6Zinc-alpha2-glycoprotein (ZAG) is a major plasma protein whose levels increase in 7 chronic energy-demanding diseases and thus serves as an important clinical biomarker 8 in the diagnosis and prognosis of the development of cachexia. Current knowledge 9 suggests that ZAG mediates progressive weight loss through β-adrenergic signaling in 10 adipocytes, resulting in the activation of lipolysis and fat mobilization. Here, through 11 crosslinking experiments, amine oxidase copper-containing 3 (AOC3) is identified as a 12 novel ZAG binding partner. AOC3 -also known as vascular adhesion protein 1 (VAP-1) 13 and semicarbazide sensitive amine oxidase (SSAO) -deaminates primary amines, 14 thereby generating the corresponding aldehyde, H2O2 and HN3. It is an ectoenzyme 15 largely expressed by adipocytes and induced in endothelial cells during inflammation. H2O2 has an insulinogenic effect. The observations described here suggest that ZAG acts 18 as an allosteric inhibitor of AOC3 and interferes with the associated pro-inflammatory 19 and anti-lipolytic functions. Thus, inhibition of the deamination of lipolytic hormone 20 octopamine by AOC3 represents a novel mechanism by which ZAG might stimulate 21 lipolysis. Furthermore, experiments involving overexpression of recombinant ZAG 22 reveal that its glycosylation is co-regulated by oxygen availability and that the pattern of 23 glycosylation affects its inhibitory potential. The newly identified protein interaction 24 2 between AOC3 and ZAG highlights a previously unknown functional relationship, which 25 may be relevant to inflammation, energy metabolism and the development of cachexia. 26 27 93 provide much-needed insight into the mechanism of ZAG function and stimulate future work 94 in basic and clinical research on ZAG.95 96 5 2 Results 97 2.1 ZAG binds to ectoenzyme AOC3 98To attempt to identify ZAG interaction partners, purified recombinant ZAG and freshly 99 prepared adipocyte plasma membranes were co-incubated and any physical interactions 100 between them were stabilized by a photoactivatable crosslinker molecule (Fig. 11). Both 101 human and murine ZAG (without leader sequence) were produced in E. coli after cloning in 102 the expression plasmid pGEX-6P-2 and affinity purified by GST (glutathione-S-transferase)-103 tag. Both purified human and mouse proteins (GST-hZAG and GST-mZAG, respectively) and 104 GST-tag aloneserving as a controlwere labeled with the photoactivatable crosslinker 105