title: Toxoid construction of AsaP1, a lethal toxic aspzincin metalloendopeptidase of Aeromonas salmonicida subsp. achromogenes, and studies of its activity and processing.
abstract: AsaP1 is a toxic aspzincin metalloendopeptidase secreted by the fish pathogen Aeromonas salmonicida subsp. achromogenes. The protease is highly immunogenic and antibodies against AsaP1 evoke a passive protection against infection with A. salmonicida subsp. achromogenes. The protease is expressed as 37 kDa pre-pro-protein and processed to an active enzyme of 19kDa in A. salmonicida subsp. achromogenes. Recombinant expression of AsaP1(rec) in E. coli results in a protease of 22 kDa that is not secreted. AsaP1(rec) induces comparable pathological changes in Atlantic salmon (Salmo salar L.) to native AsaP1(wt). The aim of the study was to construct AsaP1 toxoids by exchanging catalytically important amino acids in the active site region of the protease. Four different AsaP1 mutants (AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A), and AsaP1(Y309F)) were successfully constructed by one step site directed mutagenesis, expressed in E. coli BL21 C43 as pre-pro-proteins and purified by His-tag affinity chromatography and gel filtration. Three of the resulting mutants (AsaP1(E294A), AsaP1(E294Q), and AsaP1(Y309A)) were not caseinolytic active and are detected as unprocessed pre-pro-proteins of 37 kDa. Caseinolytic active AsaP1(rec) and a mutant with reduced activity, AsaP1(Y309F), were processed to a size of 22 kDa. Furthermore, AsaP1(rec) is able to process the inactive mutants to the mature size of 22 kDa, allowing the conclusion that AsaP1 is autocatalytically processed. All four mutants AsaP1(E294A), AsaP1(E294Q), AsaP1(Y309A) and AsaP1(Y309F) are non-toxic in fish but induce a specific anti-AsaP1 antibody response in Arctic charr (Salvelinus alpinus L.) and are therefore true toxoids and possible vaccine additives.
authors.affiliation: Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, IS-112 Reykjavík, Iceland; Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix Hausdorff-Str. 4, 17487 Greifswald, Germany. Electronic address: johanna_schwenteit@gmx.de.
authors.forename: Johanna
authors.initials: J
authors.lastname: Schwenteit
authors.affiliation: Department of Structural Biology, Greifswald University, Felix Hausdorff-Str. 4, 17487 Greifswald, Germany. Electronic address: xenia.bogdanovic@uni-greifswald.de.
authors.forename: Xenia
authors.initials: X
authors.lastname: Bogdanović
authors.affiliation: Matís ohf, Vínlandsleið 12, 113 Reykjavík, Iceland. Electronic address: olafur.h.fridjonsson@matis.is.
authors.forename: Olafur H
authors.initials: OH
authors.lastname: Fridjonsson
authors.affiliation: Matís ohf, Vínlandsleið 12, 113 Reykjavík, Iceland. Electronic address: arnthor.aevarsson@matis.is.
authors.forename: Arnthor
authors.initials: A
authors.lastname: Aevarsson
authors.affiliation: Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix Hausdorff-Str. 4, 17487 Greifswald, Germany. Electronic address: uwe.bornscheuer@uni-greifswald.de.
authors.forename: Uwe T
authors.initials: UT
authors.lastname: Bornscheuer
authors.affiliation: Department of Structural Biology, Greifswald University, Felix Hausdorff-Str. 4, 17487 Greifswald, Germany. Electronic address: winfried.hinrichs@uni-greifswald.de.
authors.forename: Winfried
authors.initials: W
authors.lastname: Hinrichs
authors.affiliation: Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, IS-112 Reykjavík, Iceland. Electronic address: bjarngud@hi.is.
authors.forename: Bjarnheidur K
authors.initials: BK
authors.lastname: Gudmundsdottir
mesh_terms: D048409:Aeromonas salmonicida / Q000201:enzymology*; D000818:Animals; D000917:Antibody Formation / Q000187:drug effects / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008666:Metalloendopeptidases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D014121:Toxoids / Q000235:genetics / Q000378:metabolism; D014337:Trout / Q000276:immunology
chemical_list: D011994:Recombinant Proteins; D014121:Toxoids; D008666:Metalloendopeptidases; C039830:peptidyl-Lys metalloendopeptidase