Continuous assays for meprin alpha and beta using prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas gingivalis

doi:10.1016/j.ab.2018.08.005

Analytical Biochemistry

Volume 559, 15 October 2018, Pages 11-16

https://doi.org/10.1016/j.ab.2018.08.005 Get rights and content

Highlights

•
Development of coupled assays to assess the endoprotease activity of meprin.
•
Establishment of a rapid protocol for the isolation of the auxiliary enzyme PtP.
•
Presentation of conditions using chromogenic or fluorogenic substrates.
•
Determination of kinetic parameters using human meprin α and meprin β.
•
Assessment of the inhibition type and potency of three inhibitors of meprin β.

Abstract

Common assays for endoprotease activity of meprin α and β are based on cleavage of internally quenched substrates. Although direct and convenient, for meprins these assays bear disadvantages such as, e.g., significant substrate inhibition or potential fluorescence quenching by compounds applied in inhibitor analysis. Here, we present a novel continuous assay by introducing an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP) and the chromogenic substrate KKGYVADAP-p-nitroanilide. We provide a quick strategy for expression and one-step-purification of the auxiliary enzyme. The enzyme kinetic data for meprin α and β suggest hyperbolic v/S-characteristics, the kinetic parameters of substrate conversion by meprin β were K_m = 184 ± 32 μM and k_cat = 20 ± 4 s⁻¹. We also present conditions for the use of the fluorogenic substrate KKGYVADAP-AMC to assess meprin β activity. The assays were applied for determination of inhibitory parameters of the natural inhibitor actinonin and two recently published hydroxamates. Hence, we present two novel methods, which can be applied to assess inhibitory mechanism and potency with the attractive current drug targets meprin α and β. Furthermore, the assay might also provide implications for analysis of other endoproteases as well as their inhibitors.

Introduction

The astacin proteases meprin α and meprin β are zinc-dependent metalloproteases of the metzincin superfamily. These enzymes gained considerable interest due to their causal involvement in different disorders such as neurodegeneration, fibrosis, inflammatory processes and impaired intestinal immune response [[1], [2], [3], [4], [5]]. Therefore, the enzymes are interesting current drug targets. Meprin α and meprin β preferentially cleave substrates with acidic amino acids in P1′-position [6]. The proteolytic degradation of the fluorogenic substrates OCK+ Abz-MGWMDEIDK(Dnp)SG-OH and S2 Abz-YVAEAPK(Dnp)G-OH has been used to characterize specific and potent inhibitors for meprin β [[7], [8], [9]]. Although the assays are convenient and direct, a detailed inhibitor characterization is hampered by the solubility of the substrate and concomitant substrate and product inhibition [8]. These kinetic characteristics generate a need for alternative assays. On our quest to develop inhibitors of astacins, we aimed at the development of a novel assay. Here, we describe a continuous coupled assay using the serine-protease prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas gingivalis [10]. The enzyme releases a tripeptide, X-X-P, from substrates exposing of a free N-terminus and a proline residue in the third position [11,12]. The cleavage of a meprin substrate leads to generation of the PtP substrate. Subsequently, the activity of PtP results in release of a chromophore or fluorophore (Scheme 1).

This study also provides a rapid protocol for the isolation of the auxiliary enzyme and conditions for assay use in characterization of inhibitors.

Section snippets

Materials

p-Nitroaniline and molecular mass standards for SDS–PAGE were provided by Sigma (Deisenhofen, Germany). His-Trap, Butyl Sepharose and HiPrep columns were obtained from GE Healthcare Life Science (Piscataway, USA).

Synthesis of KKGYVADAP-pNA

The peptide was synthesized according to a modified procedure as described by Ref. [13]. 2-Chlorotriltyl resin (410 mg, 0.5 mmol, 1 eq) was added to a solution of diisopropylethylamine (1.71 ml, 10 mmol, 20 eq) and p-Phenylendiamine hydrochloride (578 mg, 4 mmol, 8 eq) in DMF (10 ml).

Results and discussion

The astacin proteases meprin α and β are zinc-dependent metalloendoproteases with preferential specificity for acidic amino acids in P1’ position. Due to their cleavage specificity, internally quenched substrates such as OCK+ and S2 (Abz-MGWMDEIDK(Dnp)SG-OH and Abz-YVAEAPK(Dnp)G-OH, respectively) have been used in the past for determination of activity and for inhibitory characterization. Although these assays are sensitive, the substrates show conspicuous inhibition at higher concentration,

Acknowledgements

We thank Christina Schnittka, Mercedes Scharfe and Katrin Schulz for excellent technical assistance.

