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Expression of an active and inactive clip-domain Serine Protease Isolated from Signal Crayfish in an insect cell line
(Söderhäll)
(Söderhäll)
Science for Life Laboratory. (Söderhäll)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

In this study, we aimed to elucidate the cleavage specificity and endogenous substrate of a clip-domain serine protease from the crayfish Pacifastacus leniusculus (PlcSP). This serine protease can bind to white spot syndrome virus (WSSV) and is important for the entry of WSSV into the hematopoietic tissue (HPT). In order to understand its intrinsic role in the serine protease cascade, we wanted to use substrate phage display method to clarify cleavage specificity of PlcSP. Firstly, we aimed to make recombinant active and inactive forms of this PlcSP using human embryonic kidney cell line (HEK293) however PlcSP appeared to be toxic for mammalian cells and we were not able to express this crustacean protein. However, we were able to express these recombinantPlcSP both in their zymogenic and active forms in Sf9 insect cell line.

Keywords [en]
Serine protease, Pacifastacus leniusculus, invertebrate immunity, Sf9, substrate phage display
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-356482OAI: oai:DiVA.org:uu-356482DiVA, id: diva2:1236119
Available from: 2018-07-31 Created: 2018-07-31 Last updated: 2018-08-02
In thesis
1. Interaction between crayfish and some microorganisms; Effect of temperature
Open this publication in new window or tab >>Interaction between crayfish and some microorganisms; Effect of temperature
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Innate immunity, which constitutes the first line of defense in vertebrates, is the only immune system that invertebrates rely on to protect themselves from pathogens. The invertebrate immune system is composed of cellular and humoral components. Cellular immunity is phagocytosis, opsonization and encapsulation. The humoral part is mainly composed of the events taking place upon secretion of granules and the enzymes within that lead to the lysis of the pathogen by antimicrobial peptides (AMPs) and the melanization cascade. The Prophenoloxidase (proPO) activating system is an important pathway that is stored in the granules of semigranular and granular hemocytes (blood cells). These cells will degranulate and release the proPO system when activated upon pathogen recognition. This cascade results in the melanization reaction and to trap and eliminate pathogens. 

White spot syndrome virus (WSSV) is a deadly pathogen mainly targeting crustaceans and causing huge economic losses since its first emergence in 1992 in Taiwan. It is known that WSSV disables the immune system of the host by interfering with the proPO cascade. Temperature is a restricting factor for the WSSV infections however it is not known if its affects are on host immunity or on the virus itself.

With the aim of elucidating WSSV infection, we studied the virus entry mechanisms. By crosslinking WSSV with the hemocytes we showed that a new clip-domain serine protease (PlcSP) plays an important role during the WSSV infection in crayfish by means of interacting with WSSV envelope protein VP28. Moreover, we have shown that the viral entry is inhibited at cold temperatures due to temperature’s inhibitory effect on PlcSP expression. We also showed that by slowing down of the host’s metabolism hence proliferation in host tissue either by low temperature or cell cycle inhibitors, we could inhibit WSSV replication once it has entered the host cell. We tested if the temperature effects host or pathogen, or both, we investigated the mortalities, phagocytosis, bacterial clearance, total hemocyte counts, degranulation and melanization rate of crayfish under a cold and warm temperature by using two strains of gram-negative bacteria and LPS. It is apparent that the cellular immunity is more effective at low temperature while the humoral immunity can become overactivated and toxic for the host at higher temperature. Furthermore, we aimed to study the cleavage specificity for PlcSP since it is predicted to be secreted from hemocytes and takes part in the serine protease cascade during melanization reaction.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2018. p. 50
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1688
National Category
Immunology
Identifiers
urn:nbn:se:uu:diva-355147 (URN)978-91-513-0374-1 (ISBN)
Public defence
2018-09-13, Lindahlsalen, Norbyvägen 14, Uppsala, 13:00 (English)
Opponent
Supervisors
Available from: 2018-08-21 Created: 2018-06-26 Last updated: 2018-08-27

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