Sandbox Reserved 1091

Generalities

The Aeromonas Sobria Serine Protease ASP protein is a serine protease that will cut peptide bonds after specific amino acids of a target protein. It preferentially cleaves peptide bonds that follow dibasic amino-acid residues. The kexin-like serine protease belongs to the subtilisin family (Subtilase). The structure of ASP is similar to that of Kex2 (1r64), a protease of the subtilisin family from Saccharomyces cerevisiae. ^[1]

This belonging to the subtilisin serine proteases family is hypothetical. Furthermore the predicted amino acid sequence reinforces this speculation. However, the size of the ASP (MW 65 kDa) is unlike other subtilisin proteases (MW 30 kDa). Also, the amino acid residues composition is different from the family’s characteristics because ASP shows unique cysteine residues that other family members don't show. Therefore we can state that it is likely that ASP belongs to the subtilisin serine proteases family, however it remains unclear.

Moreover, ASP is the only prokaryotic memebr of the kexin-subtilisin family that is composed almost exclusively of eukaryotic proteases like Furin.^[2] ASP was shown not to be a metalloprotease because its activity is not affected by metal chelators (EDTA, EGTA, o-phenanthroline) or metalloprotease inhibitors (phosphoramidon). ^[3]

This protein is secreted by the Anaerobic bacterium Aeromonas Sobria, which can cause potentially lethal septic shock. It is a clinical syndrome of potentially fatal organ dysfunction caused by a disorder in the response to infection. In septic shock, there is a critical reduction in tissue perfusion; acute multi visceral failure, including the lungs, kidneys and liver, can be observed. ^[4]

ASP is a sepsis-related factor. It can cause several dysfunctions like by inducing vascular leakage, reducing blood pressure via the activation of the kinin system or promoting human plasma coagulation through the activation of prothrombin. Finally, it can cause the formation of pus and edema through the action of anaphylatoxin C5a (4p3a). Gastroenteritis, and in extreme cases deuteropathy, are the main syndrome caused by infection with A.sobria.

Maturation

The precursor of ASP is composed of 624 amino acids. It contains a signal peptide of 24 amino acids, a catalytic domain, similar to that of subtilisin, and a P domain. The maturation of ASP is achieved by ORF2. This protein plays the role of an external chaperone and is necessary for the construction of the stable ASP. Indeed, ASP doesn’t contain any propeptide that is involved in the proper folding of the protein. This is a major difference with an other protein, close to ASP : Kex2 (1r64) ^[5]

The ORF2 protein is composed of 152 amino-acids coded by the orf2 gene of 456 base pairs. The N-terminal extension and the C-terminal tail of the protein are implicated in the maturation of ASP. In fact, a complex ASP-ORF2 is formed. This association requires a specific organization of ASP in the space. The of ASP doesn’t bind to ORF2 but the sixth residue from the C-terminus domain of ORF2 interacts with the non-mature ASP. In the complex, the active site of ASP is blocked. This protects the protein from degradation by others.

When the complex is formed, it moves to the extracellular space and then it dissociates. The active ASP can dissociate ORF2 and exercise its virulence activity in the cell. ^[6]

Secondary structure

The structure of ASP is very similar to that of Kex2 (1r64), but it has a unique extra-occluding region close to its active site within the subtilisin domains. This extra-occluding region is unique and it could serve as a useful target to make the development of new antisepsis drugs easier.

The domain structure of ASP consists of the propeptide, the catalytic subtilisin-like domain, and the P-domain. The ASP molecule have two mean regions: an N-terminal region extending from Gly-3 to Pro-431 and forming the , and a C-terminal region extending from Leu-432 to His-595 and forming the .

Moreover, we can find three in the ASP Structure (Ca1, Ca2 and Ca3). are situated in the N-terminal domain, and is situated in the C-terminal domain. It were assigned to ASP based on electron density, counter charges, and coordination. But in contrary to Kex2 (1r64), ASP contains no Ca2+ binding sites near its catalytic site.

A schematic representation of the domains of the protein can be observed : secondary structure of ASP

We can see that Kex2 has the propeptide (in yellow) that is absent in ASP. The occluding subdomains in the C-terminal region of ASP are shown in dark blue.

Domains

The Subtilisin Domain: It contains ten helices (alpha 1 to 10) and twelve chains (beta 1 to 10 and béta 13 to 14). The N-terminal domain of ASP seems to be like the catalytic domain of Kex2 (1r64), which is similar to those of subtilisin and other subtilisin-related proteases. This ASP catalytic site contains Asp78, His115, and Ser336 residues characteristic of subtilisins. In addition, four loops (L) protrude from the N-terminal subtilisin domain of ASP : Gly3– Pro26 (), Asn221–Phe241 (), Gly300–Cys326 (), and Gln-377–Glu-397 (). L1, L2, and L3 have random coil structure, whereas L4 forms a hairpin that protrudes toward the P-domain. Moreover, two are formed between Cys4 and Cys24 in L1 and between Cys301 and Cys326 in L3, which stabilize those loops.

