An inconvenient association between granzyme A and Nicotinamide Nucleotide Transhydrogenase

ABSTRACT Granzyme A (GzmA) is a serine protease secreted by cytotoxic lymphocytes, with GzmA -/- mouse studies informing our understanding of GzmA’s physiological function. We show herein that GzmA -/- mice have a mixed C57BL/6J and C57BL/6N background and retain the full length Nicotinamide Nucleotide Transhydrogenase ( Nnt ) gene, whereas Nnt is truncated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in GzmA -/- mice; however, the presence of Nnt , rather than loss of GzmA, was responsible for this phenotype by constraining lymphocyte infiltration. A new CRISPR active site mutant C57BL/6J GzmA S211A mouse provided the first insights into GzmA’s bioactivity free of background issues, with circulating proteolytically active GzmA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of Bioprojects listing C57BL/6J as the mouse strain, had Nnt sequencing reads inconsistent with a C57BL/6J background. The Nnt issue has clearly complicated our understanding of GzmA and may similarly have influenced studies across a broad range of fields.