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
Output (sent_index, trigger,
protein,
sugar,
site):
- 0. antithrombin, , antithrombin, individual carbohydrate chains, -
- 1. glycoforms, , antithrombin, -, -
- 11. antithrombin, , antithrombin, all carbohydrate chains, -
- 5. glycosylation, , -, -, Asn residue sites
- 5. glycosylation, , -, -, sites
- 6. variants, , antithrombin variants, Heparin-agarose chromatography, -
- 7. glycoform, , heparin, -, -
- 7. glycosylation, , -, -, Asn 155
- 9. glycosylation, , -, -, site
Output(Part-Of) (sent_index,
protein,
site):
- 11. antithrombin, site
- 11. heparin, site
- 7. antithrombin, Asn 155
- 9. X-ray structure of antithrombin, site
- 9. heparin, site
*Output_Site_Fusion* (sent_index,
protein,
sugar,
site):
- 7. antithrombin, -, Asn 155