References (26)

J. Bien et al.
The metalloprotease meprin β generates amino terminal-truncated amyloid β peptide species
J. Biol. Chem.
(2012)
J. Prox et al.
Meprin alpha and meprin beta: procollagen proteinases in health and disease
Matrix Biol.: J. Int. Soc. Matrix Biol.
(2015)
G.P. Bertenshaw et al.
Marked differences between metalloproteases meprin A and B in substrate and peptide bond specificity
J. Biol. Chem.
(2001)
D. Schlenzig et al.
Expression, purification and initial characterization of human meprin β from Pichia pastoris
Protein Expr. Purif.
(2015)
D. Ramsbeck et al.
First insight into structure-activity relationships of selective meprin β inhibitors
Bioorg. Med. Chem. Lett
(2017)
A. Banbula et al.
Prolyl tripeptidyl peptidase from Porphyromonas gingivalis. A novel enzyme with possible pathological implications for the development of periodontitis
J. Biol. Chem.
(1999)
K. Ito et al.
Crystal structure and mechanism of tripeptidyl activity of prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis
J. Mol. Biol.
(2006)
Y. Xu et al.
Novel inhibitor for prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis and details of substrate-recognition mechanism
J. Mol. Biol.
(2008)
M.M. Bradford
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding
Anal. Biochem.
(1976)
S.C. Gill et al.
Calculation of protein extinction coefficients from amino acid sequence data
Anal. Biochem.
(1989)

N. Thorne et al.

Apparent activity in high-throughput screening: origins of compound-dependent assay interference

Curr. Opin. Chem. Biol.

(2010)

C. Becker-Pauly et al.

The metalloprotease meprin beta is an alternative beta-secretase of APP

Front. Mol. Neurosci.

(2016)

C. Broder et al.

The metalloproteases meprin α and meprin β: unique enzymes in inflammation, neurodegeneration, cancer and fibrosis

Biochem. J.

(2013)

Cited by (7)

Substrates incorporating unnatural amino acids are separately detectable for chymotrypsin and subtilisin with similar specificity
2023, Results in Chemistry
Protease specificity depends on the three-dimensional structure and sequence of the protease, and each protease plays a different role. Chymotrypsin and subtilisin have similar specificity for recognizing aromatic and hydrophobic amino acid residues. Previous methods using natural amino acids have difficulty in separately detecting proteases with similar specificity. To overcome this, we developed an effective substrate incorporating unnatural amino acids using a simple method that changes only one residue of a substrate. Various 7-amino-4-methylcoumarin (AMC) peptide substrates were synthesized in terms of hydrophobicity, aromaticity, and conformation, including unnatural amino acids 2-amino-4-ethylhexanoic acid (AEH) and cyclohexylalanine (Cha). Although AEH substrate is highly hydrophobic, steric hindrance based on the branched side chain of AEH residue reduced the recognition of the substrate by the two proteases. Cha substrate with a six-membered ring side chain was most preferably recognized by chymotrypsin but was slightly recognized by subtilisin. This shows that Cha substrate can separately detect the two proteases with similar specificity. Therefore, these results indicate that protease with similar specificities is separately detectable by substrates with a side chain having structural degrees of freedom that provide different steric hindrance.
Microbial proteases and their applications
2023, Frontiers in Microbiology
A glutaminyl cyclase-catalyzed α-synuclein modification identified in human synucleinopathies
2021, Acta Neuropathologica
Structure and dynamics of meprin β in complex with a hydroxamate-based inhibitor
2021, International Journal of Molecular Sciences
The effect of a peptide substrate containing an unnatural branched amino acid on chymotrypsin activity
2021, Processes
A Primary Evaluation of Potential Small-Molecule Inhibitors of the Astacin Metalloproteinase Ovastacin, a Novel Drug Target in Female Infertility Treatment
2020, ChemMedChem

View all citing articles on Scopus

View full text

Continuous assays for meprin alpha and beta using prolyl tripeptidyl aminopeptidase (PtP) from Porphyromonas gingivalis

Highlights

Abstract

Introduction

Section snippets

Materials

Synthesis of KKGYVADAP-pNA

Results and discussion

Acknowledgements

J. Biol. Chem.

Matrix Biol.: J. Int. Soc. Matrix Biol.

J. Biol. Chem.

Protein Expr. Purif.

Bioorg. Med. Chem. Lett

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

Anal. Biochem.

Anal. Biochem.

Curr. Opin. Chem. Biol.

The metalloprotease meprin beta is an alternative beta-secretase of APP

Front. Mol. Neurosci.

The metalloproteases meprin α and meprin β: unique enzymes in inflammation, neurodegeneration, cancer and fibrosis

Biochem. J.