| 1. |
- Apitanyasai, Kantamas, et al.
(författare)
-
Characterization of a hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus
- 2016
-
Ingår i: Fish and Shellfish Immunology. - : Elsevier BV. - 1050-4648 .- 1095-9947. ; 58, s. 429-435
-
Tidskriftsartikel (refereegranskat)abstract
- Hemocyte homeostasis-associated-like protein (HHAP) in the freshwater crayfish Pacifastacus leniusculus has a distinct role from that of its homolog PmHHAP in the shrimp Penaeus monodon. Knockdown of PIHHAP in vitro using double-stranded RNA (dsRNA) had no effect on the cell morphology of hematopoietic tissue (HPT) cells. The total hemocyte number and caspase activity were unchanged after PIHHAP knockdown in vivo, in contrast to the results found in shrimp. Moreover, suppression of PIHHAP both in vitro and in vivo did not change the mRNA levels of some genes involved in hematopoiesis and hemocyte homeostasis. Interestingly, bacterial count and scanning electron microscope revealed that depletion of PIHHAP in intestine by RNAi resulted in higher number of bacteria in the crayfish intestine. Together, these results suggest that PIHHAP is not involved in hemocyte homeostasis in the crayfish P. leniusculus but appears to affect the bacterial number in the intestine through an unknown mechanism. Since PIHHAP has different functions from PmHHAP, we therefore named it HHAP-like protein.
|
|
| 2. |
- Cerenius, Lage, 1956-, et al.
(författare)
-
High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans
- 2010
-
Ingår i: Developmental and Comparative Immunology. - : Elsevier. - 0145-305X .- 1879-0089. ; 34:1, s. 69-75
-
Tidskriftsartikel (refereegranskat)abstract
- Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.
|
|
| 3. |
- Donpudsa, Suchao, et al.
(författare)
-
Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus
- 2010
-
Ingår i: Journal of Invertebrate Pathology. - : Elsevier BV. - 0022-2011 .- 1096-0805. ; 104:3, s. 234-238
-
Tidskriftsartikel (refereegranskat)abstract
- The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coil expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 mu M and 3.5-8 mu M, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.
|
|
| 4. |
- Donpudsa, Suchao, et al.
(författare)
-
Proteinase inhibitory activities of two two-domain Kazal proteinase inhibitors from the freshwater crayfish Pacifastacus leniusculus and the importance of the P2 position in proteinase inhibitory activity
- 2010
-
Ingår i: Fish and Shellfish Immunology. - : Elsevier BV. - 1050-4648 .- 1095-9947. ; 29:5, s. 716-723
-
Tidskriftsartikel (refereegranskat)abstract
- Serine proteinase inhibitors are found ubiquitously in living organisms and involved in homeostasis of processes using proteinases as well as innate immune defense. Two two-domain Kazal-type serine proteinase inhibitors (KPIs), KPI2 and KPI8, have been identified from the hemocyte cDNA library of the crayfish Pacifastacus leniusculus. Unlike other KPIs from P. leniusculus, they are found specific to the hernocytes and contain an uncommon P-2 amino acid residue, Gly. To unveil their inhibitory activities, the two KPIs and their domains were over-expressed. By testing against subtilisin, trypsin, chymotrypsin and elastase, the KPI2 was found to inhibit strongly against subtilisin and weakly against trypsin, while the KPI8 was strongly active against only trypsin. With their P-1 Set and Lys residues, the KPI2_domain2 and KPI8_domain2 were responsible for strong inhibition against subtilisin and trypsin, respectively. Mutagenesis of KPI8_domain1 at P-2 amino acid residue from Gly to Pro, mimicking the P-2 residue of KPI8_domain2, rendered the KPI8_domain1 strongly active against trypsin, indicating the important role of P-2 residue in inhibitory activities of the Kazal-type serine proteinase inhibitors. Only the KPI2 was found to inhibit against the extracellular serine proteinases from the pathogenic oomycete of the freshwater crayfish, Aphanomyces astaci.
|
|
| 5. |
- Jearaphunt, Miti, et al.
