Altered dynamics of a lipid raft associated protein in a kidney model of Fabry disease.
Accumulation of globotriaosylceramide (Gb3) and other neutral glycosphingolipids with galactosyl residues is the hallmark of Fabry disease, a lysosomal storage disorder caused by deficiency of the enzyme alpha-galactosidase A (α-gal A). These lipids are incorporated into the plasma membrane and intracellular membranes, with a preference for lipid rafts. Disruption of raft mediated cell processes is implicated in the pathogenesis of several human diseases, but little is known about the effects of the accumulation of glycosphingolipids on raft dynamics in the context of Fabry disease. Using siRNA technology, we have generated a polarized renal epithelial cell model of Fabry disease in Madin-Darby canine kidney cells. These cells present increased levels of Gb3 and enlarged lysosomes, and progressively accumulate zebra bodies. The polarized delivery of both raft-associated and raft-independent proteins was unaffected by α-gal A knockdown, suggesting that accumulation of Gb3 does not disrupt biosynthetic trafficking pathways. To assess the effect of α-gal A silencing on lipid raft dynamics, we employed number and brightness (N&B) analysis to measure the oligomeric status and mobility of the model glycosylphosphatidylinositol (GPI)-anchored protein GFP-GPI. We observed a significant increase in the oligomeric size of antibody-induced clusters of GFP-GPI at the plasma membrane of α-gal A silenced cells compared with control cells. Our results suggest that the interaction of GFP-GPI with lipid rafts may be altered in the presence of accumulated Gb3. The implications of our results with respect to the pathogenesis of Fabry disease are discussed.
Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA 15261, USA; Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15261, USA; Ciência sem Fronteiras, CNPq, Brazil.
Anatália
A
Labilloy
Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Robert T
RT
Youker
Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA 15261, USA.
Jennifer R
JR
Bruns
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Ira
I
Kukic
Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Kirill
K
Kiselyov
Department of Neurobiology, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Willi
W
Halfter
Department of Human Genetics, University of Pittsburgh, Pittsburgh, PA 15261, USA.
David
D
Finegold
Immunogenetics and Molecular Biology Laboratory, Federal University of Piauí, Teresina, PI 64049-550, Brazil.
Semiramis Jamil Hadad
SJ
do Monte
Renal-Electrolyte Division, University of Pittsburgh, Pittsburgh, PA 15261, USA. Electronic address: weisz@pitt.edu.
Ora A
OA
Weisz
D000818:Animals; D004285:Dogs; D000795:Fabry Disease / Q000201:enzymology / Q000473:pathology; D015870:Gene Expression; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology / Q000473:pathology; D008247:Lysosomes / Q000201:enzymology / Q000473:pathology; D061985:Madin Darby Canine Kidney Cells; D021962:Membrane Microdomains / Q000378:metabolism* / Q000473:pathology; D008954:Models, Biological; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D014281:Trihexosylceramides / Q000096:biosynthesis; D000519:alpha-Galactosidase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D017261:Glycosylphosphatidylinositols; D034741:RNA, Small Interfering; D014281:Trihexosylceramides; D049452:Green Fluorescent Proteins; C018549:globotriaosylceramide; D000519:alpha-Galactosidase
DPPIV; EEA1; ERT; Enzyme Replacement Therapy; Fabry disease; GPI; GPI-anchored proteins; Gb3; HA; Kidney; LAMP-2; LSD; Lipid rafts; MDCK; Madin–Darby canine kidney; N&B; Number and brightness analysis; Polarized sorting; RT-PCR; dipeptidylpeptidase IV; early endosomal antigen 1; globotriaosylceramide; glycosylphosphatidylinositol; hemagglutinin; lysosomal membrane associated protein 2; lysosomal storage disorder; number and brightness; qRT-PCR; real-time quantitative reverse-transcription polymerase chain reaction; reverse transcription polymerase chain reaction; α-gal A; α-galactosidase A