Van'T Hoff, WG;
Immunolocalization of cystinosin, the protein defective in cystinosis.
J AM SOC NEPHROL
2046 - 2051.
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 carboxyterminal oligopeptide sequence from cystinosin. Antisera were screened by Western blotting and immunocytochemical analyses of transfected COS-7 cells expressing either human wildtype 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 co-localized with GFP fluorescence. Previous studies demonstrated that GFP fluorescence from this construct co-localized 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.
|Title:||Immunolocalization of cystinosin, the protein defective in cystinosis|
|Keywords:||FRACTIONS, TRANSPORT, FIBROBLASTS, LEUKOCYTES, LYSOSOMES|
|UCL classification:||UCL > School of Life and Medical Sciences
UCL > School of Life and Medical Sciences > Faculty of Population Health Sciences > Institute of Child Health
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