Detection of terminal N-linked N-acetylglucosamine residues in the Golgi apparatus using galactosyltransferase and endoglucosaminidase F/peptide N-glycosidase F: adaptation of a biochemical approach to electron microscopy.
Purified human milk beta-N-acetylglucosaminide beta 1, 4 galactosyltransferase (EC 2.4.1.38) was used to galactosylate N-acetylglucosamine (GlcNAc) residues present in ultra-thin sections of Lowicryl K4M-embedded rat and pig liver. Both endogenous galactose and galactosylated transferase products could be revealed by Ricinus communis lectin I-gold complexes (RcL I-g15). Without galactosyltransferase (GT) treatment, labeling for galactose (gal) was limited to the trans region of rat and pig hepatocyte Golgi apparatus. After exposure to GT, additional labeling was found over cis Golgi apparatus cisternae. RcL I-g15 labeling was sensitive to a purified preparation of endoglucosaminidase F/peptide N-glycosidase F (at pH 9). This indicates that endogenous gal and gal transferred by GT to terminal GlcNAc residues are present N-linked oligosaccharides. The RcL I-g15 labeling produced by GT was insensitive to extensive washing with solutions containing either EDTA and urea or SDS and 2-mercaptoethanol or 0.1 M GlcNAc. Substrate inhibition studies showed that 50 mM GlcNAc specifically inhibited the additional RcL I-g15 labeling produced by GT. The use of purified glycosyltransferases therefore appears to allow specific detection of oligosaccharide substrates and their high resolution localization in thin sections by electron microscopy.
D000117:Acetylglucosamine / Q000032:analysis*; D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism*; D005944:Glucosamine / Q000031:analogs & derivatives*; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D006596:Hexosaminidases / Q000378:metabolism*; D008099:Liver / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008722:Methods; D008854:Microscopy, Electron; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D051381:Rats; D011919:Rats, Inbred Strains; D013194:Staining and Labeling; D013552:Swine; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000378:metabolism*
D005700:Galactosyltransferases; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase; D006596:Hexosaminidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D005944:Glucosamine; D000117:Acetylglucosamine