Protein matrix effects on glycan processing by mannosidase II and sialyl transferase from rat liver.
The effect of the protein environment on the reaction sequence and the relative rates of two two-step reactions involved in the biosynthesis of complex glycans in glycoproteins has been explored by comparing the processing of biotinylated substrates either free or bound to avidin. By use of biotinyl and biotinamidohexanoyl derivatives, the display of the glycan in a proximal and distal association with the avidin surface could also be assessed. Mannosidase II removes two Man residues from the substrate GlcNAcMan5GlcNAc2-R to yield GlcNAcMAn3GlcNAc2-R. The NMR spectra of the substrate, intermediate, and product showed that the first Man is removed from the 6-arm of the substrate. The rate constants for the first and second step (estimated by direct analysis of the reactants by anion-exchange chromatography with a pulsed amperometric detector) were determined to be about 0.05 and 0.08 min-1, respectively, for the free substrates. In the proximal complex k1 was reduced 80-fold, and the k2 step could not be observed under the same conditions. In the distal complex both k1 and k2 were reduced about 8-fold. Sialyl transferases transfer Sia from CMP-Sia to the biantennary substrate Gal2GlcNAc2-Man3GlcNA2-R to yield the product Sia2Gal2-GlcNAc2Man3GlcNAc2-R with the Sia linked either 2-3 or 2-6 to the Gal residues. The NMR spectra showed that the first step involved the Gal on the 3-arm of the substrate and that both Sia residues were added 2-6.(ABSTRACT TRUNCATED AT 250 WORDS)
Shao
M C
MC
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
Krudy
G
G
Rosevear
P R
PR
Wold
F
F
D000818:Animals; D002240:Carbohydrate Sequence; D006023:Glycoproteins / Q000096:biosynthesis* / Q000235:genetics; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008361:Mannosidases / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000096:biosynthesis*; D011499:Protein Processing, Post-Translational; D051381:Rats; D012799:Sialyltransferases / Q000378:metabolism*
D006023:Glycoproteins; D011134:Polysaccharides; D012799:Sialyltransferases; D008361:Mannosidases; C038829:mannosyl-oligosaccharide 1,3 - 1,6-alpha-mannosidase