PLoS ONE 12(10): e0186392.   https://doi.org/10.1371/journal.pone.0186392 

Involvement of the Arg566 residue of Aeromonas sobria serine protease in substrate specificity

 

Hidetomo Kobayashi1, Tadamune Otsubo2, Fumiteru Teraoka2, Kiyoshi Ikeda2, Soshi Seike1, Eizo Takahashi3, Keinosuke Okamoto3, Toru Yoshida4, Hideaki Tsuge4, and Hiroyasu Yamanaka1*

1Laboratory of Molecular Microbiological Science, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima, Japan

2Laboratory of Synthetic Organic Chemistry, Faculty of Pharmaceutical Sciences, Hiroshima International University, Hiroshima, Japan

3Collaborative Research Center of Okayama University for Infectious Diseases in India, National Institute of Cholera Enteric Diseases JICA Building ID Hospital Campus, Kolkata, India

4Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kyoto, Japan

* Corresponding author

E-mail: h-yamana@ps.hirokoku-u.ac.jp

 

Abstract

Aeromonas sobria serine protease (ASP) is an extracellular serine protease secreted by the organism. Here, we identified the amino acid residue of ASP that contributes to substrate specificity by using both synthetic peptides and biological protein components. The results showed that the arginine residue at position 566 (Arg-566) of ASP, which is located in the extra occluding region of ASP close to an entrance of the catalytic cavity, is involved in the substrate specificity. A substitutional point mutation of the Arg-566 residue of ASP to Ala residue (ASP[R566A]) caused a decrease of the proteolytic efficiency for a certain substrate. In addition, ASP lost the ability to recognize the primary substrate by such a point mutation, and ASP[R566A] reacted to a wide range of synthetic substrates. It is likely that Arg-566 causes an interaction with the amino acid residue at position P3 of the substrate, which is the third amino acid residue upstream from the cleavage site. Another study using ORF2 protein, a chaperone protein of ASP, further suggested that Arg-566 could also play an important role in interaction with ORF2. We therefore conclude that the Arg-566 residue of ASP is likely responsible for the selection of substrates.

 

Supplement

Aeromonas species are Gram-negative facultative anaerobic organisms that are ubiquitous in various aquatic environments such as fresh and brackish water areas. Although more than ten Aeromonas species have been reported, only a few species including A. sobria and A. hydrophila are involved in human infectious diseases. The main symptom caused by infection with either of A. sobria and A. hydrophila is gastroenteritis, but an infected patient’s condition sometimes progresses to a more serious state, and severe acute extra-intestinal diseases such as sepsis, phlegmon and myonecrosis can be triggered by the infection, with a higher probability of such triggering in immunocompromised patients.

 

Virulent strains of Aeromonas produce a variety of extracellular toxins. These toxins might increase the virulence of Aeromonas. We have been studying the extracellular serine protease (ASP) produced by A. sobria and have reported several properties of ASP including its crystal structure [1-4]. ASP destroys not only structural and functional proteins but also the proteins essential for host defense [5,6]. It is therefore likely that ASP is involved in the onset of extra-intestinal diseases such as sepsis, phlegmon and myonecrosis. Thus, ASP contributes to the establishment of A. sobria infection as one of the major pathogenic factors.

 

To elucidate the substrate specificity of ASP is beneficial for understanding the action of ASP as a pathogenic factor and also developing a novel protease inhibitor for infection control. We had already made an initial report on the substrate specificity of ASP by using several synthetic peptides [2]. In this report, we revealed that ASP preferentially hydrolyzes the synthetic substrate whose amino acid residues at positions P1 and P2 (i.e., the first and second amino acid residues upstream from the cleavage site, respectively) are Lys residues. In addition, the crystal structure of ASP revealed that the extra occluding region, which is a structure unique to ASP among the kexin family serine proteases, may be involved in substrate selectivity through interaction with the substrate (Figure 1A). In particular, since the Arg-566 residue localized in the extra occluding region seems to be adjacent to the P3 residue of substrate (Figure 1B), we assumed that the Arg-566 residue of ASP may contribute to substrate selectivity.

