Myeloperoxidase (MPO) is a myelomonocytic granule protein responsible for generation of oxygen radicals, hydrochlorous acid, and other toxic metabolites during the respiratory burst. In addition to this microbicidal function, plasma MPO concentration is a predictor of myocardial infarction in people with chest pain, and is an autoantigen in anti-neutrophil cytoplasmic autoantibody-associated diseases. MPO has not been investigated in cats with disease as of yet, but healthy cats given propylthiouracil develop anti-MPO antibodies and a multisystemic auto-immune syndrome highly similar to a subset of hyperthyroid people given the same drug. The purpose of this study was to sequence the feline MPO gene, partially characterize the mature granulocytic protein, and compare it to the human homologue in preparation for future studies.
Total mRNA was isolated from bone marrow collected from a random-source euthanized cat using a commercial kit. cDNA was produced using both oligo(dT) and feline MPO gene-specific primers designed using published human MPO primers, but modified using the NCBI-reported feline genome. PCR resulted in six overlapping sequences which spanned the total feline MPO cDNA sequence. Sequencing of these fragments revealed a 2165 base pair coding DNA sequence with 85% homology to the human MPO sequence. Comparison of the gene to the partially sequenced feline genome shows the RNA length to be divided into a minimum of 12 exons, with 158 bp lying within a thus far non-sequenced section of the genome. The final MPO coding DNA sequence differed from the reported genome at 8 bps (1 bp deletion and 7 bp substitutions). Comparison of the predicted feline MPO amino acid sequence with the published human MPO protein supports that these bp changes are due to errors in the NCBI-reported genome rather than being single nucleotide polymorphisms or silent mutations.
Feline granulocytes were isolated from peripheral blood obtained from a healthy donor using differential centrifugation and mounted on glass slides using standard cytospin techniques. Immunofluorescent antibody staining with a FITC-conjugated anti-human MPO antibody (Dako) revealed cytoplasmic granular fluorescence similar to that seen with staining of human MPO-expressing granulocytes. MPO was presumptively isolated from the same feline donor following granulocyte isolation, cell lysis, and concentration using a concanavalin A-Sepharose column. Elution fractions were tested for peroxidase activity using the Bradley method, and the highest-activity fractions were analyzed by SDS-PAGE and Western immunoblotting. After staining with anti-human MPO antibody, 55 and 12 kD bands were detected, consistent with the molecular weights of the MPO heavy and light chains predicted by translation of the coding DNA sequence.
We were able to sequence and partially characterize the feline MPO gene and protein, and demonstrated that the feline and human DNA and amino acid sequences are significantly homologous. Knowledge of the MPO sequence will facilitate future investigations on the role of MPO in feline disease, including the suitability of human MPO in any assays developed.