Gastroenterology

Gastroenterology

Volume 117, Issue 6, December 1999, Pages 1380-1385
Gastroenterology

Liver, Pancreas, and Biliary Tract
Localization of the Wilson's disease protein in human liver,☆☆,

https://doi.org/10.1016/S0016-5085(99)70288-XGet rights and content

Abstract

Background & Aims: Wilson's disease is an autosomal-recessive disorder of copper metabolism that results from the absence or dysfunction of a copper-transporting P-type adenosine triphosphatase that leads to impaired biliary copper excretion and disturbed holoceruloplasmin synthesis. To gain further insight into the role of the Wilson's disease protein in hepatic copper handling, its localization in human liver was investigated. Methods: By use of a specific antibody, localization of the Wilson's disease protein was studied in liver membrane fractions and liver sections by immunoblotting, immunohistochemistry, and double-label confocal scanning laser microscopy. Results: The 165-kilodalton protein, found by immunoblotting, was most abundant mainly in isolated plasma membrane fractions enriched in canalicular domains. Immunohistochemistry revealed intracellular punctuate staining of hepatocytes in certain regions of the liver, whereas a canalicular membrane staining pattern was observed in other regions. Double-labeling studies showed that in the latter regions the transporter is present mainly in vesicular structures just underneath the canalicular membrane that are positive for markers of the trans-Golgi network. A weak staining of the canalicular membrane, identified by staining for P-glycoprotein, was observed. Conclusions: These results show that in human liver the Wilson's disease protein is predominantly present in trans-Golgi vesicles in the pericanalicular area, whereas relatively small amounts of the protein appear to localize to the canalicular membrane, consistent with a dual function of the protein in holoceruloplasmin synthesis and biliary copper excretion.

GASTROENTEROLOGY 1999;117:1380-1385

Section snippets

Materials

The C219 antibody to P-glycoproteins was purchased from Dako (Hamburg, Germany); the Golgi markers 58K and AP-1 were obtained from Sigma Chemical Co. (St. Louis, MO); rabbit polyclonal antibodies against the human WND protein8, 13 and the human sodium taurocholate–cotransporting polypeptide (NTCP) were kindly provided by Jonathan D. Gitlin (Washington University School of Medicine, St. Louis, MO) and Peter Meier (University Hospital Zürich, Switzerland), respectively. Secondary antibodies

Detection of WND protein in canalicular enriched membrane fractions

On Western blotting (Figure 1), the WND protein was detectable as a 165-kilodalton protein in both blLPM and cLPM fractions, with a clearly greater abundance in the canalicular fraction (Figure 1, lanes 1 and 2).

. Immunoblot analysis of the WND protein in normal human liver. Immunoblot of enriched cLPM and blLPM incubated sequentially with WND antibody (WD, lanes 1 and 2), P-glycoprotein antibody (pgp, lanes 3 and 4), and NTCP (lanes 5 and 6).

To confirm that these fractions were enriched in

Discussion

The role of the WND protein in hepatic copper handling and its exact localization within the liver remains unclear. The present study shows endogenous hepatocellular expression of the WND protein in the human liver. Immunoblotting experiments, using material from multiple normal human livers, indicate that the WND protein is abundantly present in a canalicular membrane-enriched fraction and much less in blLPM fractions. In liver membrane fractions of a patient with WND and an unknown mutation,

Acknowledgements

The authors thank Dr. Jonathan D. Gitlin for providing the antibody against the human Wilson's Disease protein and Dr. Peter Meier for providing the antibody against human Na+/taurocholate- cotransporting polypeptide.

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    Address requests for reprints to: Roel J. Vonk, M.D., Center for Liver, Digestive and Metabolic Diseases, Department of Pediatrics, University Hospital Groningen; P.O. Box 30001, 9700 RB Groningen, The Netherlands. e-mail: [email protected]; fax: (31) 50-3611746.

    ☆☆

    Supported by German Research Council grant DFG STR 216/6-1 (to W.S.), The Netherlands Digestive Diseases Foundation grant WS96-70 (to R.J.V.), and Biomed and Health Research Program grant PL 931436 (to F.K. and W.S.). Mark Schaefer is the recipient of young scientist education grant SCHA 781/1-1 from the German Research Council/DFG.

    Drs. Schaefer and Roelofsen contributed equally to this work.

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