate cystinosis (with late-onset renal disease) and ocular cysti- discovered only two decades before (11). Subsequent work nosis (limited to corneal involvement), are much more rare and showed that cystine was transported out of normal lysosomes result from the combination of one severe (nephropathic) mu- by a carrier system that exhibited countertransport, saturation tation and one mild mutation in CTNS (30,33). kinetics, and stereospecificity (12,13); this system was defec- Isolation of the cystinosis gene has confirmed previous tive in cystinosis lysosomes (12,14,15) and displayed a gene- findings concerning cystinosin and has revealed new facts dosage effect in heterozygotes for cystinosis (16). about integral lysosomal membrane proteins with transport The major therapeutic breakthrough for cystinosis children function. Cystinosin contains 367 amino acids, including seven ␤ came with the discovery that cysteamine ( -mercaptoethyl- transmembrane regions, eight potential N-linked glycosylation amine) could deplete cells of cystine (17) by combining with sites, and a lysosomal-targeting motif at the carboxy terminus cystine to produce cysteine and cysteine-cysteamine mixed (26). A second targeting motif necessary for proper placement disulfide, which exits cystinotic lysosomes via a functional of cystinosin in the lysosomal membrane was later demon- lysine carrier (18). This finding made meaningful therapy strated using green fluorescent protein (GFP)–tagged normal possible, and cysteamine was subsequently shown to retard and mutant cystinosin fusion proteins (37). Other experiments renal deterioration and enhance growth in cystinosis children showed that normal cystinosin localized in lysosomes and (19). Patients treated early (i.e., before 1 yr of age) and dili- colocalized with a lysosomal marker protein, LAMP-2 (37). gently with oral cysteamine are expected to preserve their Recent studies have demonstrated that cystinosin acts as a kidney function for several decades and might even avoid proton-driven lysosomal cystine transporter, which confirms requiring a renal allograft altogether (20). In fact, oral cysteam- previous findings (38). Kalatzis et al. (39) deleted the lysoso- ine therapy has become the treatment of choice for cystinosis mal targeting motif of cystinosin at its carboxy terminus, (6) and was approved by the United States Food and Drug causing misrouting to the plasma membrane. Incorporation of Administration on August 15, 1994, for the treatment of pre- the mutant but still functional cystinosin protein into the plasma membrane allowed cystine transport studies to be per- formed at the whole-cell level. These studies showed pH Correspondence to Dr. William A. Gahl, 10 Center Drive, MSC 1830, Building 10, Room 9S-241, NICHD, NIH, Bethesda, MD 20892-1830. Phone: 301-496- dependence, substrate specificity, and kinetic data that con- 9101; Fax: 301-402-0234; E-mail: [email protected] firmed previous results obtained using crude lysosome-rich 1046-6673/1308-2189 granular fractions (12-14). Other investigators incubated fibro- Journal of the American Society of Nephrology blasts from cystinosis patients carrying a nonsense mutation Copyright © 2002 by the American Society of Nephrology with gentamicin, which can cause read-through of nonsense DOI: 10.1097/01.ASN.0000027098.90648.F2 mutations; this led to correction of the mutation and depletion 2190 Journal of the American Society of Nephrology J Am Soc Nephrol 13: 2189–2191, 2002 of intracellular cystine (40). In addition, GFP fused to the 3. Beumer H, Wepler W: U¨ ber die Cystinkrankheit der ersten N-terminal and C-terminal ends of cystinosin verified the ly- lebenszeit. Klin Wochenschr 16: 8–10, 1937 sosomal location of this protein (40), again confirming previ- 4. Fanconi G, Bickel H: Die chronische aminoacidurie (aminosa¨u- ous work with newer and more sophisticated techniques. rediabetes oder nephrotisch-glukosurischer zwergwuchs) bei der In this issue of the JASN, Haq et al. (41) report that they glykogenose und der cystinkrankheit. Helv Paed Acta 4: 359– 396, 1949 have developed a powerful antibody directed against the car- 5. Bickel H: Die Entwicklung der biochemischen la¨sion bei der boxy terminal portion of cystinosin. Although endogenous lignac-fanconischen krankheit. Helv Paed Acta 10: 259–268, levels of cystinosin in normal and cystinotic cells in culture 1955 were not examined, transfected COS-7 cells expressing wild- 6. Gahl WA, Thoene JG, Schneider JA: Cystinosis: A disorder of type cystinosin showed an antibody reaction whereas cells lysosomal membrane transport. In: The Metabolic and Molecular expressing mutant cystinosin did not. The antibody also cor- Bases of Inherited Disease, 8th ed., edited by Scriver CR, Beau- rectly identified the lysosomal location of cystinosin and its det AL, Valle D, Sly WS, New York, McGraw-Hill, 2001, presence in the renal tubular cells of normal kidneys. Indeed, 5085–5108 the signal was missing from the kidneys of cystinosis patients 7. Gahl WA: Cystinosis coming of age. Adv Pediatr 33: 95–126, homozygous for the common 57-kb deletion. 