EFFECT OF INDIVIDUAL CARBOHYDRATE CHAINS OF RECOMBINANT ANTITHROMBIN ON HEPARIN AFFINITY AND ON THE GENERATION OF GLYCOFORMS DIFFERING IN HEPARIN AFFINITY

Two major glycoforms of recombinant antithrombin which differ 10-fold in their affinity for the effector glycosaminoglycan, heparin, were pr eviously shown to be expressed in BHK or CHO mammalian cell lines (I. Bjork, et al., 1992, Biochem. J. 286, 793-800; B. Fan ct al., 1993, J. Biol. Chem. 268, 17588-17596). To determine the source of the glycosy lation heterogeneity responsible for these different heparin-affinity forms, each of the four Asn residue sites of glycosylation, residues 9 6, 135, 155, and 192, was mutated to Gln to block glycosylation at the se sites. Heparin-agarose chromatography of the four antithrombin vari ants revealed that Gln 96, Gln 135, and Gln 192 variants still display ed the two functional heparin-affinity forms previously observed with the wild-type inhibitor, whereas the Gin 155 variant showed only a sin gle functional high heparin affinity form. These results demonstrate t hat heterogeneous glycosylation of Asn 155 of recombinant antithrombin is responsible for generating the low heparin affinity glycoform. Ana lysis of heparin binding to the higher heparin affinity forms of the f our variants showed that all exhibited increased heparin affinities of two- to sevenfold compared to wild-type higher heparin affinity form or to plasma antithrombin, with the Gln 135 variant showing the larges t effect on this affinity. The extent of heparin-affinity enhancement was correlated with the distance of the mutated glycosylation site to the putative heparin-binding site in the X-ray structure of antithromb in. All variants displayed normal kinetics of thrombin inhibition in t he absence and presence of saturating heparin, indicating that the car bohydrate chains solely affected heparin binding and not heparin-activ ation or proteinase-binding functions. These results indicate that all carbohydrate chains of recombinant antithrombin adversely affect hepa rin-binding affinity to an extent that correlates with their relative proximity to the putative heparin-binding site in antithrombin. (C) 19 97 Academic Press.