N-glycan analysis of human α1-antitrypsin produced in Chinese hamster ovary cells.
Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1 - 6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2 - 3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2 - 6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.
Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 305-806, Korea.
Kyung Jin
KJ
Lee
Sang Mee
SM
Lee
Jin Young
JY
Gil
Ohsuk
O
Kwon
Jin Young
JY
Kim
Soon Jae
SJ
Park
Hye-Shin
HS
Chung
Doo-Byoung
DB
Oh
D000818:Animals; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003412:Cricetulus; D057075:Enzyme Assays; D006031:Glycosylation; D006207:Half-Life; D006801:Humans; D007700:Kinetics; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D013552:Swine; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D011134:Polysaccharides; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D019272:Leukocyte Elastase