Neoglycoproteins. Preparation and in vivo clearance of serum albumin derivatives containing ovalbumin oligosaccharides.

To explore the specificity determinants involved in lectin-sugar interactions, we have prepared 6 neoglycoproteins, each containing a single ovalbumin-derived oligosaccharide unit linked to bovine serum albumin (BSA), and compared their concanavalin A (Con A) binding and their in vivo circulatory clearance rate and tissue specificity in the rat. The neoglycoproteins were prepared using the glycosyl-Asn derivatives obtained by pronase digestion of ovalbumin as starting materials. By treatment with ninhydrin under carefully controlled conditions, the Asn moiety was deaminated and decarboxylated to the malonamide semialdehyde derivative, which in turn was coupled to BSA by reductive (NaCNBH3) amination. With a large excess of BSA in the reaction mixture, the only product was monoglycosylated BSA; it was separated from unreacted BSA by virtue of its binding to a Con A-Sepharose affinity column and from unreacted oligosaccharide by gel filtration. The yield of neoglycoprotein was 15-20% based on the original amount of glycosyl-Asn subjected to ninhydrin activation. The following 6-neoglycoproteins were produced with 6 different oligosaccharide units: I, GalGlcNAc3Man6GlcNAc2-BSA; IIB, GlcNAc3ManSGlcNAc2-BSA; IIIB, GlcNAczMansGlcNAcz-BSA; IIIC, Man7GlcNAc2-BSA; IV, MansGlcNAcz-BSA; V, MansGlcNAc2-BSA, and in addition neoglycoprotein V was treated with endoglycosidase H to give neoglyprotein V-H, GlcNAc-BSA. All but BSA (control) and neoglycoprotein in V-H bound to the Con A-Sepharose column and could be eluted with 0.1 M a-methylmannoside, but no significant difference in the binding affinity of the 6 neoglycoproteins could be detected by this method. For the in vivo studies, the protein were labeled with C 3 H ] raffinose and their lifetime in circulation and their tissue distribution after 24-48 h were determined. The in vivo circulatory clearance times (2 X tllz from 2.5-14 h) for the 6 neoglycoproteins as compared to 14.3 h for neoglycoprotein V-H and 19.6 h for BSA together with a corresponding shift in the tissue clearance sites from muscle and hide for BSA to primarily liver and kidney for the neoglycoproteins strongly suggest that specific receptors are involved in the clearance and that these receptors can distinguish the different oligosaccharide structures.