Postdoctoral Fellow Vanderbilt University Medical Center
Deepika Nambiar (Vanderbilt University Medical Center)| Mark Athanason (Vanderbilt University Medical Center)| Mohamed Rafi (Vanderbilt University Medical Center)| Roberto Vanacore (Vanderbilt University Medical Center)
Lysyl oxidase like 2 (LOXL2) has emerged as an attractive target for the development of therapies for different forms of tissue fibrosis including liver, lung, kidney and cancer-associated fibrosis. LOXL2 is a copper dependent enzyme that catalyzes the oxidative deamination of lysine residues to form lysyl aldehydes (allysine) in collagens. Allysines spontaneously condense into lysyl-derived crosslinks which are important for the deposition of collagens in the extracellular matrix (ECM). In our previous studies, we have established that LOXL2 promotes the formation of lysine-derived crosslinks in the 7S dodecamer, a triple-helical crosslinking domain formed by the overlap of four collagen IV molecules. Although the specific location lysine residues targeted by lysyl oxidase have been described for fibrillar collagens, the equivalent information for 7S dodecamer of collagen IV is unknown.
To better understand the mechanism by which LOXL2 mediates the crosslinking of collagen IV, we developed a fluorometric assay to detect amine oxidase activity using 7S dodecamer as substrate. We characterized the ability of LOXL2 to deaminate lysine residues in two forms of 7S substrate: the wild-type crosslinked 7S substrate and an uncrosslinked form purified from ECM derived from cells lacking LOXL2. Surprisingly, the enzyme showed little to no activity with crosslinked 7S dodecamer whereas a pronounced enzyme activity was observed with the uncrosslinked form of the 7S substrate. As trypsin digestion indicated that not all lysine residues present in the wild type 7S dodecamer take part in crosslinking, we hypothesized that the LOXL2 targets specific lysine residues.
To identify specific lysine residues, uncrosslinked 7S dodecamers were oxidized with LOXL2 and newly formed allysines were labeled with a biotin tag. Optimization of biotinylation reaction was monitored by Streptavidin-HRP followed by enhanced chemiluminescence (ECL). After trypsin digestion of 7S dodecamers, biotinylated peptides containing allysine residues were identified by mass spectrometry. We discuss the potential rol of the identified allysines in the alignment of the four triple-helical molecules forming the 7S dodecamer. A better understanding of the mechanism by which LOXL2 promotes collagen IV crosslinking will provide the foundations for the development of effective therapies against tissue fibrosis.