Biochemistry of invertebrate peroxidasins

Background.

Members of the peroxidase-cyclooxygenase superfamily (Zamocky et al., 2008) catalyse biochemical reactions essential to a broad spectrum of biological processes, including host defense, thyroid hormone biosynthesis, and modification of the extracellular matrix, as well as contributing to the pathogenesis of chronic inflammatory diseases. Subfamily 2 of this superfamily is comprised of multidomain oxidoreductases called peroxidasins and can be subdivided in five distinct clades. In general peroxidasins (Pxd) are glycosylated secreted heme peroxidases having in addition leucine-rich repeat domains (LRRs), C-like immunoglobulin (Ig) domains as well as a von Willebrand factor C (VWC) module (Soudi et al., 2012). These are typical motifs of extracellular proteins that mediate protein-protein interactions. Peroxidasin was initially discovered as a basement membrane constituent and later on was shown to be involved in innate immune reactions, extracellular matrix (ECM) formation, tissue development, tumor progression and oxidative reactions that lead to induction of cell apoptosis. Very recently it was demonstrated that human peroxidasin forms sulfilimine bonds in collagen IV by hypohalous acids thus catalysing a reaction that is important in tissue development and human diseases (Bhave et al., 2012).
Despite the upcoming biological importance of this protein family the biochemical knowledge is very poor. Neither high-resolution structural nor detailed mechanistic and kinetic data are available. The few groups (including mine) working on these novel multi-domain enzymes focus on the two human enzymes hPxd01 and hPxd02 (Clade 5). By contrast, in this PhD project, we aim at the investigation of the biochemistry and biophysics of thePxd from Drosophila melanogaster (Clade 3) and two peroxidasins of Clade 2 (CelPxd02 and CelPxd03) from Caenorhabditis elegans.

Sequence alignment suggests the presence of five LRRs, four Ig-domains, one peroxidase domain and one C-terminal VWC in DmPxd. This was actually the first peroxidasin that was described and shown to be associated with the function of insect hemocytes and plasmatocytes (Nelson et al., 1994). Nevertheless, a comprehensive biochemical characterization is still lacking. Caenorhabditis elegans apparently has six peroxidasins. Two of these are homologous and multidomain enzymes (CelPxd01 and CelPxd02) but lack the von Willebrand factor and have a different number of immunoglobulin domains compared to the homologous human Pxds. In addition, C. elegans seems to have four “short peroxidasins”, CelPxd03-CelPxd06, that only consist of the peroxidase domain. There is one report on the putative in vivo role of peroxidasin in C. elegans (Gotenstein et al., 2010). CelPxd02 was identified upon screening for mutants defective in embryonic worm development. It was found to be essential for specific stages of morphogenesis and muscle-epidermal attachment as well as post-embryonically for basement membrane integrity. The peroxidase activity was shown to be responsible for these developmental roles. In adult worms loss of CelPxd02 promotes regrowth of axons after injury, providing evidence that C. elegans’ extracellular matrix can play an inhibitory role in axon regeneration. By contrast, loss of CelPxd01 did not cause developmental effects.

Aims and methods.

Recombinant production of DmPxd, CelPxd02 and CelPxd03 as well as truncated forms of DmPxd and CelPxd02 that lack distinct domains will be performed at first in insect cell lines (cooperation with GRABHERR); production of hsPxd01 in mammalian cell cultures is already established in our lab in cooperation with MACH and BORTH. Biochemical/physical analyses of recombinant proteins will include (i) UV-vis-, fluorescence-, electronic circular dichroism-, multi-angle light scattering-, resonance Raman- (SMULEVICH) and electronic paramagnetic resonance spectroscopy (VAN DOORSLAER), (ii) multi-mixing stopped-flow spectroscopy and polarography, (iii) mass-spectrometry (ALTMANN, WILSON) and X-ray crystallography (DJINOVIC-CARUGO), (iv) site-directed mutagenesis, (v) spectroelectrochemistry (BATTISTUZZI), (vi) differential scanning calorimetry and (vii) molecular modelling and simulation (OOSTENBRINK).

Those methods will help to elucidate the structure and enzymatic activity of these metalloproteins including (i) oligomeric state and architecture of substrate access channel(s) and heme cavity, (ii) domain interaction and (un)folding pathway(s), (iii) chemistry of the prosthetic group including oxidation and spin-state(s), ligation and posttranslational modification(s), (iv) substrate pattern and specificity, accessibility and binding site of substrate molecules and ligands as well as the nature of reaction products, (v) chemistry, reactivity and relevance of enzymatic redox intermediates (ferrous and ferric forms, Compounds I, II and III) and of their redox thermodynamics and (vi) the role of (conserved) active site amino acids in substrate/ligand binding and conversion.

It is the clear goal of this project to understand the molecular basis of substrate oxidation and conversion of peroxidasins at all relevant redox steps and to present the first high-resolution structures of these multi-domain oxidoreductases. Moreover, functional and structural comparison of these proteins will give new insights into the evolution of this protein family as well as in its proposed roles in innate immunity and/or extracellular matrix formation.

Bhave, G., Cummings, C.F., Vanacore, C., Kumagai-Cresse, Ero-Tolliver, I.A., Rafi, M., Kang, J.-S., Pedchenko, V., Fessler, L.I., Fessler, J.H., Hudson, B.G. (2012) Peroxidasin forms sulfilimine chemical bonds using hypohylous acids in tissue genesis. Nature Chem. Biol. 8, 784-790
Gotenstein, J.R., Swale, R.E., Fukuda, T., Wu, Z., Giurumescu, C. A., Goncharov, A., Jin, Y., Chisholm, A.D. (2010) The C. elegans peroxidasin PXN-2 is essential for embryonic morphogenesis and inhibits adult axon regeneration. Development 137, 3603-3636
Nelson, R.E., Fessler, L.I., Takagi, Y., Blumberg, B., Keene, B.D.R., Olson, P.F., Parker, C., Fessler, J.H. (1994) Peroxidasin: a novel enzyme-matrix protein of Drosophila development. EMBO J. 13, 3438-3447
Soudi, M., Zamocky, M., Jakopitsch, C., Furtmueller, P.G., Obinger, C. (2012) Molecular evolution, structure and function of peroxidasins. Chem. Biodivers. 9, 1776-1793
Zamocky, M., Jakopitsch, C., Furtmueller, P.G., Dunand, C., Obinger, C. (2008) The peroxidase-cyclooxygenase superfamily. Reconstructed evolution of critical enzymes of the innate immune defense system. Proteins 71, 589-605