(författare)
-
Caspase-1-like regulation of the proPO-system and role of ppA and caspase-1-like cleaved peptides from proPO in innate immunity
- 2014
-
Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 10:4, s. e1004059-
-
Tidskriftsartikel (refereegranskat)abstract
- Invertebrates rely on innate immunity to respond to the entry of foreign microorganisms. One of the important innate immune responses in arthropods is the activation of prophenoloxidase (proPO) by a proteolytic cascade finalized by the proPO-activating enzyme (ppA), which leads to melanization and the elimination of pathogens. Proteolytic cascades play a crucial role in innate immune reactions because they can be triggered more quickly than immune responses that require altered gene expression. Caspases are intracellular proteases involved in tightly regulated limited proteolysis of downstream processes and are also involved in inflammatory responses to infections for example by activation of interleukin 1ß. Here we show for the first time a link between caspase cleavage of proPO and release of this protein and the biological function of these fragments in response to bacterial infection in crayfish. Different fragments from the cleavage of proPO were studied to determine their roles in bacterial clearance and antimicrobial activity. These fragments include proPO-ppA, the N-terminal part of proPO cleaved by ppA, and proPO-casp1 and proPO-casp2, the fragments from the N-terminus after cleavage by caspase-1. The recombinant proteins corresponding to all three of these peptide fragments exhibited bacterial clearance activity in vivo, and proPO-ppA had antimicrobial activity, as evidenced by a drastic decrease in the number of Escherichia coli in vitro. The bacteria incubated with the proPO-ppA fragment were agglutinated and their cell morphology was altered. Our findings show an evolutionary conserved role for caspase cleavage in inflammation, and for the first time show a link between caspase induced inflammation and melanization. Further we give a more detailed understanding of how the proPO system is regulated in time and place and a role for the peptide generated by activation of proPO as well as for the peptides resulting from Caspase 1 proteolysis.
|
|
| 6. |
- Prapavorarat, Adisak, et al.
(författare)
-
A Novel Viral Responsive Protein Is Involved in Hemocyte Homeostasis in the Black Tiger Shrimp, Penaeus monodon
- 2010
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 285:28, s. 21467-21477
-
Tidskriftsartikel (refereegranskat)abstract
- A novel viral responsive protein, namely hemocyte homeostasis-associated protein (HHAP), was characterized for its role in the response of shrimp to white spot syndrome virus infection. The full-length cDNAs of HHAP from the black tiger shrimp (PmHHAP), Penaeus monodon, and the fresh water crayfish (PlHHAP), Pacifastacus leniusculus, were obtained and showed high sequence identity to a hypothetical protein from various organisms, with the highest identity to the hypothetical protein TcasGA2_TC006773 from the red flour beetle, Tribolium castaneum (54% amino acid sequence identity). Transcripts of PmHHAP were expressed in various shrimp tissues with the highest expression in hematopoietic tissue, whereas the transcripts of PlHHAP were found in the hematopoietic and nerve tissues. Upon white spot syndrome virus infection, a high up-regulation level of shrimp hemocytic HHAP mRNA and protein was observed by real-time reverse transcription-PCR and immunofluorescence microscopy, respectively. Gene silencing of PmHHAP by RNA interference resulted in a significant decrease in the number of circulating hemocytes and 100% shrimp mortality within 30 h of the double-stranded PmHHAP RNA injection (but not in control shrimp), indicating that HHAP is essential for shrimp survival. Interestingly, severe damage of hemocytes was observed in vivo in the PmHHAP knockdown shrimp and in vitro in shrimp primary hemocyte cell culture, suggesting that PmHHAP plays an important role in hemocyte homeostasis. Thus, it is speculated that the up-regulation of PmHHAP is an important mechanism to control circulating hemocyte levels in crustaceans during viral infection.
|
|
| 7. |
- Söderhäll, Kenneth, et al.
