The regulation of glycan processing in glycoproteins. The effect of avidin on individual steps in the processing of biotinylated glycan derivatives.

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)