Copper transportion of WD protein in hepatocytes from Wilson disease patients in vitro

Abstract

Aim: To study the effect of copper transporting P-type ATPase in copper metabolism of hepatocyte and pathogenesis of Wilson disease (WD).

Methods: WD copper transporting properties in some organelles of the cultured hepatocytes were studied from WD patients and normal controls.These cultured hepatocytes were incubated in the media of copper 15 mg x L(-1) only, copper 15 mg x L(-1) with vincristine (agonist of P-type ATPase) 0.5mg x L(-1), or copper 15 mg x L(-1) with vanadate (antagonist of P-type ATPase) 18.39 mg x L(-1) separately. Microsome (endoplasmic reticulum and Golgi apparatus), lysosome, mitochondria, and cytosol were isolated by differential centrifugation. Copper contents in these organelles were measured with atomic absorption spectrophotometer, and the influence in copper transportion of these organelles by vanadate and vincristine were comparatively analyzed between WD patients and controls. WD copper transporting P-type ATPase was detected by SDS-PAGE in conjunction with Western blot in liver samples of WD patients and controls.

Results: The specific WD proteins (M(r)155,000 lanes) were expressed in human hepatocytes, including the control and WD patients. After incubation with medium containing copper for 2 h or 24 h, the microsome copper concentration in WD patients was obviously lower than that of controls, and the addition of vanadate or vincristine would change the copper transporting of microsomes obviously. When incubated with vincristine, levels of copper in microsome were significantly increased, while incubated with vanadate, the copper concentrations in microsome were obviously decreased. The results indicated that there were WD proteins, the copper transportion P-type ATPase in the microsome of hepatocytes. WD patients possessed abnormal copper transporting function of WD protein in the microsome, and the agonist might correct the defect of copper transportion by promoting the activity of copper transportion P-type ATPase.

Conclusion: Copper transportion P-type ATPase plays an important role in hepatocytic copper metabolism. Dysfunction of hepatocytic WD protein copper transportion might be one of the most important factors for WD.

Figure 1

Western blotting analysis of WD protein separated from cultured human hepatocytes. M: Protein molecular mass markers; WD3: The hepatocytes of one WD patient; VE: Blood vessel endotheliocyte; S: Normal human hepatocytes.

Figure 2

Cu²⁺ levels in hepatic cytosol at different culturing conditions.

Figure 3

Cu²⁺ levels in hepatic microsome under different culturing conditions

Figure 4

Cu²⁺ levels in hepatic lysosome under different culturing conditions.

Figure 5

Cu²⁺ levels in hepatic mitochondria under different culturing conditions.