(författare)
-
Prophenoloxidase-activating Enzyme
- 2013. - 3 uppl.
-
Ingår i: Handbook of Proteolytic Enzymes. - : Elsevier. - 9780123822192 - 9780123822208 ; , s. 6675-6687
-
Bokkapitel (refereegranskat)
|
|
| 8. |
- Tharntada, Sirinit, et al.
(författare)
-
Role of anti-lipopolysaccharide factor from the black tiger shrimp, Penaeus monodon, in protection from white spot syndrome virus infection
- 2009
-
Ingår i: Journal of General Virology. - : Microbiology Society. - 0022-1317 .- 1465-2099. ; 90:Part 6, s. 1491-1498
-
Tidskriftsartikel (refereegranskat)abstract
- The anti-lipopolysaccharide factor (ALF) from the black tiger shrimp, Penaeus monodon, has been shown previously to exhibit a broad spectrum of activity against various strains of bacteria and fungi. Herein, the recombinant ALFPm3 (rALFPm3) protein was examined for its role in the defence against white spot syndrome virus (WSSV) infection in haematopoietic (Hpt) cell cultures of the freshwater crayfish, Pacifastacus leniusculus, as well as in live P. monodon shrimps. Incubation of Hpt cell cultures with a mixture of WSSV and rALFPm3 resulted in a dose-dependent decrease in VP28 gene expression levels, compared with those incubated with WSSV alone, with an rALFPm3 IC(50) value lower than 2.5 muM. However, pre-treatment of Hpt cells with 5 muM rALFPm3 showed no induced protection against subsequent WSSV infection, whereas the synthetic crayfish ALF peptide could protect cells at a higher concentration (10 muM). The in vivo role of ALFPm3 was examined by injection of P. monodon with WSSV pre-treated with rALFPm3 protein. The results clearly showed that rALFPm3 was able to reduce WSSV propagation and prolong the survival of shrimps.
|
|
| 9. |
- Wu, Chenglin, et al.
(författare)
-
An MBL-like protein may interfere with the activation of the proPO-system, an important innate immune reaction in invertebrates
- 2013
-
Ingår i: Immunobiology. - : Elsevier BV. - 0171-2985 .- 1878-3279. ; 218:2, s. 159-168
-
Tidskriftsartikel (refereegranskat)abstract
- An important characteristic of the innate immune systems of crayfish and other arthropods is the activation of a serine proteinase cascade in the hemolymph, which results in the activation of prophenoloxidase and subsequently leading to the formation of toxic quinones and melanin. Although no true complement homologues have been detected in crayfish or crustaceans, several proteins with similarities to vertebrate pattern recognition receptors (PRRs), which are involved in the lectin pathway of complement activation in vertebrates, are present. One is a C-type lectin, a mannose-binding lectin (Pl-MBL), which is secreted from granular hemocytes. Here we report that Pl-MBL has LPS-binding capacity and is dependent upon high Ca(2+) for its solubility and Pl-MBL interferes with proPO activation in vitro when HLS is prepared at high Ca(2+). The proPO-activating system is efficiently activated by microbial polysaccharides and it has to be neatly regulated to avoid activation in places where it is inappropriate and the active enzyme PO should be prevented from spreading throughout the body of the animal. This may be particularly important during molting when proPO is involved in hardening of a new cuticle and the animal is vulnerable to microbes. The presence of high amount of Pl-MBL in the granular hemocytes may play a role in this process. Since a hemocyte lysate supernatant (HLS) prepared at 100mM Ca(2+) could become activated when the concentration of LPS was increased up to 3mg/ml, this may indicate that Pl-MBL acts as a scavenger for LPS to prevent spreading of LPS in the hemolymph to avoid further activation of the proPO-system.
|
|