The P-domain: The core of the P-domain in ASP contains eight beta-strands (beta 16 18 23 and 26). The is comprised of two parts, (Gly521–Thr525, beta 5, 6, and 12) and (Gly557–Asn578, béta 25), and it is situated close to Asp78,His115,and Ser336.

All these domains are represented schematically in the article ^[7] : representation 2D of ASP

On these figures, we can see the different domains of the protein in A and also a superposition with the Kex2. We clearly see the resemblance between both serine protease, and the extra occluding region in the C-terminal region of ASP.

Active site

The catalytic triad: The catalytic triad of ASP is composed of Asp78, His115 and Ser336. These amino acids are the base is the active site of the protein, where the mode of action of the serine protease takes place. A peptide can be inserted in the space of the active site. There, the amino acids of will interact together and the mechanism will lead to a cut in the polypeptide.

This triad can be observed in a 2D representation of the protein : catalytic triad of ASP

Mechanism: The mechanism of the reaction is the following : The histidine will react with the serine and deprotonate it. The deprotonated hydroxyl group of the serine will act as a nucleophilic species and attack the carbon from the carbonyl function on the peptide. This will lead to the formation of a tetrahedral intermediate. The regeneration of the carbonyl group will be followed by the release of one part of the peptide, with an amine group at its extremity. Then, a second tetrahedral intermediate will be formed, but with the attack of a deprotonated water molecule. In the end, the regeneration of the active site will be done with the release of the part of the peptide with a carboxyl extremity. The polypeptide is also cut in two parts and the target protein isn't functional anymore. ^[8]

A schematic representation of the mechanism with the involved amino acids can be found under the following link : mechanism of the reaction

The peptide bonds were shown to be cleaved when two basic residues were in sequence. A Lys residue at positions P1 and P2 relative to the cleavage site is prefered. If an Arg residue is at P4 position the substrate cleavage will be enhanced. ^[9]

Properties

ASP has its highest activity at pH 7,5 and loses it after heating at 60° for 10 minutes. ^[10]

Experiments have been done in order to establish the sensitivity of ASP to proteases. In has been found that the ASP protease activity was strongly attenuated by serine protease inhibitors (DFP, AEBSF). Moreover, a soybean trypsin inhibitor was shown not to block the proteolytic action of ASP itself but could inhibit the vascular permeability enhancing activity that follows after injection of ASP into epithelial cells. ^[11]

This experimental finding suggests that epithelial trypsin-like proteases mediate the reaction causing enhanced vascular permeability. It is likely that ASP stimulates the secretion and maturation of epithelial trypsin proteases, thus enhancing vascular permeability. ASP could stimulate the bradykinin-releasing pathway, thus stimulating mast cells to release histamine and further enhance the vascular permeability.

Antihistaminic agents (diphenhydramine and pyrilamine) were shown to efficiently inhibit vascular permeability enhancing the activity of the ASP. It is very likely that the vascular permeability enhancement is related to the release of histamine from mast cells. Through histopathological examinations it was shown that mast cells appeared around the injection site, confirming the role of histamine as a key factor.^[12]

Impact on human body

The predominant infection vector is through exposure to water harbouring Aeromonas sobria. ^[13] The most common form of disease is one where A.sobria pathogen adheres to the surface of the intestine causing painful diarrhea, also known as gastroenteritis. The enterotoxin activity of the hemolysin virulence factors of A.sobria contributes to those symptoms. However, the mortality due to intestinal disease type of infection is low compared to the non-intestinal diseases caused by the A.sobria infection. Pili facilitate the adherence of Aeromonas to human buccal cells. ^[14] Once invaded the intestine epithelial cells, Aeromonas can reach any organ via the blood. Multiple virulence factor than promote their pathogenicity.

The nonintestinal form of the disease reports symptoms such as septicemia, lesions of skin and soft tissues as well as meningitis, often ending fatally. That leads to a crucial reduction in tissue perfusion followed by fatal organ disfunction.

The ASP induced proteolysis digestion of proteins like kininogen, prothrombin, fibrinogen or prekallikrein at restricted sites generates fragments, expressing their own activity and therefore inducing specific physiological reactions. The kinin system activation, for example, reduces the blood pressure while the prothrombin system promotes plasma coagulation.

In the kallikrein/kinin system, the ASP induce the production of kinin from kininogen (low MW or high MW). The kinin also release caused edema at the infection site and shock in the circulation. The schematic representation of this action is represented : acting points of ASP in the kinin system

Experiments have been done in order to try to reduce the virulence activity of ASP. It has been demonstrated that the α2-macroglobulin, a plasma protein, can limit ASP activity. This protein can bind to ASP which is also inactivated. ^[15]