Functional Conversion of Hemocyanin to Phenoloxidase by Horseshoe Crab Antimicrobial Peptides

Nagai, Taku; Osaki, Tsukasa; Kawabata, Shun Ichiro

doi:10.1074/jbc.m102596200

Cited by 180 publications

(117 citation statements)

References 56 publications

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“…Nagai et al documented that hemocyanin of horseshoe crab Tachypleus tridentatus could be functionally converted into a phenoloxidase-like enzyme by the clotting enzyme and by chitin-binding antimicrobial peptides [4,5]. Treating hemocyanins from crustacean with SDS resulted in an opening of the entrance to the active site of the protein for bulkyphenolic compounds [6].…”

Section: Introductionmentioning

confidence: 99%

SNPs of hemocyanin C‐terminal fragment in shrimp Litopenaeus vannamei

et al. 2012

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a b s t r a c tIn this study, we identified a variable region in the C-terminus of hemocyanin from the shrimp Litopenaeus vannamei (2288-2503 bp, HcSC) by sequence alignments. A total of 13 SNPs were identified by PCR-SSCP and HcSC clone sequencing. The SSCP patterns of HcSC could be modulated in Vibro parahaemolyticus-treated shrimps. A novel SSCP band with four SNP sites was identified in V. parahaemolyticus-resistant shrimps. More importantly, three of these four SNPs introduced variations in amino acid sequence and possibly secondary structure of the HcSC polypeptide and resulted in a higher agglutinative activity against seven pathogenic bacteria. These results suggest that the C-terminus of shrimp L. vannamei hemocyanin possesses SNPs, which may be related to shrimp resistance to different pathogens.

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Section: Introductionmentioning

confidence: 99%

SNPs of hemocyanin C‐terminal fragment in shrimp Litopenaeus vannamei

et al. 2012

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“…Besides being an important respiratory protein, the HMC plays a crucial role in frontline innate immune response during which it is proteolytically processed by serine proteases to activate its prophenoloxidase (PPO) activity. 29 Our recent study has shown , unpublished). Indeed, we have also identified a prophenoloxidase activating enzyme, PPOA (HpF149), which was strongly upregulated in the hepatopancreas from 3 hpi to reach a peak at 6 hpi ( Figure 4b).…”

Section: à3mentioning

confidence: 99%

“…The resulting PPO activity of HMC catalyses the oxidation of phenols to quinones, thereby inducing melanisation to oxidatively kill the invading microbe. 29 Furthermore, HMC also releases a C-terminal peptide that has antimicrobial activity. and tissue remodelling.…”

Section: à3mentioning

confidence: 99%

Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda

et al. 2005

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Knowledge on how genes are turned on/off during infection and immunity is lacking. Here, we report the coregulation of diverse clusters of functionally related immune response genes in a horseshoe crab, Carcinoscorpius rotundicauda. Expressed sequence tag (EST) clusters for frontline immune defense, cell signalling, apoptosis and stress response genes were expressed or repressed spatio-temporally during the acute phase of Pseudomonas infection. An infection time course monitored by virtual Northern evaluation indicates upregulation of genes in blood cells (amebocytes) at 3-h postinfection, whereas most of the hepatopancreas genes remained downregulated over 72 h of infection. Thus, the two tissues orchestrate a coordinated and timely response to infection. The hepatopancreas probably immunomodulates the expression of other genes and serves as a reservoir for later response, if/when chronic infection ensues. On the other hand, being the first to encounter pathogens, we reasoned that amebocytes would respond acutely to infection. Besides acute transactivation of the immune genes, the amebocytes maintained morphological integrity, indicating their ability to synthesise and store/secrete the immune proteins and effectors to sustain the frontline innate immune defense, while simultaneously elicit complement-mediated phagocytosis of the invading pathogen. Our results show that the immune response against Pseudomonas infection is spatially and temporally coordinated.

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“…detergents and alcohols) or polypeptides (e.g. antimicrobial peptides, serine proteinase precursors) (Asada et al, 1993;Decker et al, 2001;Nagai et al, 2001;Nagai and Kawabata, 2000), PAP-1 and SPHs could simply bind to proPO, change its conformation, and yield PO activity without any proteolysis. To test this possibility, we first incubated PAP-1 with an irreversible serine proteinase inhibitor, APMSF, to block its amidase activity.…”

Section: Significance Of Proteolytic Cleavage In Propo Activationmentioning

confidence: 99%

Manduca sexta prophenoloxidase (proPO) activation requires proPO-activating proteinase (PAP) and serine proteinase homologs (SPHs) simultaneously

Gupta

Wang

Jiang

2005

Insect Biochemistry and Molecular Biology

104

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In the tobacco hornworm Manduca sexta, proteolytic activation of prophenoloxidase (proPO) is mediated by three proPO-activating proteinases (PAPs) and two serine proteinase homologs (SPHs) (Proceedings of the National Academy of Sciences, USA 95 (1998) (2004) 731-742), roles of these clip-domain proteins (i.e. PAPs and SPHs) in proPO activation are poorly defined. To better understand this process, we further characterized the activation reaction using proPO, PAP-1 and SPHs. PAP-1 itself cleaved nearly 1/3 of proPO at Arg 51 without generating much phenoloxidase (PO) activity. In the presence of SPHs, the cleavage of proPO became more complete while the increase in PO activity was over 20-fold, indicating that the extent of cleavage does not directly correlate with PO activity. Since SPHs and p-amidinophenyl methanesulfonyl fluoride (APMSF)-treated PAP-1 did not generate active PO by interacting with proPO, proteolytic cleavage is critical for proPO activation. After 1/5 of proPO was processed by PAP-1 alone which was then inactivated by M. sexta serpin-1J or APMSF, further incubation of the reaction mixture with SPHs failed to generate active PO either. Thus, SPHs cannot generate PO activity by simply binding to cleaved proPO. M. sexta proPO activation requires active PAP-1 and SPHs at the same time-one for limited proteolysis and the other as a cofactor, perhaps. Gel filtration chromatography and native gel electrophoresis revealed the PAP-SPH, proPO-PAP, and SPH-proPO associations, essential for generating high M r , active PO at the site of infection.

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Functional Conversion of Hemocyanin to Phenoloxidase by Horseshoe Crab Antimicrobial Peptides

Cited by 180 publications

References 56 publications

SNPs of hemocyanin C‐terminal fragment in shrimp Litopenaeus vannamei

SNPs of hemocyanin C‐terminal fragment in shrimp Litopenaeus vannamei

Spatial and temporal coordination of expression of immune response genes during Pseudomonas infection of horseshoe crab, Carcinoscorpius rotundicauda

Manduca sexta prophenoloxidase (proPO) activation requires proPO-activating proteinase (PAP) and serine proteinase homologs (SPHs) simultaneously

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