Activity of ulilysin, an archaeal PAPP-A-related gelatinase and IGFBP protease

Abstract

Human growth and development are conditioned by insulin-like growth factors (IGFs), which have also implications in pathology. Most IGF molecules are sequestered by IGF-binding proteins (IGFBPs) so that exertion of IGF activity requires disturbance of these complexes. This is achieved by proteolysis mediated by IGFBP proteases, among which the best characterised is human PAPP-A, the first member of the pappalysin family of metzincins. We have previously identified and studied the only archaeal homologue found to date, Methanosarcina acetivorans ulilysin. This is a proteolytically functional enzyme encompassing a pappalysin catalytic domain and a pro-domain involved in maintenance of latency of the zymogen, proulilysin. Once activated, the protein hydrolyses IGFBP-2 to -6 and insulin chain b in vitro. We report here that ulilysin is also active against several other substrates, viz (azo)casein, azoalbumin, and extracellular matrix components. Ulilysin has gelatinolytic but not collagenolytic activity. Moreover, the proteolysis-resistant skeletal proteins actin and elastin are also cleaved, as is fibrinogen, but not plasmin and a1-antitrypsin from the blood coagulation cascade. Ulilysin develops optimal activity at pH 7.5 and strictly requires peptide bonds preceding an arginine residue, as determined by means of a novel fluorescence resonance energy transfer assay, thus pointing to biotechnological applications as an enzyme complementary to trypsin.These two authors contributed equally to this work. a

Catalytic Domain Architecture of Metzincin Metalloproteases

“…It is coordinated by the N ⑀2 atoms of the three consensus histidines (two histidine N ⑀2 atoms and one aspartate O ␦2 atom in snapalysin) and the catalytic solvent molecule, substituted by other ligands in the enzyme/inhibitorproduct complexes reported (supplemental Table 1). Some metzincins display an additional protein ligand at a slightly greater distance from the catalytic cation in the form of a tyrosine O atom, as seen in unbound astacin and serralysins and as hypothesized in ulilysin (21). This tyrosine residue lies two positions ahead of the Met-turn methionine.…”

“…The CSD shows two disulfide bonds and a unique two-calcium site. This site is a molecular switch for activity, as the proteinase can be reversibly inhibited through calcium chelators (15,20,21). Finally, in the absence of an unbound structure, ulilysin may possess a fifth zinc-binding tyrosine ligand provided by the Met-turn that is swung out upon substrate binding.…”

On the Relevance of the Met-turn Methionine in Metzincins

“…Both 38-kDa proulilysin C269A and its selenomethionine variant undergo calcium-mediated autoactivation at two sites, one upstream and one downstream of the catalytic moiety, to render mature 29-kDa ulilysin quantitatively after 24 h at room temperature (16,17,27 Fig. 1 in 27), respectively, and agreed well with the specificity of the enzyme for basic residues in subsite P 1Ј .…”

“…Site-directed Mutagenesis-Plasmids based on pET28a, which were modified to encode for both M. acetivorans proulilysin wild-type and variant C269A (16,17), were used as a template to mutate Met 290 to alanine, leucine, valine, phenylalanine, glycine, serine, cysteine, aspartate, lysine, and histidine (M290X mutants). The vector employed attached a His 6 tag to the protein N terminus, followed by a thrombin protease cleavage site, and conferred resistance to kanamycin.…”

“…Protease Assays-Purified M290X and C269A ulilysin mutants and the wild-type enzyme were tested (at 1 mg/ml in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5 mM CaCl 2 ) for proteolytic activity against the specific quenched fluorescent peptide substrate, DABCYL-Leu-Ala-Arg-Val-Glu-EDANS (Bachem) at 37°C for 2 h (16,17). The increase in fluorescence intensity ( ex ϭ 355 nm; em ϭ 460 nm) upon substrate cleavage was monitored on a fluorescence microplate reader (FLx800; BIOTEK), and kinetic parameters (K m and k cat ) were derived by using different substrate concentrations (10, 25, 50, 100, 150, and 175 M).…”