Immunocytochemical localization of the Golgi apparatus using protein-specific antibodies to galactosyltransferase.
Polyclonal antisera to human milk galactosyltransferase have been widely used as immunocytochemical reagent to visualize the Golgi apparatus. Immunochemical analysis revealed partial specificity of these antisera to glycan epitopes expressed on galactosyltransferase and other gene products (R. A. Childs et al., Biochem. J. 238, 605-611 (1986)). Since glycan-specific reagents such as lectins are known to label the Golgi apparatus by virtue of their exclusive glycan specificity, Golgi localization of galactosyltransferase by use of these polyclonal antisera remained elusive. In order to demonstrate authentic Golgi localization of the galactosyltransferase peptide, we expressed the enzyme in Escherichia coli as non-glycosylated beta-galactosidase-fusion proteins and used them as affinity matrix to protein-epitope purified polyclonal antibodies from antisera to the milk enzyme, and to induce protein-specific antisera by injecting them into rabbits, respectively. A short fusion protein comprising most of the substrate binding sites and a protein which included the stem region of galactosyltransferase were expressed. The short fusion protein was poorly immunogenic whereas the long fusion protein induced a high-titer antiserum. Protein-epitope purified antibodies derived from polyclonal antisera to the milk enzyme as well as antibodies to the long fusion protein cross-reacted with milk galactosyltransferase and with fusion protein as demonstrated by immunoblotting and enzyme-linked immunosorbent assay (ELISA) and produced typical Golgi morphologies in both HeLa and CaCo2 cells when used for immunocytochemical localization of galactosyltransferase. We conclude that previous immunolocalization studies have correctly been interpreted as Golgi localization of the galactosyltransferase protein and that antigenicity to the protein moiety is confirmed to the N-terminal third of the enzyme.
Watzele
G
G
Institute of Physiology, University of Zürich, Switzerland.
Bachofner
R
R
Berger
E G
EG
D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D000939:Epitopes / Q000276:immunology; D005700:Galactosyltransferases / Q000032:analysis* / Q000276:immunology / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000201:enzymology / Q000276:immunology / Q000648:ultrastructure*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008892:Milk / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000276:immunology
D000939:Epitopes; D011993:Recombinant Fusion Proteins; D005700:Galactosyltransferases