To further our knowledge of intracellular copper transport, we used a proteomics strategy to search for hepatic proteins with copper-binding ability. Hep G2 cytosolic and microsomal fractions were applied to a copper(II)-loaded immobilized metal-affinity chromatography (IMAC) column. Protein identification was performed with 2-D gel electrophoresis and mass spectrometry. We identified 48 cytosolic proteins and 19 microsomal proteins displaying copper-binding ability. These proteins are diverse in function. Fifty-two of the 67 proteins contain putative metal-binding domains. We have identified many components of the Hep G2 copper metalloproteome including a large number of proteins not previously known to bind copper.

The metalloproteome is defined as the set of proteins that have metal-binding capacity by being metalloproteins or having metal-binding sites. A different metalloproteome may exist for each metal. Mass spectrometric characterization of metalloproteomes provides valuable information relating to cellular disposition of metals physiologically and in metal-associated diseases. We examined the Cu and Zn metalloproteomes in three human hepatoma lines: Hep G2 and Mz-Hep-1, which retain many functional characteristics of normal human hepatocytes, and SK-Hep-1, which is poorly differentiated. Additionally we studied a single specimen of normal human liver and Hep G2 cells depleted in vitro of cellular copper. We used matrix-assisted laser desorption ionization and electrospray ionization quadrupole time-of-flight mass spectrometry to analyze peptide sequences of tryptic digests obtained by either in-gel digestion of metal-binding proteins or peptides on an immobilized metal affinity chromatography column loaded with either Cu or Zn. Mainly high abundance proteins were identified. Cu-binding proteins identified included enolase, albumin, transferrin, and alcohol dehydrogenase as well as certain intracellular chaperone proteins. The Cu metalloproteome was not identical to the Zn metalloproteome. Peptide binding experiments demonstrated that Cu coordination prefers the order of residues histidine > methionine > cysteine. Although the Cu metalloproteome was similar from line to line, subtle differences were apparent. Gel profiling showed more extensive variation in expression of annexin II in SK-Hep-1 and Mz-Hep-1 than in Hep G2 and normal liver tissue. Glycerylphosphorylethanolamine was identified as a post-translational modification at residue Glu-301 of elongation factor 1-alpha in Hep G2. Intracellular copper depletion was associated with loss of the glycerylphosphoryl side group. These findings suggest that post-translational modification could be affected by intracellular actions of copper. Comparison of the Cu and Zn metalloproteomes in Hep G2 with a published general proteome of Hep G2 disclosed little overlap (Seow, T. K., et al. (2001) Proteomics 1, 1249-1263). Proteins in the metalloproteomes of human hepatocytes can be identified by these methods. Variations in these metalloproteomes may have important physiological relevance.

Upregulating hepatocyte function in proliferating human liver cell lines could provide cells for a bio-artificial liver. Ideally, a means of mimicking the biological extracellular matrix with a relatively inert, bio-compatible matrix is required. Alginate encapsulation of primary hepatocytes is biocompatible. This study aimed to characterize cells grown in a 3D configuration in alginate. A human-derived liver cell line encapsulated in 1% alginate was assessed for synthetic and detoxification functions. Secreted proteins measured (e.g., albumin, fibrinogen, alpha-1-antitrypsin etc.) were increased in alginate compared with monolayers. Cytochrome P450 1A1 activity increased three- to fourfold, whilst urea synthesis, undetectable in monolayer cultures, was synthesized by cells in alginate at levels approaching in vivo production. TEM revealed good ultrastructure reminiscent of normal hepatocytes. Alginate promotes 3D colonies of proliferating cells with upregulated liver functions. Rapid recovery of function of cryopreserved cells (< 18 h) provides added advantages for this system to support the biological component of an artificial liver for patients with fulminant hepatic failure.