Subcellular localization and N-glycosylation of human ABCC6, expressed in MDCKII cells

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Abstract

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.

Section snippets

Materials and methods

Materials. Tunicamycin, trypsin (type XIII), and soybean trypsin inhibitor (type 1-S) were from Sigma, N-Glycosydase F from Boehringer–Mannheim GmbH. The polyclonal antibody K14 was a kind gift from Stieger, see [21]. The chicken polyclonal anti-Na,K-ATPase antibody was obtained from Chemicon International (Temecula, CA), the Alexa Fluor 488 goat anti-rat IgG (H + L), and the Alexa Fluor 594 goat anti-chicken IgG (H + L) were obtained from Molecular Probes (Eugene, USA).

Expression of human ABCC6 in

Expression and localization of human ABCC6 in polarized cells

In order to provide expression of human ABCC6 in a polarized monolayer, a retroviral transduction system and kidney-derived MDCKII cells have been utilized. Cell lines with various expression levels were produced by growing individual cell-derived ABCC6-transduced cells. By using this method, we obtained stable MDCKII clones with high yield of ABCC6 expression.

For the detection of the subcellular localization of human ABCC6, the polarized cultures of transduced MDCKII cells were immunostained

Acknowledgements

The technical help by Györgyi Demeter, Judit Kis, and Ilona Zombori is gratefully acknowledged. Thanks are due to Dr. Spat of the Department of Physiology, Semmelweis University of Budapest for making the confocal microscope available for the present study. K14 polyclonal antibody was a kind gift of Dr. B. Stieger. This work has been supported by grants from OTKA (T31952, T35926, T38337, and F 034247) by FKFP 0130/2001 and by NKFP 1/047 of Hungary. A. Váradi and B. Sarkadi are awardees of the

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