 

 

Figure 1. Close-up view of the catalytic site of ASP with a bound substrate model (Lys-Lys-Glu-Arg). (A) The structure of ASP and a bound substrate model (Lys-Lys-Glu-Arg) are depicted by a ribbon model and a stick model, respectively. The positions of the four amino acid residues of a substrate (P1; Lys, P2; Lys, P3; Glu, and P4; Arg) and those of several amino acid residues of ASP[S336A] are shown. Among them, D78, H115, and A336 (S336 in wild-type ASP) constitute the catalytic triad. The Arg-566 residue located in the extra occluding region of ASP is also displayed in large letters. (B) Schematic representation of the subsites of ASP with a bound substrate model. The Arg-566 residue seems to be adjacent to the P3 residue of substrate. (Data adapted from Kobayashi, H. et al. (2009). J. Biol. Chem. 284 (40): 27655-27663)

 

 

To verify this possibility, we first conducted experiments using synthetic substrates in the present study reported in PLoS ONE 12(10): e0186392 [7]. We synthesized several substrates composed of three amino acid residues whose amino acid residue at the P3 position varied but amino acid residues at both P1 and P2 positions were Lys residues. We then examined the ability of ASP to cleave these substrates, in order to determine how the reactivity of ASP to the substrate is changed by the difference in the P3 residue. We also created mutant ASP in which the Arg-566 residue was substituted with an alanine residue, and we then examined the ability of the mutant ASP to cleave these substrates.

 

As shown in Figure 2A, the results have obviously revealed that ASP most efficiently hydrolyzed the substrate of which the P3 residue was Glu. Such substrate specificity was lost by replacing the Arg-566 residue of ASP with Ala (ASP[R566A]) (Figure 2B). Thus the Arg-566 residue of ASP is thought to play an important role in the selection of a substrate by recognizing the structure of the side chain at P3. Analysis of kinetic parameters obtained in this study also suggested the involvement of the Arg-566 residue of ASP in specific substrate recognition.

 

 

Figure 2. Cleavage of various fluorogenic peptide substrates by wild-type ASP and the mutant ASP (ASP[R566A]). Kcat/Km values were calculated from the reaction of wild-type ASP (A) and ASP[R566A] (B) with the various substrates.

(Data adapted from Kobayashi, H. et al. (2017). PLoS ONE 12(10): e0186392. https://doi.org/10.1371/journal.pone.0186392)

 

 

Furthermore, to determine whether the Arg-566 residue is related to the proteolytic action of ASP against biological protein components, we examined the proteolytic action of both ASP and the mutant ASP against fibrinogen and high-molecular-weight kininogen which are already known as the biological protein substrate for ASP [5]. As shown in Figure 3, ASP hydrolyzes both fibrinogen and high-molecular-weight kininogen more efficiently than ASP[R566A]. These results mean that the Arg-566 residue of ASP also plays an important role in the proteolytic action against the biological protein components.

 

 

Figure 3. The cleavage of various proteins by wild-type ASP and the mutant ASP (ASP[R566A]). (A) Cleavage of fibrinogen by wild-type ASP and by ASP[R566A]. Human fibrinogen (200 nM) was incubated with wild-type ASP or ASP[R566A] (0.4 nM) for various periods of time (min). The locations of fibrinogen Aa, Bb, and g chains are indicated by the arrows. (B) Cleavage of high-molecular-weight kininogen by wild-type ASP and by ASP[R566A]. High-molecular-weight kininogen (200 nM) was incubated with wild-type ASP or ASP[R566A] (0.4 nM) for various periods of time (min). The location of high-molecular-weight kininogen (HK) is indicated by the arrow.

(Data adapted from Kobayashi, H. et al. (2017). PLoS ONE 12(10): e0186392. https://doi.org/10.1371/journal.pone.0186392)

 

 

In the present study, we observed the ability of ASP to recognize substrates selectively was lost by the mutation of ASP from Arg-566 to Ala-566. These results indicate that the Arg-566 residue of ASP contributes to the association of the primary substrate with ASP. Since the Arg-566 residue is located in the extra occluding region of ASP, we propose that the extra occluding region of ASP may play an important role in the process of selecting and binding appropriate substrates.

 

Extracellular proteases are often closely related to the development of bacterial infections. From this point view, it is considered that investigating substrate specificity of ASP in detail is an important issue in controlling the bacterial infection. Further studies are in progress in our laboratory.

 

 

References

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