Effect of glycosylation on the in vivo circulating half-life of ribonuclease

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The circulating half-lives of the four isozymes of bovine pancreatic ribonuclease (RNases A, B, C, and D) have been determined in normal and in nephrectomized rats. The isozymes differ only in their glycosyl content. While A contains no sugars, B has a simple oligosaccharide (GlcNAc2 Man4-5),and C and D each have a complex oligosaccharide (GlcNAc4 Man 2-3 Gal2 Fuc NeuAc2, and GlcNAc4 Man3 Gal2 Fuc NeuAc4, respectively) attached to Asn-34 of the polypeptide chain. All four isozymes were cleared rapidly in normal rats (t 1/2 = 2 to 3 min), as expected on the basis of the established role of the kidneys in removing low molecular weight proteins from circulation. In nephrectomized rats, however, a much slower clearance was observed, thus permitting the evaluation of the role of the carbohydrate chains in the catabolism of the isozymes. The clearance curves can be analyzed in terms of two processes, a rapid initial one, shown to represent the equilibration of the injected enzyme into extravascular space, and a second one which is interpreted as the catabolic clearance of the enzyme. The haf-life of the RNase isozymes was calculated from this second process and found to be in the range 528 to 577 min for RNase A, 15 min for RNase B, 681 to 862 min for RNase C, and 839 to 941 min for RNase D. The rapidly cleared RNase B was treated with alpha-mannosidase to remove 3 of the 4 mannosyl residues, leaving only a trisaccharide (GlcNAc2-betaMan) attached to the protein. The half-life of this RNase B derivatives was found to be in the range 616 to 733 min. From these results it is concluded (a) that the addition of complex oligosaccharides to a protein does not have any significant direct effect on its circulating half-life (RNases C and D compared to RNase A), and (b) that in the rat there exists a mechanism for clearing glycoproteins based on specific recognition of exposed alpha-mannosyl residues (RNase B compared to the other isozymes and to alpha-mannosidase-treated RNase B).