Foster, M. W., Thompson, J. W., Ledford, J. G., Dubois, L. G., Hollingsworth, J. W., Francisco, D., Tanyaratsrisakul, S., Voelker, D. R., Kraft, M., Moseley, M. A., & Foster, W. M. (2014). Identification and Quantitation of Coding Variants and Isoforms of Pulmonary Surfactant Protein A. Journal of proteome research, 13(8), 3722-32.
Pulmonary surfactant protein A (SP-A), a heterooligomer of SP-A1 and SP-A2, is an important regulator of innate immunity of the lung. Nonsynonymous single nucleotide variants of SP-A have been linked to respiratory diseases, but the expressed repertoire of SP-A protein in human airway has not been investigated. Here, we used parallel trypsin and Glu-C digestion, followed by LC-MS/MS, to obtain sequence coverage of common SP-A variants and isoform-determining peptides. We further developed a SDS-PAGE-based, multiple reaction monitoring (GeLC-MRM) assay for enrichment and targeted quantitation of total SP-A, the SP-A2 isoform, and the Gln223 and Lys223 variants of SP-A, from as little as one milliliter of bronchoalveolar lavage fluid. This assay identified individuals with the three genotypes at the 223 position of SP-A2: homozygous major (Gln223/Gln223), homozygous minor (Lys223/Lys223), or heterozygous (Gln223/Lys223). More generally, our studies demonstrate the challenges inherent in distinguishing highly homologous, copurifying protein isoforms by MS and show the applicability of MRM mass spectrometry for identification and quantitation of nonsynonymous single nucleotide variants and other proteoforms in airway lining fluid.
Jimenez-Preitner, M., Berney, X., Uldry, M., Vitali, A., Cinti, S., Ledford, J. G., & Thorens, B. (2011). Plac8 is an inducer of C/EBPβ required for brown fat differentiation, thermoregulation, and control of body weight. Cell metabolism, 14(5), 658-70.
Brown adipocytes oxidize fatty acids to produce heat in response to cold or to excessive energy intake; stimulation of brown fat development and function may thus counteract obesity. Brown adipogenesis requires activation of the transcription factor C/EBPβ and recruitment of the zinc finger protein Prdm16, but upstream inducers of these proteins are incompletely defined. Here, we show that genetic inactivation of Plac8, a gene encoding an evolutionarily conserved protein, induces cold intolerance, and late-onset obesity, as well as abnormal morphology and impaired function of brown adipocytes. Using brown preadipocyte lines we show that Plac8 is required for brown fat differentiation, that its overexpression induces C/EBPβ and Prdm16, and that upon induction of differentiation Plac8 associates with C/EBPβ and binds to the C/EBPβ promoter to induce its transcription. Thus, Plac8 is a critical upstream regulator of brown fat differentiation and function that acts, at least in part, by inducing C/EBPβ expression.
Ledford, J. G., Kovarova, M., & Koller, B. H. (2007). Impaired host defense in mice lacking ONZIN. Journal of immunology (Baltimore, Md. : 1950), 178(8), 5132-43.
ONZIN is a small, cysteine-rich peptide of unique structure that is conserved in all vertebrates examined to date. We show that ONZIN is expressed at high levels in epithelial cells of the intestinal tract, the lung, and in cells of the immune system including macrophages and granulocytes. Because this pattern of expression is suggestive of a role in innate immune function, we have generated mice lacking this protein and examined their ability to respond to challenge with infectious agents. Onzin(-/-) mice show a heightened innate immune response after induction of acute peritonitis with Klebsiella pneumoniae. This increased response is consistent with an increased bacterial burden in the Onzin(-/-) mice. Ex vivo studies show that, whereas phagocytosis is not altered in Onzin(-/-) neutrophils, phagocytes lacking this protein kill bacteria less effectively. This result identifies ONZIN as a novel class of intracellular protein required for optimal function of the neutrophils after uptake of bacteria.
Hsia, B. J., Ledford, J. G., Potts-Kant, E. N., Nikam, V. S., Lugogo, N. L., Foster, W. M., Kraft, M., Abraham, S. N., & Wright, J. R. (2012). Mast cell TNF receptors regulate responses to Mycoplasma pneumoniae in surfactant protein A (SP-A)-/- mice. The Journal of allergy and clinical immunology, 130(1), 205-14.e2.
Mycoplasma pneumoniae (Mp) frequently colonizes the airways of patients with chronic asthma and likely contributes to asthma exacerbations. We previously reported that mice lacking surfactant protein A (SP-A) have increased airway hyperresponsiveness (AHR) during M pneumoniae infection versus wild-type mice mediated by TNF-α. Mast cells (MCs) have been implicated in AHR in asthma models and produce and respond to TNF-α.
Ledford, J. G., Addison, K. J., Francisco, D., Foster, M. W., Voelker, D. R., Que, L. G., & Kraft, M. (2016). Genetic Variation in Surfactant Protein-A2 Results in Altered Regulation of Eosinophil Activities and Enhanced Eosinophilia in Patients with Asthma. Annals of the American Thoracic Society, 13 Suppl 1, S101.
