Post-Golgi apparatus localization and regional expression of rat intestinal sialyltransferase detected by immunoelectron microscopy with polypeptide epitope-purified antibody.
During studies on the Golgi apparatus immunolocalization of beta-galactoside alpha 2,6-sialyltransferase in intestinal cells, immunostaining of a number of post-Golgi apparatus structures including mucus droplets and plasma membrane were observed. In order to determine if this labeling was in fact due to sialyltransferase and not carbohydrate-specific antibodies in the polyclonal antiserum preparation, fusion protein to sialyltransferase was used to epitope purify polypeptide-specific antibodies. The affinity purification was performed on a column containing a beta-galactosidase-sialyltransferase fusion protein expressed in Escherichia coli. Using such antibodies we present evidence that in intestinal cells sialyltransferase is not only present in the Golgi apparatus cisternal stack but also its transtubular network and various post-Golgi apparatus structures. In absorptive enterocytes, post-Golgi apparatus vesicles, the brush border and basolateral plasma membrane, multivesicular bodies, and lysosome-like structures were labeled. In goblet cells the limiting membrane and lumen of forming and mature mucus droplets as well as the plasma membrane exhibited label for sialyltransferase. The results provide evidence for "ecto-sialyltransferase" in the plasma membranes of these cells, and suggest that most of the sialyltransferase is released from the Golgi membranes and becomes secreted with the goblet cell mucus. In addition, the polypeptide epitope-purified antibody was also used to examine regional expression of sialyltransferase in the rat intestinal epithelium. Immunolabel was restricted to the large intestine and not found in duodenum, jejunum, and ileum. Direct measurement of the enzyme activity was found to correlate with the immunoelectron microscopic data. This observation suggests that there is regional specific expression of the beta-galactoside alpha 2,6-sialyltransferase.
D000818:Animals; D002846:Chromatography, Affinity; D000939:Epitopes / Q000032:analysis*; D006056:Golgi Apparatus / Q000201:enzymology*; D007150:Immunohistochemistry; D007422:Intestines / Q000166:cytology / Q000201:enzymology*; D008854:Microscopy, Electron; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012799:Sialyltransferases / Q000032:analysis* / Q000235:genetics; D013347:Subcellular Fractions / Q000201:enzymology; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase
D000939:Epitopes; D011993:Recombinant Fusion Proteins; D012799:Sialyltransferases; D000094802:beta-D-Galactoside alpha 2-6-Sialyltransferase