A Novel Serine Protease Inhibitor Acts as an Immunomodulatory Switch while Maintaining Homeostasis

Jiang, Naxin; Thangamani, Saravanan; Chor, Cui Fang; Wang, S. Y.; Winarsih, Imelda; Du, Ruijuan; Sivaraman, J.; Ho, Bow; Ding, Jeak Ling

doi:10.1159/000209224

Cited by 15 publications

(21 citation statements)

References 90 publications

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“…It is also plausible that S. africanus plasma contained prophenoloxidase (PPO); PPO conversion to active enzyme can be brought about by miniscule amounts of LPS (Söderhäll and Cerenius, 1998). In the horseshoe crab (Carcinoscorpius rotundicauda), there are agents (i.e., serine proteases) that transiently exist in the plasma (Jiang et al, 2009) that are key players in PPO conversion to PO. Whether or not there is diPO or there is active PPO conversion in S. africanus plasma remains to be seen.…”

Section: Discussionmentioning

confidence: 99%

Endotoxin-induced coagulation reactions and phenoloxidase activity modulation inSudanonautes africanushemolymph fractions

Salawu

Oloyede

2011

Journal of Immunotoxicology

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Section: Discussionmentioning

confidence: 99%

Endotoxin-induced coagulation reactions and phenoloxidase activity modulation inSudanonautes africanushemolymph fractions

Salawu

Oloyede

2011

Journal of Immunotoxicology

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“…Our previous studies showed that full length as well as domain 2 of CrSPI-1 is a specific inhibitor of subtilisin, however the specificity of domain-1 is not yet established [10]. An analysis of P3 to P4′ residues of the RSLs of various substrates like binding serine protease inhibitors such as for subtilisin, thrombin, trypsin, chymotrypsin and furin was performed to identify the minimum side chains of CrSPI-1-D1 to be mutated to alter the selectivity (Table 2).…”

Section: Resultsmentioning

confidence: 97%

“…Although the sequence of the reactive-site loop (RSL) is different in several families of serine protease inhibitors, the conformation of the RSL is similar [10], [11]. Like other Kazal-type inhibitors, the disulfide bonds formed by cysteine residues at the P3 and P5′ positions (Cys1 and Cys9 in CrSPI-1-D1) hold the RSL in a relatively rigid conformation.…”

Section: Resultsmentioning

confidence: 99%

“…The full length and domain 2 of CrSPI-1 have been shown to contain full inhibitory activities against subtilisin. However, the function of the domain 1 of CrSPI (hereafter referred to as CrSPI-1-D1) is not yet characterized [10]. Analysis of the CrSPI-1-D1 sequence shows that it is significantly homologous to that of rhodniin-D1 from Rhodnius prolixus , which is a thrombin inhibitor [11].…”

Section: Introductionmentioning

confidence: 99%

“…In spite of several studies on serine protease inhibitors, CrSPIs are relatively new and potent [10]. There are several unexplored potentials and unanswered questions about CrSPIs, for example, what is the structural homology of the CrSPI domains, among themselves and other SPIs?…”

Section: Introductionmentioning

confidence: 99%

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Modifying the Substrate Specificity of Carcinoscorpius rotundicauda Serine Protease Inhibitor Domain 1 to Target Thrombin

et al. 2010

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Protease inhibitors play a decisive role in maintaining homeostasis and eliciting antimicrobial activities. Invertebrates like the horseshoe crab have developed unique modalities with serine protease inhibitors to detect and respond to microbial and host proteases. Two isoforms of an immunomodulatory two-domain Kazal-like serine protease inhibitor, CrSPI-1 and CrSPI-2, have been recently identified in the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. Full length and domain 2 of CrSPI-1 display powerful inhibitory activities against subtilisin. However, the structure and function of CrSPI-1 domain-1 (D1) remain unknown. Here, we report the crystal structure of CrSPI-1-D1 refined up to 2.0 Å resolution. Despite the close structural homology of CrSPI-1-D1 to rhodniin-D1 (a known thrombin inhibitor), the CrSPI-1-D1 does not inhibit thrombin. This prompted us to modify the selectivity of CrSPI-1-D1 specifically towards thrombin. We illustrate the use of structural information of CrSPI-1-D1 to modify this domain into a potent thrombin inhibitor with IC50 of 26.3 nM. In addition, these studies demonstrate that, besides the rigid conformation of the reactive site loop of the inhibitor, the sequence is the most important determinant of the specificity of the inhibitor. This study will lead to the significant application to modify a multi-domain inhibitor protein to target several proteases.

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Endotoxin Detection – from Limulus Amebocyte Lysate to Recombinant Factor C

Ding

2010

Subcellular Biochemistry

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Gram negative bacterial endotoxin is a biological pyrogen that causes fever when introduced intravenously. The endotoxin, also known as lipopolysaccharide (LPS), is found in the outer membrane of Gram-negative bacteria. During Gram-negative sepsis, endotoxin stimulates host macrophages to release inflammatory cytokines. However, excessive inflammation causes multiple organ failure and death. Endotoxins, which are ubiquitous pathogenic molecules, are a bane to the pharmaceutical industry and healthcare community. Thus early and sensitive detection of endotoxin is crucial to prevent endotoxaemia. The limulus amebocyte lysate (LAL) has been widely used for ~30 years for the detection of endotoxin in the quality assurance of injectable drugs and medical devices. The LAL constitutes a cascade of serine proteases which are triggered by trace levels of endotoxin, culminating in a gel clot at the end of the reaction. The Factor C, which normally exists as a zymogen, is the primer of this coagulation cascade. In vivo, Factor C is the perfect biosensor, which alerts the horseshoe crab of the presence of a Gram-negative invader. The hemostatic end-point entraps the invader, killing it and limiting further infection. However, as an in vitro endotoxin detection tool, variations in the sensitivity and specificity of LAL to endotoxin, and the dwindling supply of horseshoe crabs are posing increasing challenges to the biotechnology industry. This has necessitated the innovation of an alternative test for endotoxin. Thus, Factor C became the obvious, albeit tricky target for the recombinant technology effort. This chapter documents the backwater of mining the natural blood lysate of the endangered species to the monumental effort of genetic engineering, to produce recombinant Factor C (rFC). The rFC is a 132 kDa molecule, which was produced as a proenzyme inducible by the presence of trace levels of endotoxin. The rFC forms the basis of the "PyroGene" kit, which is a novel micro-enzymatic endotoxin diagnostic assay for high-throughput screens of endotoxin. Using the rFC, Lonza Inc. has spawned the "PyroSense" which serves as checkpoints of the biotechnology production line. Thus, from cloning to commercial applications, the rFC has initiated a new era in endotoxin-testing for the quality assurance of biomedical products and for the healthcare industry, whilst sparing the endangered horseshoe crabs.

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A Novel Serine Protease Inhibitor Acts as an Immunomodulatory Switch while Maintaining Homeostasis

Cited by 15 publications

References 90 publications

Endotoxin-induced coagulation reactions and phenoloxidase activity modulation inSudanonautes africanushemolymph fractions

Endotoxin-induced coagulation reactions and phenoloxidase activity modulation inSudanonautes africanushemolymph fractions

Modifying the Substrate Specificity of Carcinoscorpius rotundicauda Serine Protease Inhibitor Domain 1 to Target Thrombin

Endotoxin Detection – from Limulus Amebocyte Lysate to Recombinant Factor C

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