Mammalian augmenter of liver regeneration protein is a sulfhydryl oxidase.

Abstract

Augmenter of Liver Regeneration is an important secondary hepatic growth factor. Augmenter of liver regeneration protein has been shown to control mitochondrial gene expression and the lytic activity of liver-resident Natural Killer cells through the levels of interferon-gamma, but the precise enzymatic function of this protein is unknown. To define the enzymatic activity of augmenter of liver regeneration protein. The carboxy terminus of augmenter of liver regeneration protein contains a special CXXC motif characteristic for redox proteins and with faint homologies to the redox-active site of sulfhydryl oxidases. Tests were, therefore, carried out to establish whether isolated augmenter of liver regeneration protein can also function in the formation of sulfur bridges. Purified augmenter of liver regeneration proteins from rat and human were tested in enzyme assays for the ability to introduce disulfide bonds into protein substrates. The isolated proteins were tested for the formation of dimers and the presence of bound FAD was investigated spectroscopically. The function of the conserved CXXC motif was investigated by in vitro mutagenesis experiments and subsequent enzyme assays. In this study, we demonstrate that rat and human augmenter of liver regeneration protein are flavin-linked sulfhydryl oxidases that catalyze the formation of disulfide bonds in reduced protein substrates. A flavin moiety is firmly but not covalently attached to the protein. In human cell cultures augmenter of liver regeneration protein is expressed in a long and short form that both exist as covalently linked dimers. The active site of the enzyme is associated with a conserved CXXC motif in the carboxy-terminal domain, that is present in the homologous proteins from yeast to humans and also in the human Q6 growth regulator protein. In vitro mutagenesis of one cysteine residue in the CXXC motif results in loss of enzymatic function and the mutated protein no longer binds FAD. For the first time, these data assign an enzymatic activity to the important hepatic growth factor augmenter of liver regeneration protein. The finding that augmenter of liver regeneration protein acts as a FAD-linked sulfhydryl oxidase is essential to identify the molecular targets inside liver cells and to elucidate the precise role of mammalian augmenter of liver regeneration protein in hepatic cell growth, liver disease and regeneration.