Title : Effect of
individual carbohydrate chains of recombinant
antithrombin on
heparin affinity and on the generation of glycoforms differing in
heparin affinity
Abstract :
- Two major glycoforms of recombinant antithrombin which differ 10-fold in their affinity for the effector glycosaminoglycan, heparin , were previously shown to be expressed in BHK or CHO mammalian cell lines (I. Björk, et al., 1992, Biochem
- J. 286, 793-800; B. Fan et al., 1993, J. Biol
- Chem
- 268, 17588-17596)
- To determine the source of the glycosylation heterogeneity responsible for these different heparin-affinity forms, each of the four Asn residue sites of glycosylation, residues 96, 135, 155, and 192 , was mutated to Gln to block glycosylation at these sites
- Heparin-agarose chromatography of the four antithrombin variants revealed that Gln 96, Gln 135, and Gln 192 variants still displayed the two functional heparin-affinity forms previously observed with the wild-type inhibitor, whereas the Gln 155 variant showed only a single functional high heparin affinity form
- These results demonstrate that heterogeneous glycosylation of Asn 155 of recombinant antithrombin is responsible for generating the low heparin affinity glycoform
- Analysis of heparin binding to the higher heparin affinity forms of the four 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 largest 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 antithrombin
- All variants displayed normal kinetics of thrombin inhibition in the absence and presence of saturating heparin , indicating that the carbohydrate chains solely affected heparin binding and not heparin-activation or proteinase-binding functions
- These results indicate that all carbohydrate chains of recombinant antithrombin adversely affect heparin-binding affinity to an extent that correlates with their relative proximity to the putative heparin-binding site in antithrombin