IMMUNOCYTOCHEMICAL LOCALIZATION OF ALPHA-2,3(N)-SIALYLTRANSFERASE (ST3GAL-III) IN CELL-LINES AND RAT-KIDNEY TISSUE-SECTIONS - EVIDENCE FOR GOLGI AND POST-GOLGI LOCALIZATION

Sialylation is a biosynthetic process occurring in the trans compartme nts of the Golgi apparatus. Corresponding evidence is based on localiz ation and biochemical studies of alpha 2,6(N)-sialyltransferase (ST6Ga l I) as previously reported. Here we describe generation and character ization of polyclonal antibodies to recombinant rat alpha 2,3(N)-sialy ltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells o r as a beta-galactosidase-human-ST3Gal III fusion-protein from E.coli, respectively. These antibodies were used to localize ST3Gal III by im munofluorescence in various cell lines and rat kidney tissue sections, In transiently transfected COS cells the antibodies directed to solub le sialyltransferase or the sialyltransferase portion of the fusion-pr otein only recognized the recombinant antigen retained in the endoplas mic reticulum. However, an antibody fraction crossreactive with beta-g alactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta 1,4-galactosyltransferase in the Golgi app aratus of several cultured cell lines. Antibodies affinity purified on the beta-galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion- fixed rat kidney sections. We found strong staining of the Golgi appar atus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H(+)ATPase. This subcel lular localization was not observed for ST6Gal I which localized to th e Golgi apparatus. These data show colocalization in the Golgi apparat us and different post-Golgi distributions of the two sialyltransferase s.