Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells.
Mutations in the gene coding for a human ABC transporter protein, ABCC6 (MRP6), are responsible for the development of pseudoxanthoma elasticum. Here, we demonstrate that human ABCC6, when expressed by retroviral transduction in polarized mammalian (MDCKII) cells, is exclusively localized to the basolateral membrane. The human ABCC6 in MDCKII cells was found to be glycosylated, in contrast to the underglycosylated form of the protein, as expressed in Sf9 cells. In order to localize the major glycosylation site(s) in ABCC6, we applied limited proteolysis on the fully glycosylated and underglycosylated forms, followed by immunodetection with region-specific antibodies for ABCC6. Our results indicate that Asn15, which is located in the extracellular N-terminal region of human ABCC6, is the only N-glycosylation site in this protein. The polarized mammalian expression system characterized here provides a useful tool for further examination of routing, glycosylation, and function of the normal and pathological variants of human ABCC6.
Sinkó
Emese
E
Institute of Enzymology, Hungarian Academy of Sciences, Budapest, Hungary.
Iliás
Attila
A
Ujhelly
Olga
O
Homolya
László
L
Scheffer
George L
GL
Bergen
Arthur A B
AA
Sarkadi
Balázs
B
Váradi
András
A
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D016764:Cell Polarity; D004285:Dogs; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D011561:Pseudoxanthoma Elasticum / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D013347:Subcellular Fractions / Q000378:metabolism; D014161:Transduction, Genetic; D014357:Trypsin; D014415:Tunicamycin / Q000494:pharmacology
D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; D014357:Trypsin; D014415:Tunicamycin; C102029:ABCC6 protein, human