Apolactoferrin inhibits the catalytic domain of matrix metalloproteinase-2 by zinc chelation

<jats:p> Lactoferrin (LTF) is a multifunctional iron-binding protein that is also capable of binding other divalent metal cations, especially Zn<jats:sup>2+</jats:sup>. Recent investigations indicate that lactoferrin levels are elevated in many disease conditions in which matrix metalloproteinases (MMPs), particularly MMP-2, are also elevated, suggesting that the 2 proteins may interact. This possibility was examined by determining the effect of LTF in its holo (metal-bound) and apo (metal-free) forms on the proteolytic activity of MMP-2 and other similar zinc metalloproteases. Pre-incubation with apolactoferrin, but not hololactoferrin, greatly reduced the hydrolysis of a peptide substrate by MMP-2, but not by MMP-1, -8, -9, or -13. This inhibition was specific for the 42 kDa catalytic domain fragment of MMP-2 lacking the hemopexin domain, since the 66 kDa form was poorly inhibited by apolactoferrin. The inhibition of the MMP-2 catalytic domain was strongly temperature sensitive, indicating that the conformation of one or both proteins is crucial to this interaction. To ascertain the mechanism of inhibition, increasing concentrations of ZnCl<jats:sub>2</jats:sub> and FeCl<jats:sub>2</jats:sub> were added to the reaction. While addition of Fe<jats:sup>2+</jats:sup> did not reverse inhibition, the addition of Zn<jats:sup>2+</jats:sup> resulted in a recovery of MMP-2 activity, and furthermore, zinc-saturated LTF did not inhibit MMP-2. Together, these data strongly suggest that apolactoferrin is capable of removing the catalytic zinc from the active site of MMP-2, although an exosite-based interaction between the 2 proteins cannot be fully ruled out. This inhibitory activity suggests a novel function for LTF and may represent a novel regulatory mechanism that regulates proteolysis by MMP-2 in vivo. </jats:p>