HETEROGENEITY OF RECOMBINANT HUMAN ANTITHROMBIN-III EXPRESSED IN BABYHAMSTER-KIDNEY CELLS - EFFECT OF GLYCOSYLATION DIFFERENCES ON HEPARIN-BINDING AND STRUCTURE

To determine the effects of differences in glycosylation on the struct ure and functional properties of recombinant human antithrombin (rHAT) , we have characterized the properties of the recombinant protein over expressed by baby hamster kidney cells. Three forms of rHAT, I-III, we re isolated which differed in affinity for heparin. Form I had the low est affinity and contained a high proportion of highly branched comple x carbohydrate. Form II had higher affinity and contained both complex and high mannose-type chains. Form III had the highest affinity and w as similar to form II in the type of carbohydrate present, but had a l ower level of glycosylation, consistent with the absence of carbohydra te at one of the four glycosylation sites. H-1 NMR spectra of plasma H AT and rHAT forms I-III suggested very similar protein structures for all forms. Heparin pentasaccharide produced almost identical NMR pertu rbation difference spectra. The only functional difference found was i n the rates of inactivation of factor Xa. Forms II and III gave second order rate constants similar to that of plasma HAT, whereas form I ga ve a biphasic inhibition, with the first phase having a rate about fou r times that of the other forms. We conclude that carbohydrate heterog eneity does not alter the structure of the HAT polypeptide or the hepa rin-induced conformational change, but does affect the heparin affinit y and can alter the rate of proteinase inhibition.