HISTIDINE-RICH GLYCOPROTEIN BINDS TO HUMAN-IGG AND C1Q AND INHIBITS THE FORMATION OF INSOLUBLE IMMUNE-COMPLEXES

Purification of the complement component C1q from human serum using an established method resulted in the copurification of two 30 kDa prote ins with an N-terminal sequence identical to human histidine-rich glyc oprotein(HRG). Therefore, to explore the possibility that HRG can inte ract with C1q, we examined the ability of 81 kDa (native) and the 30 k Da proteins (presumably proteolytic N-terminal fragments of HRG) to bi nd to C1q, using both ELISA and optical biosensor techniques. Both for ms of HRG were found to bind to the human complement component C1q and also to purified human and rabbit IgG by ELISA. Kinetic analyses of t he HRG-C1q and HRG-IgG interactions using the IAsys biosensor indicate two distinct binding sites with affinities K-d1 0.78 x 10(-8) M and K -d2 3.73 x 10(-8) M for C1q, and one binding site with affinity K-d 8. 5 x 10(-8) M for IgG. Moreover, the fact that both native and 30 kDa H RG bind to C1q and to IgG suggests that the IgG and C1q binding region s on HRG are located in the 30 kDa N-terminal region of the HRG molecu le. The Fab region of IgG is likely to be involved in the HRG-IgG inte raction since HRG also bound to F(ab')(2) fragments with an affinity s imilar to that seen with the complete IgG molecule. Interestingly, the binding between HRG and IgG was significantly potentiated (K-d reduce d from 85.0 to 18.9 nM) by the presence of physiological concentration s of Zn2+ (20 mu M). Conversely, the presence of Zn2+ weakened the bin ding of HRG to C1q (Kd increased from 7.80 to 29.3 nM). Modulation of these interactions by other divalent metal cations was less effective with relative potencies being Zn2+ > Ni2+ > Cu2+. A, examination of th e effect of native and 30 kDa HRG on the formation of insoluble immune complexes (IIC) between ovalbumin and polyclonal rabbit anti-ovalbumi n IgG revealed that physiological concentrations of HRG can markedly i nhibit IIC formation in vitro. The results show that human HRG binds t o C1q and to IgG in a Zn2+-modulated fashion, and that HRG can regulat e the formation of IIC in vitro, thus indicating a new functional role for HRG in vivo.