HETEROGENEITY OF RECOMBINANT HUMAN ANTITHROMBIN-III EXPRESSED IN BABYHAMSTER-KIDNEY CELLS - EFFECT OF GLYCOSYLATION DIFFERENCES ON HEPARIN-BINDING AND STRUCTURE
Bq. Fan et al., HETEROGENEITY OF RECOMBINANT HUMAN ANTITHROMBIN-III EXPRESSED IN BABYHAMSTER-KIDNEY CELLS - EFFECT OF GLYCOSYLATION DIFFERENCES ON HEPARIN-BINDING AND STRUCTURE, The Journal of biological chemistry, 268(23), 1993, pp. 17588-17596
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.