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Application of a Dual Internally Quenched Fluorogenic Substrate in Screening for D-Arginine Specific Proteases.

ABSTRACT: The application of D-stereospecific proteases (DSPs) in resolution of racemic amino acids and in the semisynthesis of proteins has been a successful strategy. The main limitation for a broader application is, however, the accessibility of suitable DSPs covering multiple substrate specificities. To identify DSPs with novel primary substrate preferences, a fast specificity screening method using the easily accessible internally quenched fluorogenic substrate aminobenzoyl-D-arginyl-D-alanyl-p-nitroanilide was developed. By monitoring both UV/vis-absorbance and fluorescence signals at the same time it allows to detect two distinct D-amino acid substrate specificities simultaneously and separately with respect to the individual specificities. In order to identify novel DSP specificities for synthesis applications, DSPs specific for D-arginine were of special interest due to their potential ability as catalysts for substrate mimetics-mediated peptide and protein ligations. D-alanine in the substrate served as positive control and reference based on its known acceptance by numerous DSPs. In silico analysis suggested that DSPs are predominantly present in gram-positive microorganisms, therefore this study focused on the bacilli strains Bacillus thuringiensis and Bacillus subtilis as potential hosts of D-Arg-specific DSPs. While protease activities toward D-alanine were found in both organisms, a novel and so far unknown D-arginine specific DSP was detected within the culture supernatant of B. thuringiensis. Enrichment of this activity via cation exchange and size exclusion chromatography allowed isolation and further characterization of this novel enzyme consisting of a molecular mass of 37.7 kDa and an enzymatic activity of 8.3 U mg^-1 for cleaving the D-Arg|D-Ala bond in the detecting substrate. Independent experiments also showed that the identified enzyme shows similarities to the class of penicillin binding proteins. In future applications this enzyme will be a promising starting point for the development of novel strategies for the semisynthesis of all-L-proteins.

SUBMITTER: Simon AH

PROVIDER: S-EPMC6456654 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Application of a Dual Internally Quenched Fluorogenic Substrate in Screening for D-Arginine Specific Proteases.

Simon Andreas H AH Liebscher Sandra S Aumüller Tobias H TH Treblow Dennis D Bordusa Frank F

Frontiers in microbiology 20190403

The application of D-stereospecific proteases (DSPs) in resolution of racemic amino acids and in the semisynthesis of proteins has been a successful strategy. The main limitation for a broader application is, however, the accessibility of suitable DSPs covering multiple substrate specificities. To identify DSPs with novel primary substrate preferences, a fast specificity screening method using the easily accessible internally quenched fluorogenic substrate aminobenzoyl-D-arginyl-D-alanyl-<i>p</i ...[more]

PMID: 31001242

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Project description:Internally quenched fluorogenic (IQF) peptides bearing the fluorescence donor/acceptor pair o-aminobenzoic acid (Abz)/N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) at N- and C-terminal ends were synthesized containing heparin-binding sites from the human serpins kallistatin and antithrombin, as well as consensus heparin-binding sequences (Cardin clusters). The dissociation constant (K(d)), as well as the stoichiometry for the heparin-peptide complexes, was determined directly by measuring the decrease in fluorescence of the peptide solution. Experimental procedures were as sensitive as those used to follow the fluorescence change of tryptophan in heparin-binding proteins. The conformation of the peptides and the heparin-peptide complexes were obtained from measurements of time-resolved fluorescence decay and CD spectra. Kallistatin (Arg(300)-Pro(319))-derived peptide (HC2) and one derived from antithrombin III helix D [(AT3D), corresponding to Ser(112)-Lys(139)], which are the heparin-binding sites in these serpins, showed significant affinity for 4500 Da heparin, for which K(d) values were 17 nM and 100 nM respectively. The CD spectra of the heparin-HC2 peptide complex did not show any significant alpha-helix content, different from the situation with peptide AT3D, for which complex-formation with heparin resulted in 24% alpha-helix content. The end-to-end distance distribution and the time-resolved fluorescence-decay measurements agree with the CD spectra and K(d) values. The synthetic alpha-methyl glycoside pentasaccharide AGA*IA(M) (where A represents N,6-O-sulphated alpha-d-glucosamine; G, beta-d-glucuronic acid; A*, N,3,6-O-sulphated alpha-d-glucosamine; I, 2-O-sulphated alpha-l-iduronic acid; and A(M), alpha-methyl glycoside of A) also binds to AT3D and other consensus heparin-binding sequences, although with lower affinity. The interaction of IQF peptides with 4500 Da heparin was displaced by protamine. In conclusion, IQF peptides containing Abz/EDDnp as the donor/acceptor fluorescence pair are very promising tools for structure-activity relationship studies on heparin-peptide complexes, as well as for the development of new peptides as heparin reversal-effect compounds.

Project description:The fourth member of Ca2+-dependent mammalian secretory subtilase, PC4 (proprotein convertase 4), is primarily expressed in testicular germ cell and ovarian macrophage. Its role in sperm fertilization and in early embryonic development has been demonstrated earlier through several studies, including those with PC4 null mice. A number of physiological substrates found in reproductive tissues have been postulated or identified for PC4 by various biochemical studies. These include growth factors IGF-1 (insulin-like growth factor-1) and IGF-2, hormonal polypeptide proPACAP (where PACAP stands for pituitary adenylate cyclase-activating polypeptide) and a number of surface proteins of ADAM (ADisintegrin And Metalloproteinase-like) family such as ADAM-1 (fertilin a), ADAM-2 (fertilin b), ADAM-3 (procyritestin) and ADAM-5. To provide further evidence in support of this notion and also to study the substrate specificity and bioassay of PC4, a series of intramolecularly quenched fluorogenic peptides containing the cleavage sites and several mutants were prepared. A comparative kinetic analysis and measurement of Vmax (app)/Km (app) ratio of these fluorogenic substrates against PC4 and PC7 revealed that the mutant variants of h (human) proPACAP and m (mouse) ADAM-5 derived peptides Q-PACAP141-151-mutant [Abz-141RVKNKGRRI150P151SY(NO2)-A-CONH2] (150A151Y replaced by PS) and Q-ADAM-5380-388-mutant [Abz-380E381PKPARRP388RY(NO2)A-CONH2] (381R replaced by P) are most efficiently and selectively cleaved by PC4. Using these two and Q-IGF-263-71 peptides, we showed that the sperm extract of normal adult mice is much higher when compared with that of PC4-null mice. This suggests that these fluorogenic peptides are useful for specific bioassay of PC4 activity. In addition, kinetic studies with various peptidyl-MCA indicate that the hexapeptide Ac-KTKQLR-MCA (where MCA stands for 4-methyl coumaryl-7-amide) is most efficiently and selectively cleaved by PC4 at RMCA, making it another effective agent for bioassay of PC4 activity. The study concludes that the most probable sequence motif for recognition by PC4 is KXKXXR or KXXR, where X is any amino acid other than cysteine and that it prefers proline at P3, P5 and/or P2' positions. It was also revealed that PC4 is a good candidate processing enzyme for growth factors IGF-1 and -2, neuropeptide proPACAP and several ADAM proteins such as ADAM-1, -2, -3 and -5.

Dataset Information

Application of a Dual Internally Quenched Fluorogenic Substrate in Screening for D-Arginine Specific Proteases.

Publications

Application of a Dual Internally Quenched Fluorogenic Substrate in Screening for D-Arginine Specific Proteases.

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