Decreased affinity of recombinant antithrombin for heparin due to increased glycosylation

Abstract

Recombinant antithrombin produced by baby hamster kidney (BHK) or Chinese hamster ovary (CHO) cells was separated into two fractions, containing comparable amounts of protein, by affinity chromatography on matrix-linked heparin. Fluorescence titrations showed that the more tightly binding fraction had a heparin affinity similar to that of plasma antithrombin (Kd 20 nM), whereas the affinity of the more weakly binding fraction was nearly 10-fold lower (Kd 175 nM). Analyses of the heparin-catalysed rate of inhibition of thrombin further showed that the fractions differed only in their affinity for heparin and not in the intrinsic rate constant of either the uncatalysed or the heparin-catalysed inactivation of thrombin. The recombinant antithrombin fraction with lower heparin affinity migrated more slowly than both the fraction with higher affinity and plasma antithrombin in SDS/PAGE under reducing conditions, consistent with a slightly higher apparent relative molecular mass. This apparent size difference was abolished by the enzymic removal of the carbohydrate side chains from the proteins. Such removal also increased the heparin affinity of the weakly binding fraction, so that it eluted from matrix-linked heparin at a similar position to the deglycosylated tightly binding fraction or plasma antithrombin. Analyses of N-linked carbohydrate side chains showed that the weakly binding fraction from CHO cells had a higher proportion of tetra-antennary and a lower proportion of biantennary oligosaccharides than the tightly binding fraction. We conclude that the recombinant antithrombin produced by the two cell lines is heterogeneously glycosylated and that the increased carbohydrate content of a large proporti6n of the molecules results in a substantial decrease in the affinity of these molecules for heparin. These findings are of particular relevance for studies aimed at characterizing the heparin-binding site of recombinant antithrombin by site-directed mutagenesis.

Lysine 114 of Antithrombin Is of Crucial Importance for the Affinity and Kinetics of Heparin Pentasaccharide Binding

“…Second order rate constants for inhibition of human ␣-thrombin or factor Xa by the N135A, K114A/N135A, N135Q, and K114M/N135Q variants in the absence and presence of pentasaccharide or full-length heparin were measured under pseudo-first order conditions, essentially as in earlier work (11,19,20,22,25,31,33). Uncatalyzed reactions with thrombin and factor Xa were analyzed at I 0.15 and 0.05, pH 7.4, and such reactions with factor Xa were also studied at I 0.025, pH 6.0.…”

“…Second order rate constants for uncatalyzed reactions were obtained by dividing k obs with the antithrombin concentration. Most such rate constants for pentasaccharide-or full-length heparincatalyzed reactions were derived from the least-squares slope of the linear dependence of k obs on the concentration of the antithrombinsaccharide complex, calculated from measured dissociation constants (11,22,31,33). Alternatively, the rate constants for some catalyzed reactions were calculated from k obs measured at a single saccharide concentration by first subtracting k obs for the uncatalyzed reaction and then dividing by the calculated concentration of the antithrombinsaccharide complex (22,31,33), with several such values averaged.…”

“…Most such rate constants for pentasaccharide-or full-length heparincatalyzed reactions were derived from the least-squares slope of the linear dependence of k obs on the concentration of the antithrombinsaccharide complex, calculated from measured dissociation constants (11,22,31,33). Alternatively, the rate constants for some catalyzed reactions were calculated from k obs measured at a single saccharide concentration by first subtracting k obs for the uncatalyzed reaction and then dividing by the calculated concentration of the antithrombinsaccharide complex (22,31,33), with several such values averaged. Uncatalyzed rate constants for thrombin inhibition by the N135A and K114A/N135A variants at I 0.025, pH 6.0, and I 0.05, pH 7.4, were obtained from the intercepts on the ordinate of the plots of k obs versus the concentration of the antithrombin-heparin complex from which also the heparin-catalyzed rate constants were derived.…”

Structural aspects of serpin inhibition

“…A heparin pentasaccharide interacts with serpins via four of its sulfate groups, probably at helix D in AT111 and HCII ( [25] ATIII, accelerates thrombin inhibition and promotes interaction with factor Xa, attributed to the induction of a conformational change in AT111 [32]. Heparin binding also depends on the degree of glycosylation of AT111 [33], and involves the N-terminus and other regions of the protein [34]. There is also evidence of heparin dependent domain rearrangement or unfolding [35].…”

Identification of the Antithrombin III Heparin Binding Site

“…Antithrombins expressed in BHK and CHO cell lines are heterogeneously glycosylated due to nonuniformity in host cell post-translational modification processes. This kind of glycosylation heterogeneity, in addition to ␣-ATIII/␤-ATIII glycosylation heterogeneity caused by the wild type Asn 135 NXS consensus sequence (31), causes the expressed antithrombin molecules to display a broad range of affinities for heparin (32,35,36). Consequently, it is problematic to determine the heparin affinities of ATIII variants produced in these expression systems, and they are not optimal for in vitro mutagenesis studies of the ATIII heparin binding site (35).…”