1986 This antibody represents an important advance for future 8. Schneider JA, Bradley K, Seegmiller JE: Increased cystine in leukocytes from individuals homozygous and heterozygous for investigations into cystinosis because it can help answer a cystinosis. Science 157: 1321–1322, 1967 litany of questions. If missense mutations produce cystinosin 9. Schulman JD, Bradley KH, Seegmiller JE. Cystine: Compart- proteins that remain antigenic, the antibody can be used to mentalization within lysosomes in cystinotic leukocytes. Science follow the intracellular location of the abnormal proteins. The 166: 1152–1154, 1969 mutations characterizing ocular (33) and intermediate (30) 10. Patrick AD, Lake BD: Cystinosis: Electron microscopic evidence cystinosis will be of special interest. The antibody can be used of lysosomal storage of cystine in lymph node. J Clin Pathol 21: to ascertain the expression level of the cystinosin protein in 571–575, 1968 normal human tissues, as well as in ocular cystinosis tissues 11. De Duve C, Pressman BC, Gianetto R, Wattiaux R, Appelmans such as the kidney, if they become available. It may also F: Tissue fractionation studies. 6. Intracellular distribution pat- crossreact with murine cystinosin and could prove useful in terns of enzymes in rat-liver tissue. Biochem J 60: 604–617, mouse studies, including those involving a knockout mouse. 1955 Pulse-chase experiments using metabolic labeling and poly- 12. Gahl WA, Bashan N, Tietze F, Bernardini I, Schulman JD: Cystine transport is defective in isolated leukocyte lysosomes acrylamide gel electrophoresis-autoradiography will determine from patients with cystinosis. Science 217: 1263–1265, 1982 the extent and time course of glycosylation and processing of 13. Gahl WA, Tietze F, Bashan N, Bernardini I, Raiford D, Schul- cystinosin. Finally, the antibody may help answer the question man JD: Characteristics of cystine counter-transport in normal of why I-cells store cystine (42,43). I-cells, or mucolipidosis-II and cystinotic lysosome-rich leukocyte granular fractions. Bio- cells, have deficient mannose-6-phosphate targeting of en- chem J 216: 393–400, 1983 zymes to lysosomes (44). One hypothesis is that a deficient 14. Gahl WA, Tietze F, Bashan N, Steinherz R, Schulman JD: contingent of lysosomal enzymes could cause cystinosin to be Defective cystine exodus from isolated lysosome-rich fractions improperly processed and therefore dysfunctional. of cystinotic leucocytes. J Biol Chem 257: 9570–9575, 1982 Cystinosis has taught us volumes about lysosomal mem- 15. Jonas AJ, Smith ML, Schneider JA: ATP-dependent lysosomal brane transport and its genes and will reveal more about cystine efflux is defective in cystinosis. J Biol Chem 257: 13185– trafficking and processing of integral lysosomal membrane 13188, 1982 16. Gahl WA, Bashan N, Tietze F, Schulman JD. Lysosomal cystine proteins. It may even inform us of the various subsets of counter-transport in the detection of heterozygotes for cystinosis. lysosomes that must exist and of how they form. Cystinosin Am J Hum Genet 36: 277–282, 1984 antibodies will assist in these academic endeavors; however, 17. Thoene JG, Oshima RG, Crawhall JC, Olson DL, Schneider JA: for the children and adults who cope daily with a devastating Cystinosis. Intracellular cystine depletion by aminothiols in vitro systemic disease, the main goal is to endure and prevail. Unlike and in vivo. J Clin Invest 58: 180–189, 1976 those in previous generations who succumbed to dehydration 18. Pisoni RL, Thoene JG, Christensen HN: Detection and charac- or uremia, today’s cystinosis patients have cysteamine as a terization of carrier mediated cationic amino acid transport in disease-altering therapy. This treatment should be universally lysosomes of normal and cystinotic human fibroblasts. Role in embraced for pretransplant patients and strongly considered for therapeutic cystine removal? J Biol Chem 260: 4791–4798, 1985 posttransplant use. In addition, the medical profession must 19. Gahl WA, Reed GF, Thoene JG, Schulman JD, Rizzo WB, Jonas work diligently to promote early treatment and earlier diagno- AJ, Denman DW, Schlesselman JJ, Corden BJ, Schneider JA: sis, complete with attempts at newborn screening. Cysteamine therapy for children with nephropathic cystinosis. N Engl J Med 316: 971–977, 1987 20. Markello TC, Bernardini IM, Gahl WA: Improved renal function References in children with cystinosis treated with cysteamine. New Engl 1. Abderhalden E: Familia¨re cystindiathese. Hoppe Seylers Zeitschr J Med 328: 1157–1162, 1993 f physiol Chemie 38: 557–561, 1903 21. Kaiser-Kupfer MI, Caruso RC, Minckler DS, Gahl WA: Long- 2. Lignac GOE: U¨ ber sto¨rung des cystinstoffwechsels bei kindern. term ocular manifestations in nephropathic cystinosis-post-renal Deut Arch Klin Med 145: 139–150, 1924 transplantation. Arch Ophthalmol 104: 706–711, 1986 J Am Soc Nephrol 13: 2189–2191, 2002 Cystonosis: Antibodies and Healthy Bodies 2191

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See related article, “’Immunolocalization of Cystinosin, the Protein Defective in Cystinosis,” on pages 2046–2051.