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Peptidoglycan activation of the proPO-system without a peptidoglycan receptor protein (PGRP)?
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Comparative Physiology.
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2011 (English)In: Developmental and Comparative Immunology, ISSN 0145-305X, E-ISSN 1879-0089, Vol. 35, no 1, 51-61 p.Article in journal (Refereed) Published
Abstract [en]

Recognition of microbial polysaccharide by pattern recognition receptors triggers the prophenoloxidase (proPO) cascade, resulting in melanin synthesis and its deposition on the surface of invading pathogens. Several masquerade-like proteins and serine proteinase homologues have been shown to be involved in the proPO activation in insects. In this study, a novel serine proteinase homologue, Pl-SPH2, was found and isolated as a 30 kDa protein from hemocytes of the freshwater crayfish, Pacifastocus leniusculus, by its binding property to a partially lysozyme digested or TCA-treated insoluble Lysine (Lys)-type pepticloglycan (PGN) and soluble polymeric Lys-type PGN. Two other proteins, the Pl-SPH1 and lipopolysaccharide- and beta-1,3-glucan-bincling protein (LGBP) were also found in the several different PGN-binding assays. However no PGRP homologue was detected. Neither was any putative PGRP found after searching available crustacean sequence databases. If RNA interference of Pl-SPH2, Pl-SPH1 or LGBP in the crayfish hematopoietic tissue cell culture was performed, it resulted in lower PO activity following activation of the proPO-system by soluble Lys-type PGN. Taken together, we report for the first time that Lys-type PGN is a trigger of proPO-system activation in a crustacean and that two Pl-SPlis are involved in this activation possibly by forming a complex with LGBP and without a PGRP.

Place, publisher, year, edition, pages
2011. Vol. 35, no 1, 51-61 p.
Keyword [en]
Innate immunity, Lys-type PGN, PGRP, proPO, SPHs
National Category
Immunology
Research subject
Biology with specialization in Comparative Physiology
Identifiers
URN: urn:nbn:se:uu:diva-140158DOI: 10.1016/j.dci.2010.08.005ISI: 000285024400007PubMedID: 20713082OAI: oai:DiVA.org:uu-140158DiVA: diva2:383100
Available from: 2011-01-04 Created: 2011-01-04 Last updated: 2017-12-11
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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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, KennethSöderhäll, Irene

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