title: The regulation of glycan processing in glycoproteins. The effect of avidin on individual steps in the processing of biotinylated glycan derivatives.
abstract: The effect of the protein matrix on glycan processing by rat liver Golgi enzymes has been evaluated by a direct comparison of substrate----products conversion of a free glycan and of the same glycan linked to a protein. The glycan substrates had the general structure R-glycan where R represented either biotinyl-Asn-GlcNAc2- or 6-(biotinamido)hexanoyl-Asn-Glc-NAc2- and the protein used was avidin; the extension arm in one of the glycan substrates permitted the additional comparison of two avidin-biotin-glycan complexes. By the use of different glycans as substrates, by the presence or absence of donor substrates (UDP-GlcNAc, UDP-Gal, and CMP-sialic acid (Sia) and/or the inhibitor, swainsonine, it was possible to dissect the individual steps involved in the conversion of R-Man6 (or R-Man5) to a biantennary complex glycan, R-Man3-GlcNAc2-Gal2-Sia2 or to the hybrid glycan R-Man5-GlcNAc-Gal-Sia. Using fast atom bombardment-mass spectrometry to identify and quantify the substrates and products of each parallel incubation of free and avidin-bound substrates, the following observations were made. With the substrate without the extension arm, avidin-binding inhibited mannosidase I, GlcNAc transferase I, and the second step of the reaction catalyzed by mannosidase II (R-Man4-GlcNAc----R-Man3-GlcNAc); the second step of the reaction catalyzed by Gal-transferase was also inhibited to a lesser extent. This inhibition was greatly reduced or absent with the substrates with the extension arm and was consequently referred to as the short range effect. A long range effect of avidin binding expressed by both substrates with and without extension arm was observed for Gal-transferase acting in the hybrid glycan pathway (R-Man5-GlcNAc----R-Man5-GlcNAc-Gal) in the presence of swainsonine and also for Sia-transferase in the catalysis of the incorporation of the second Sia residue into the complex product (R-Man3-GlcNA2-Gal2-Sia----R-Man3-GlcNAc2- Gal2-Sia2) and to a lesser extent in the hybrid pathway (R-Man5-GlcNAc-Gal----R-Man5-GlcNAc-Gal-Sia). GlcNAc transferase II did not appear to be affected by avidin. Based on the information available on the biotin-binding site in avidin, it is proposed that the short range effect reflects the masking of the core chitobiose unit in the avidin-glycan complexes in the absence of the extension arm, but not in the presence of the arm, and that the early processing enzymes thus may require a fully exposed chitobiose for full activity.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D001360:Avidin / Q000378:metabolism / Q000494:pharmacology*; D005700:Galactosyltransferases / Q000037:antagonists & inhibitors; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors; D006023:Glycoproteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000201:enzymology*; D007425:Intracellular Membranes / Q000201:enzymology; D008099:Liver / Q000648:ultrastructure; D008361:Mannosidases / Q000037:antagonists & inhibitors; D013058:Mass Spectrometry; D017351:N-Acetylglucosaminyltransferases; D019158:N-Acetylneuraminic Acid; D011134:Polysaccharides / Q000378:metabolism*; D051381:Rats; D012794:Sialic Acids / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D017026:Swainsonine
chemical_list: D000470:Alkaloids; D006023:Glycoproteins; D011134:Polysaccharides; D012794:Sialic Acids; D001360:Avidin; D005700:Galactosyltransferases; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C024850:alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase I; D008361:Mannosidases; D019158:N-Acetylneuraminic Acid; D017026:Swainsonine