Purification and characterization of recombinant human antithrombin containing the prelatent form in Chinese hamster ovary cells.
Antithrombin (AT) is a serine proteinase inhibitor and a major regulator of the blood coagulation cascade. AT in human plasma has two isoforms, a predominant alpha-isoform and a minor beta-isoform; the latter lacks N-glycosylation at Asn 135 and has a higher heparin affinity. From the difference in its folding states, the AT molecule can be separated into three forms: a native form, a denatured and inactive form known as the latent form, and a partially denatured form called the prelatent form. In this study, we purified and characterized recombinant human AT (rAT) containing the prelatent form produced by Chinese hamster ovary (CHO) cells. When rAT was purified at physiological pH, its specific activity was lower than that of plasma-derived human AT (pAT). The latent and prelatent forms were detected in rAT by using hydrophobic interaction chromatography analysis. However, when rAT was purified at alkaline pH, the prelatent form was reversibly folded to the native form and the inhibitory activity of rAT increased to a value similar to that of pAT. Highly purified rAT was analyzed and compared with pAT by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, amino acid composition, N-terminal sequence, monosaccharide composition, peptide mapping, and heparin-binding affinity. From these analyses, rAT was found to be structurally identical to pAT, except for carbohydrate side-chains. rAT in CHO cells had a high beta-isoform content and it caused a higher heparin affinity than by pAT and also pH-dependent reversible inhibitory activity.
Mochizuki
Shinobu
S
Protein Research Laboratory, Pharmaceuticals Research Unit, Research and Development Division, Mitsubishi Pharma Corporation, 2-25-1 Shodai-ohtani, Hirakata, Osaka 573-1153, Japan. Mochizuki.Shinobu@mb.m-pharma.co.jp
Miyano
Kenmi
K
Kondo
Masahide
M
Hirose
Masaaki
M
Masaki
Atsushi
A
Ohi
Hideyuki
H
D000818:Animals; D000991:Antithrombins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D016466:CHO Cells; D002241:Carbohydrates / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D006493:Heparin / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
D000991:Antithrombins; D002241:Carbohydrates; D011994:Recombinant Proteins; D006493:Heparin