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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)

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Abstract

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.

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Acknowledgements

We thank Drs. Dorothy Hudig, Dieter Jenne, Jan Potempa, and James Powers for generously providing reagents and valuable suggestions.

This work was supported by BARD # US-3159-99C and Veterinary Medical Experimental Station Grants at the College of Veterinary Medicine, University of Georgia.

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  1. Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Agriculture Drive, Athens, GA, 30602, USA

    Kesavannair Praveen, John H. Leary III, Donald L. Evans & Liliana Jaso-Friedmann

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  1. Kesavannair Praveen
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  2. John H. Leary III
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  3. Donald L. Evans
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  4. Liliana Jaso-Friedmann
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Correspondence to Liliana Jaso-Friedmann.

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Praveen, K., Leary, J.H., Evans, D.L. et al. 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) . Immunogenetics 58, 41–55 (2006). https://doi.org/10.1007/s00251-005-0063-4

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