Effect of individual carbohydrate chains on the in vitro and in vivo biological function of rabbit antithrombin

Abstract

<p>Antithrombin (AT) is the principal anticoagulant protein that inhibits thrombin and factor Xa in plasma. AT is a glycoprotein with a molecular weight of 58 KDa and contains four consensus sequences for N-linked glycosylation, located at Asn 96, Asn 135, Asn 155, and Asn 192. To investigate the effect of individual glycan side chain on the biological function of AT, each of the four Asn residue sites of glycosylation was mutated to Gln to block glycosylation at these sites. The resulting constructs were initially expressed in COS cells. Permanent cell lines were generated by transfecting CHO cells with the constructs, encoding either the wild-type rabbit AT (AT-WT) or one of the four underglycosylated mutant variants (AT-N96Q, AT-N135Q, AT-N155Q, and AT-N155Q. The results demonstrated that neither the amount of AT secreted nor the kinetics of secretion differed significantly between cell lines expressing AT-WT and any of the mutant AT variants. Purification and radioiodination of AT-WT and each of the four mutant variants permitted pharmacokinetic analysis of their individual catabolism in rabbits. The rate constants for protein catabolism were significantly higher for underglycosylated mutant variants than that of AT-WT. However, no significant difference was identified among the catabolic half-lives of the underglycosylated mutant variants. These results suggest that the extent of glycosylation is more important than its location for determining AT catabolism in vivo. Further in vivo studies of double and triple underglycosylated forms of AT as well as totally deglycosylated AT are needed before a final conclusion can be made. The AT-WT and all forms of mutant variants were capable of forming complexes with thrombin. However, compared with AT-WT, the second order rate constant was significantly reduced for the AT-N 155Q variant, by 2 and 20 fold in the absence and presence of heparin, respectively. No statistically significant differences in second order rate constant were observed between AT-WT and other three underglycosylated variants, AT-N96Q, AT-N135Q, and AT-N192Q. These results suggest that a failure to glycosylate Asn 155 in AT results in reduction of its ability to inhibit thrombin. In comparison with AT-WT, AT-N135Q and AT-N192Q appeared to have slightly higher heparin affinity, but no statistically significant difference was identified. The AT-N96Q showed a small increase in Kd. Compared with AT-WT, however, there was significant reduction in heparin affinity for AT-N155Q (6-fold increase in Kd). These results suggest that a failure to glycosylate Asn 155 in AT results in significant reduction of its ability to bind to heparin.</p>