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 sections of porcine lymph node with antibodies specific for CMP-N-acetylneuraminic acid hydroxylase (h-3 antibody) and glycoconjugate-bound N-glycolylneuraminic acid (Neu5Gc), which appears as a result of the hydroxylase reaction (a-Gc antibody). The observed localization of the enzyme in cells of the perifollicular zone, including lymphocytes, was reflected in a similar distribution of glycoconjugate-bound Neu5Gc. This result confirms previous biochemical investigations on the role of the hydroxylase in regulating Neu5Gc biosynthesis in vitro on a histological level. An analysis of lymphocytes isolated from porcine thymus, spleen, lymph node and peripheral blood revealed differences in the amount of Neu5Gc in the various lymphocytes that correlated 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, respectively. Ultrastructural immunocytochemistry with the h-3 antibody and immunogold 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 those close to the nucleus. The antigen was also detected on cytoplasmic tubular structures. In addition, a weak labelling of the Golgi apparatus was also observed occasionally. The possibility that this localization may be related to the availability of the substrate CMP-Neu5Ac and the redox partner cytochrome b5 is discussed.