Temperature and salts effects on the peptidase activities of the recombinant metallooligopeptidases neurolysin and thimet oligopeptidase

Abstract

We report the recombinant neurolysin and thimet oligopeptidase (TOP) hydrolytic activities towards internally quenched fluorescent peptides derived from the peptide Abz-GGFLRRXQ-EDDnp (Abz, ortho-aminobenzoicacid; EDDnp, N-(2,4-dinitrophenyl) ethylenediamine), in which X was substituted by 11 different natural amino acids. Neurolysin hydrolyzed these peptides at R-R or at R-X bonds, and TOP hydrolyzed at R-R or L-R bonds, showing a preference to cleave at three or four amino acids from the C-terminal end. The kinetic parameters of hydrolysis and the variations of the cleavage sites were evaluated under different conditions of temperature and salt concentration. The relative amount of cleavage varied with the nature of the substitution at the X position as well as with temperature and NaCl concentration. TOP was activated by all assayed salts in the range 0.05-0.2 M for NaCl, KCl, NH 4 Cl and NaI, and 0.025-0.1 M for Na 2 SO 4 . Concentration higher than 0.2 N NH 4 Cl and NaI reduced TOP activity, while 0.5 N or higher concentration of NaCl, KCl and Na 2 SO 4 increased TOP activity. Neurolysin was strongly activated by NaCl, KCl and Na 2 SO 4 , while NH 4 Cl and NaI have very modest effect. High positive values of enthalpy (DH*) and entropy (DS*) of activation were found together with an unusual temperature dependence upon the hydrolysis of the substrates. The effects of low temperature and high NaCl concentration on the hydrolytic activities of neurolysin and TOP do not seem to be a consequence of large secondary structure variation of the proteins, as indicated by the far-UV CD spectra. However, the modulation of the activities of the two oligopeptidases could be related to variations of conformation, in limited regions of the peptidases, enough to modify their activities.Keywords: protease; peptide; metalloprotease; fluorescence; enthalpy of activation in proteolysis; entropy of activation in proteolysis.Thimet oligopeptidase (TOP, EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are zinc-dependent peptidases, members of the metallopeptidase M3 family and contain in their primary sequence the HEXXH motif [1,2]. Rat neurolysin has been the first member of the M3 family of which the 3D structure has been determined [3]. As it has been shown that this enzyme and thermolysin have common ancestors, the M3 family was thus included in the clan MA [4]. It is interesting to note that in the structure of neurolysin, the catalytic center is located in a deep channel [3], which limits the access only to short peptides [5,6]. This selectivity toward hydrolysis of oligopeptides was also verified for TOP [5][6][7][8][9]. The high primary sequence identity found for these related peptidases, which is about 65% [2], allows hypothesizing that they may share a similar folding including the deep channel that constitutes the neurolysin active site. This feature seems to prevent the unspecific cleavage of other proteins and it is of particular relevance for TOP and neurolysin as they are not expressed as inactive precursors and they are...

Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes

“…TOP efficiently liberated the epitope's C-terminus in a variety of systematically substituted precursors of the ELFSYLIEK epitope and other epitopes, consistent with its known flexible capacity to remove three to five C-terminal residues from the substrate [25][26][27]31 . Purified TOP, A CTL epitope from M. Tuberculosis Hsp65 with undefined aa sequence has previously been shown to rely on TOP 36 .…”

“…Thus, TOP released three or four C-terminal residues, in accordance with its cleavage preference 25,26 , thereby efficiently producing the exact nonameric epitope (Fig. 3b).…”

“…TOP preferentially releases, with broad sequence specificity 27,31 , three to five C-terminal residues 25,26 from substrates extending up to 17 aa 27 , endowing TOP with the potential to generate the Cterminus of epitopes. We assessed the flexibility of TOP in producing different epitope sequences by systematically substituting the positions (P1 and P1') surrounding the scissile bond after the Cterminus of ELFSYLIEK (ELFSYLIEP1-P1'KRK).…”

“…123) anti-PRA [190][191][192][193][194][195][196][197][198] (ELFSYLIEK) and CTL clone (no. 313) anti-MART-1 [26][27][28][29][30][31][32][33][34][35] (EAAGIGILTV) were generated by in vitro stimulation against synthetic peptides exogenously loaded on antigen presenting cells, according to procedures described previously 20 , using HLA class I-typed PBMCs obtained with prior informed consent from an anonymous blood bank donor or a melanoma patient, respectively. The CTL (clone RT.c38) anti-EBNA-3C (aa 258-266) (RRIYDLIEL) was generated previously 30 .…”

Role of the Novel Metallopeptidase MoP112 and Saccharolysin for the Complete Degradation of Proteins Residing in Different Subcompartments of Mitochondria

“…Several oligopeptidases have been identified but their function has been exclusively studied in vitro. These include mammalian neurolysin (18,19) and yeast saccharolysin (20,21), both metallopeptidases localized in the mitochondrial intermembrane space, and a novel metallopeptidase, zinc-MP, which is present in mitochondria and chloroplasts and was shown to degrade targeting peptides of nuclear-encoded preproteins in vitro (22). Moreover, recent characterization of the mitochondrial proteome of various organisms revealed the presence of additional oligopeptidases in mitochondria that include the bleomycin hydrolase Lap3 (23), however, their functional characterization remains to be elucidated.…”