Colocalization of leukotriene C synthase and microsomal glutathione S-transferase elucidated by indirect immunofluorescence analysis.

We have previously shown that the two membrane bound enzymes leukotriene C synthase and microsomal glutathione S-transferase interact in vitro and in vivo. Rat basophilic leukemia cells and murine mastocytoma cells, two well-known sources of leukotriene C synthase, both expressed microsomal glutathione S-transferase as determined by Western blot analyses. Several human tissues were found to contain both leukotriene C synthase and microsomal glutathione S-transferase mRNA. These data suggest that the interaction may be physiologically important. To study this further, expression vectors encoding the two enzymes were cotransfected into mammalian cells and the subcellular localization of the enzymes was determined by indirect immunofluorescence using confocal laser scanning microscopy. The results showed that leukotriene C synthase and microsomal glutathione S-transferase were both localized on the nuclear envelope and adjacent parts of the endoplasmic reticulum. Image overlay demonstrated virtually identical localization. We also observed that coexpression substantially reduced the catalytic activity of each enzyme suggesting that a mechanism involving protein-protein interaction may contribute to the regulation of LTC4 production.

Demonstration of different forms of the anti-inflammatory proteins lipocortin-1 and Clara cell protein-16 in human nasal and bronchoalveolar lavage fluids.

The anti-inflammatory proteins lipocortin-1 and Clara cell protein-16 (CC-16) were studied in two-dimensional gel electrophoresis (2-DE) protein patterns of human nasal lavage fluids (NLFs) and bronchoalveolar lavage fluids (BALFs). Seven forms of lipocortin-1 were detected with Western immunoblots: three isoforms with an apparently normal Mr of 38 kDa and pI of 5.9, 6.0 and 6.1, and four truncated variants with pI/kDa 6.0/36, 6.4/36, 7.0/33, and 7.4/34. Four 6 kDa isoforms of CC-16 were found with pI 4.6, 4.8, 4.9, and 5.2. Lipocortin-1 and CC-16 were expressed in all individuals tested although not all variants were found in each individual. The overall levels of lipocortin-1 were higher in BALF than NLF and there were significant differences in the distribution of the different lipocortin-1 forms between BALFs and NLFs. One patient with occupational asthma and four children with rhinitis had increased levels of one of the truncated lipocortin-1 forms in NLF (pI/kDa: 7.4/34) and decreased levels of the major CC-16 form (pI/kDa: 4.8/6). The levels of CC-16 but not of lipocortin-1 were higher in BALF from smokers than from nonsmokers. These results indicate that the levels of lipocortin-1 and CC-16 in NLF and BALF may be altered in inflammatory airway disorders. Furthermore, the identification of different forms of the two proteins makes possible more detailed studies on the role of these proteins in inflammatory disease processes and anti-inflammatory therapies.

Protein patterns of human nasal and bronchoalveolar lavage fluids analyzed with two-dimensional gel electrophoresis.

We have previously described the protein patterns of human nasal lavage fluid (NLF) and bronchoalveolar lavage fluid (BALF) after two-dimensional gel electrophoresis (2-DE). We now report the identification of a number of additional proteins in these 2-DE patterns. Several plasma proteins (alpha2-macroglobulin, haptoglobin alpha1-chain, IgA S chain, ceruloplasmin, alpha1-microglobulin, amyloid P and apolipoprotein A-1) could be included both in the BALF and NLF spot pattern data bases by matching with a master plasma 2-DE pattern (SWISS-2DPAGE). Furthermore, lysozyme, lactoferrin and the antiinflammatory proteins lipocortin-1 and Clara cell protein 16 (CC-16) were identified by matching with reference proteins and Western immunoblots. Significant differences in the levels of some of the identified proteins were found between NLF and BALF, and between BALF from smokers and nonsmokers. Transferrin, hemopexin and haptoglobin alpha1 were lower in NLF than BALF, while IgA, lysozyme and lactoferrin were higher in NLF than BALF. One form of alpha1-microglobulin was more abundant in NLF than in BALF, while the opposite was found for a second form of the same protein. Moreover, the levels of IgA, ceruloplasmin and the pro-form of apolipoprotein A-1 in BALF were lower in smokers than in nonsmokers. The possibility to describe and analyze differences in NLF and BALF 2-DE patterns at the protein spot level may have wide clinical applications.