Zn-alpha(2)-glycoprotein (ZAG) is a member of the major histocompatibility complex (MHC) class I family of
proteins and is identical in amino acid sequence to a
tumor-derived
lipid-mobilizing factor associated with
cachexia in
cancer patients. ZAG is present in plasma and other body fluids, and its natural function, like
leptin's, probably lies in
lipid store homeostasis. X-ray crystallography has revealed an open groove between the helices of ZAG's alpha(1) and alpha(2) domains, containing an unidentified small
ligand in a position similar to that of
peptides in MHC
proteins (Sanchez, L. M., Chirino, A. J., and Bjorkman, P. J. (1999) Science 283, 1914-1919). Here we show, using serum-derived and bacterial
recombinant protein, that ZAG binds the fluorophore-tagged
fatty acid 11-(dansylamino)undecanoic acid (DAUDA) and, by competition, natural
fatty acids such as arachidonic, linolenic, eicosapentaenoic, and
docosahexaenoic acids. Other MHC class I-related
proteins (FcRn, HFE, HLA-Cw*0702) showed no such evidence of binding. Fluorescence and isothermal calorimetry analysis showed that ZAG binds DAUDA with K(d) in the micromolar range, and differential scanning calorimetry showed that
ligand binding increases the thermal stability of the
protein. Addition of
fatty acids to ZAG alters its intrinsic (
tryptophan) fluorescence emission spectrum, providing a strong indication that
ligand binds in the expected position close to a cluster of exposed
tryptophan side chains in the groove. This study therefore shows that ZAG binds small hydrophobic
ligands, that the natural
ligand may be a
polyunsaturated fatty acid, and provides a fluorescence-based method for investigating ZAG-
ligand interactions.