Neoglycoproteins: preparation of noncovalent glycoproteins through high-affinity protein- (glycosyl) ligand complexes.
This work was undertaken as part of a search for well-characterized glycoprotein models in which both the oligosaccharide structure, the number of oligosaccharide chains, and the precise location of these chains in the protein are known. On the basis of the fact that high-affinity ligand binding sites have been defined precisely for several proteins in terms of both number and relative location, the hypothesis to be tested was that if oligosaccharide chains were covalently attached to such high-affinity ligands, they would be specifically bound in the ligand sites of the appropriate protein, thus permitting the preparation of neoglycoproteins of precise predetermined oligosaccharide valency and topography. To test this hypothesis, pyridoxal 5'-phosphate was reductively (NaB3H4) aminated with the alpha-amino group of the asparagine oligosaccharide Man6-GlcNAc2-Asn from ovalbumin. When the resulting phosphopyridoxylated oligosaccharide (PG) was added to the apo form of aspartate aminotransferase (AAT; EC 2.6.1.1, the cytosolic enzyme from pig heart, consisting of two subunits and containing two coenzyme binding sites), a 2:1 (PG-AAT) complex was formed which could be characterized on the basis of tritium content, the absorbance and fluorescence of the pyridoxamine phosphate moiety of PG, and the concanavalin A binding properties acquired by AAT through the incorporation of the oligosaccharide. As expected from the established properties of the holoenzyme, the AAT-PG complex is stable in the absence of phosphate or vitamin B6 derivatives and can be dialyzed for 24 h without any significant loss of PG. According to the three-dimensional model of AAT, the oligosaccharide chain of PG should be partially masked in the coenzyme binding pocket.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001219:Aspartate Aminotransferases / Q000378:metabolism*; D001665:Binding Sites; D008956:Models, Chemical; D008970:Molecular Weight; D011732:Pyridoxal Phosphate / Q000378:metabolism; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000378:metabolism; D013552:Swine; D014961:X-Ray Diffraction
D001051:Apoenzymes; D001059:Apoproteins; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; C010627:pyridoxamine phosphate