Neoglycoproteins: preparation and properties of complexes of biotinylated asparagine-oligosaccharides with avidin and streptavidin

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Abstract
Neoglycoproteins in which the oligosaccharide moieties are attached noncovalently to the protein through a high-affinity ligand have been prepared from biotinylated oligosaccharides and avidin or the nonglycosylated microbial analogue streptavidin. One of the asparagine-oligosaccharides purified from Pronase-digested ovalbumin (Man6-GlcNAc2-Asn) was reacted with an excess of the hydroxysuccinimide ester of biotin or, for the purpose of quantitation, [3H]biotin. Derivatives were also prepared with an extension arm, a 6-aminohexanoyl group, between biotin and asparagine. When the purified biotinyl-Asn-oligosaccharide was added to avidin or streptavidin, a complex was formed containing 3 mol of oligosaccharide/mol of protein. The complexes were stable at neutral pH in the absence of biotin and could be dialyzed for 2 weeks without any significant loss of ligand. In the presence of biotin, or under denaturing conditions, the oligosaccharide derivatives was released and could be quantitatively recovered. To assess the influence of the protein matrix on the reactivity of the oligosaccharide units, free biotinyl-Asn-oligosaccharide and the corresponding avidin and streptavidin complexes were exposed to .alpha.-mannosidase in parallel experiments. The rate of hydrolysis of the free derivative was severalfold faster than that of the two protein complexes, and at the time when about 90% of the free derivatives had all five .alpha.-mannosyl residues removed, the majority of the protein-bound derivatives contained two to four undigested .alpha.-mannosyl residues and also had a significant amount of undigested starting material. The case of preparation and the properties of these neoglycoproteins suggest that they should be excellent models for the study of glycoprotein-receptor binding and glycoprotein processing.

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