Heterogeneity of recombinant human antithrombin III expressed in baby hamster kidney cells. Effect of glycosylation differences on heparin binding and structure.
To determine the effects of differences in glycosylation on the structure and functional properties of recombinant human antithrombin (rHAT), we have characterized the properties of the recombinant protein overexpressed by baby hamster kidney cells. Three forms of rHAT, I-III, were isolated which differed in affinity for heparin. Form I had the lowest affinity and contained a high proportion of highly branched complex carbohydrate. Form II had higher affinity and contained both complex and high mannose-type chains. Form III had the highest affinity and was similar to form II in the type of carbohydrate present, but had a lower level of glycosylation, consistent with the absence of carbohydrate at one of the four glycosylation sites. 1H NMR spectra of plasma HAT and rHAT forms I-III suggested very similar protein structures for all forms. Heparin pentasaccharide produced almost identical NMR perturbation difference spectra. The only functional difference found was in 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 gave a biphasic inhibition, with the first phase having a rate about four times that of the other forms. We conclude that carbohydrate heterogeneity does not alter the structure of the HAT polypeptide or the heparin-induced conformational change, but does affect the heparin affinity and can alter the rate of proteinase inhibition.
Fan
B
B
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Crews
B C
BC
Turko
I V
IV
Choay
J
J
Zettlmeissl
G
G
Gettins
P
P
D000818:Animals; D000990:Antithrombin III / Q000737:chemistry / Q000378:metabolism*; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D065427:Factor Xa Inhibitors; D006031:Glycosylation; D006493:Heparin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D002241:Carbohydrates; D065427:Factor Xa Inhibitors; D011994:Recombinant Proteins; D000990:Antithrombin III; D006493:Heparin