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A novel protein acts as a negative regulator of prophenoloxidase activation and melanization in the freshwater crayfish Pacifastacus leniusculus
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Physiology and Developmental Biology, Comparative Physiology. (Jämförande Fysiologi)
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2009 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 284, no 10, 6301-6310 p.Article in journal (Refereed) Published
Abstract [en]

Melanization is an important immune component of the innate immune system of invertebrates and is vital for defense as well as for wound healing. In most invertebrates melanin synthesis is achieved by the prophenoloxidase-activating system, a proteolytic cascade similar to vertebrate complement. Even though melanin formation is necessary for host defense in crustaceans and insects, the process needs to be tightly regulated because of the hazard to the animal of unwanted production of quinone intermediates and melanization in places where it is not suitable. In the present study we have identified a new melanization inhibition protein (MIP) from the hemolymph of the crayfish, Pacifastacus leniusculus. Crayfish MIP has a similar function as the insect MIP molecule we recently discovered in the beetle Tenebrio molitor but interestingly has a completely different sequence. Crayfish MIP as well as Tenebrio MIP do not affect phenoloxidase activity in itself but instead interfere with the melanization reaction from quinone compounds to melanin. Importantly, crayfish MIP in contrast to Tenebrio MIP contains a fibrinogen-like domain, most similar to the substrate recognition domain of vertebrate l-ficolins. Surprisingly, an Asp-rich region similar to that found in ficolins that is likely to be involved in Ca2+ binding is present in crayfish MIP. However, crayfish MIP did not show any hemagglutinating activity as is common for the vertebrate ficolins. A mutant form of MIP with a deletion lacking four Asp amino acids from the Asp-rich region lost most of its activity, implicating that this part of the protein is involved in regulating the prophenoloxidase activating cascade. Overall, a new negative regulator of melanization was identified in freshwater crayfish that shows interesting parallels with proteins (i.e. ficolins) involved in vertebrate immune response.

Place, publisher, year, edition, pages
2009. Vol. 284, no 10, 6301-6310 p.
Keyword [en]
Pacifastacus leniusculus, melanization inhibiting protein, crayfish, phenoloxidase
National Category
Immunology
Research subject
Immunology; Biology with specialization in Comparative Physiology
Identifiers
URN: urn:nbn:se:uu:diva-111473DOI: 10.1074/jbc.M806764200ISI: 000263742700033PubMedID: 19129195OAI: oai:DiVA.org:uu-111473DiVA: diva2:281317
Available from: 2009-12-15 Created: 2009-12-15 Last updated: 2017-12-12
In thesis
1. Innate Immune Proteins in a Crustacean Pacifastacus leniusculus
Open this publication in new window or tab >>Innate Immune Proteins in a Crustacean Pacifastacus leniusculus
2011 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Hemocytes (blood cells) are important in the immune defense against pathogens in invertebrates. In crusteacean, the hemocytes and plasma components mount a strong innate immune response against different pathogens including bacteria and virus. This thesis is aimed to identify marker proteins associated with development of different hemocyte types, and to find a protein involved in the phenoloxidase-induced melanization and other innate immune reactions in freshwater crayfish Pacifastacus leniusculus.

In crustaceans, the hemocytes are produced and partly differentiated in the hematopoietic tissue (Hpt) before they are released into the hemolymph circulation. To investigate the connection between semigranular cells, granular cells and precursor cells in Hpt of P. leniusculus and possibly also in other crustaceans, two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS) analysis was used to identify specific proteins expressed in different hemocytes. The specific expression was analyzed by RT-PCR and western blot. Moreover, RNA interference was used to study the hemocyte differentiation in vivo and in vitro.

Melanin formation is essential for host defence in arthopods, and it needs to be tightly regulated since unwanted production of quinone intermediates or melanization is also dangerous to the animal. By using western blot, 2-DE and MS, a melanization inhibiting protein (MIP) was found to have similar function as mealworm Tenebrio molitor MIP. Both of them interfere with the melanization reaction, but do not affect phenoloxidase activity.

In order to reveal the mechanism by which peptidoglycan (PGN) induces activation of the prophenoloxidase activating system in P. leniusculus, different forms of Lys-type PGN were used to pull down PGN recognition proteins (PGRPs) from plasma or hemocyte lysate supernatant of crayfish. The binding proteins were separated and then analyzed with MS. Results showed that two serine protease homologues are involved in this activation possibly by forming a complex with lipopolysaccharide and β-1,3-glucan binding protein (LGBP) and without a PGRP.

Besides, two ficolin-like proteins (FLPs) have been found from crayfish plasma by using different bacteria including Staphylocuccus aureus as an affinity matrix to pull down bacterial binding proteins, followed by the analysis with 2-DE and MS. Two FLPs can bind to bacteria, and may help crayfish to clear Gram-negative bacteria, but not Gram-positive bacteria injected into the crayfish hemolymph, which suggests that FLPs may function as pattern recognition receptors in the immune response of crayfish.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2011. 72 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 807
Keyword
2-DE, hematopoiesis, marker protein, melaniztion inhibiting protein, proPO-system, pattern recognition protein, PGN, ficolin-like protein
National Category
Immunology
Research subject
Biology with specialization in Comparative Physiology
Identifiers
urn:nbn:se:uu:diva-147600 (URN)978-91-554-8015-8 (ISBN)
Public defence
2011-04-08, Lindahlsalen, Evolutionary Biology Centre, Norbyvägen 18A,, Uppsala, 10:00 (English)
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Note
Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 737Available from: 2011-03-16 Created: 2011-02-26 Last updated: 2011-05-04

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Söderhäll, IreneSöderhäll, Kenneth

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