caa a22 4500 467906815 CHVBK 20180406152836.0 cr unu---uuuuu 170328e20060201xx s 000 0 eng 10.1007/s00251-005-0063-4 doi (NATIONALLICENCE)springer-10.1007/s00251-005-0063-4 Molecular characterization and expression of a granzyme of an ectothermic vertebrate with chymase-like activity expressed in the cytotoxic cells of Nile tilapia (Oreochromis niloticus) [Elektronische Daten] [Kesavannair Praveen, John Leary III, Donald Evans, Liliana Jaso-Friedmann] We have identified the gene coding for a novel serine protease with close similarities to mammalian granzymes from nonspecific cytotoxic cells of a teleost fish Oreochromis niloticus. The genomic organization of tilapia granzyme-1 (TLGR-1) has the signature five-exon-four-intron structure shared by all granzymes and similar hematopoietic Ser proteases. Molecular modeling studies suggested a granzyme-like structure for this protein with four disulfide linkages and two additional Cys residues. The expression of this gene is found to be restricted to cytotoxic cell populations with a low level of constitutive expression when compared to similar granzymes in other teleost species. High levels of transcriptional activation of TLGR-1 with different stimuli suggested that this gene is highly induced during immune reactions. Triplet residues around the active site Ser of TLGR, which determines the primary substrate specificity of granzymes, differ significantly from that of other granzymes. Recombinant TLGR-1 was expressed in the mature and proenzyme forms using pPICZ-alpha vector in the Pichia pastoris expression system. Recombinant TLGR-1 was used to determine the primary substrate specificity of this protease using various synthetic thiobenzyl ester substrates. In vitro enzyme kinetics assays suggested a preference for residues with bulky side chains at the P1 site, indicating a chymase-like activity for this protease. These results indicate the presence of novel granzymes in cytotoxic cells from ectothermic vertebrates. Springer-Verlag, 2006 Cytotoxicity nationallicence Natural killer cells nationallicence Granzyme nationallicence Teleosts nationallicence Nonspecific cytotoxic cells nationallicence Praveen Kesavannair Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Agriculture Drive, 30602, Athens, GA, USA aut Leary III John Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Agriculture Drive, 30602, Athens, GA, USA aut Evans Donald Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Agriculture Drive, 30602, Athens, GA, USA aut Jaso-Friedmann Liliana Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Agriculture Drive, 30602, Athens, GA, USA aut Immunogenetics Springer-Verlag 58/1(2006-02-01), 41-55 0093-7711 58:1<41 2006 58 251 https://doi.org/10.1007/s00251-005-0063-4 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s00251-005-0063-4 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Praveen Kesavannair Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Agriculture Drive, 30602, Athens, GA, USA aut NATIONALLICENCE 700 1- Leary III John Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Agriculture Drive, 30602, Athens, GA, USA aut NATIONALLICENCE 700 1- Evans Donald Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Agriculture Drive, 30602, Athens, GA, USA aut NATIONALLICENCE 700 1- Jaso-Friedmann Liliana Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Agriculture Drive, 30602, Athens, GA, USA aut NATIONALLICENCE 773 0- Immunogenetics Springer-Verlag 58/1(2006-02-01), 41-55 0093-7711 58:1<41 2006 58 251 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer