Detection and cellular localization of 12R-lipoxygenase in human tonsils

Formation of the 12R-lipoxygenase product, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE), has been detected previously only in human skin (Boeglin ct al. (1998) Proc. Natl. Acad. Sci. USA 95, 6744). The unexpected appearan ce of an EST sequence (AA649213) for human 12R-lipoxygenase from germinal c enter B lymphocytes purified from human tonsils prompted our search for the existence of the enzyme in this novel source. Incubation of [1-C-14]arachi donic acid with homogenates of human tonsillar tissue yielded mixtures of r adiolabeled 12-HETE and 15-HETE. Stereochemical analysis showed varying rat ios of 12S- and 12R-HETE, while 15-HETE was exclusively of the S-configurat ion. Using stereospecifically labeled [10S-H-3]- and [10R-H-3]arachidonic a cid substrates we detected pro-R hydrogen abstraction at carbon 10 associat ed with formation of 12R-HETE. This mechanist ic evidence implicates a 12R- lipoxygenase in the biosynthesis of 12R-HETE. The mRNA for the enzyme was i dentified in tonsils by RT-PCR and Northern analysis. The cellular distribu tion was established by in situ hybridization. Unexpectedly, hybridization was not observed in the lymphocytes of the germinal centers. Specific react ion was restricted to squamous epithelial cells, including the epithelium l ining the tonsillar crypts. Ln this location the 12R-lipoxygenase might hel p regulate differentiation of the epithelium or participate in lymphocyte-e pithelial cell interactions. (C) 2001 Academic Press.