Distribution and localization of CMP-N-acetylneuraminic acid hydroxylase and N-glycolylneuraminic acid-containing glycoconjugates in porcine lymph node and peripheral blood lymphocytes

An immunohistochemical analysis was performed on paraplast-embedded section s of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneurami nic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a -Gc antibody). The observed localization of the enzyme in cells of the peri follicular zone, including lymphocytes, was reflected in a similar distribu tion of glycoconjugate-bound NeuSGc. This result confirms previous biochemi cal investigations on the role of the hydroxylase in regulating NeuSGc bios ynthesis in vitro on a histological level. An analysis of lymphocytes isola ted from porcine thymus, spleen, lymph node and peripheral blood revealed d ifferences in the amount of NeuSGc in the various lymphocytes that correlat ed well with the activity of the hydroxylase determined in these cells. The largest amount of Neu5Gc and highest activity of the enzyme were detected in the peripheral blood lymphocytes (PBL). Immunohistochemical studies with a-Gc and h-3 antibodies on sections of paraplast-embedded PBL showed that these antigens were located at the cell surface and in the cytosol, respect ively, Ultrastructural immunocytochemistry with the h-3 antibody and immuno gold labelling was used to investigate the subcellular localization of the hydroxylase, The enzyme was detected in the cytosol in the vicinity of the nuclear membrane and the outer membrane of mitochondria, in particular thos e close to the nucleus, The antigen was also detected on cytoplasmic tubula r structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally, The possibility that this localization may be relat ed to the availability of the substrate CMP-Neu5Ac and the redox partner cy tochrome b(5) is discussed.