Apolactoferrin inhibits the catalytic domain of matrix metalloproteinase-2 by zinc chelation.
Lactoferrin (LTF) is a multifunctional iron-binding protein that is also capable of binding other divalent metal cations, especially Zn2+. 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 ZnCl2 and FeCl2 were added to the reaction. While addition of Fe2+ did not reverse inhibition, the addition of Zn2+ 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.
Newsome
Anthony L
AL
Department of Biology, Middle Tennesee State University, 1301 E. Main Street, Murfreesboro, TN 37132, USA.
Johnson
Jon Paul
JP
Seipelt
Rebecca L
RL
Thompson
Michael W
MW
D001059:Apoproteins / Q000494:pharmacology*; D020134:Catalytic Domain; D002614:Chelating Agents / Q000737:chemistry / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007781:Lactoferrin / Q000494:pharmacology*; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D013696:Temperature; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism
D001059:Apoproteins; D002614:Chelating Agents; D061965:Matrix Metalloproteinase Inhibitors; C039223:apolactoferrin; C492629:hololactoferrin, human; D007781:Lactoferrin; D020778:Matrix Metalloproteinase 2; D015032:Zinc