Structural characterization of recombinant alpha-1-antitrypsin expressed in a human cell line

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• 作者：Zihao Wang ; ^{Zihao.Wang@grifols.com} ; Thomas L. Hilder ; Koen van der Drift ; Jessica Sloan ; Kevin Wee
• 关键词：Alpha-1-antitrypsin ; Recombinant ; Liquid chromatography ; Mass spectrometry ; Glycosylation ; Sialylation
• 刊名：Analytical Biochemistry
• 出版年：2013
• 出版时间：1 June, 2013
• 年：2013
• 卷：437
• 期：1
• 页码：20-28
• 全文大小：973 K

文摘

Human alpha-1-antitrypsin (A1PI) is a plasma protein with the function of protecting lung tissues from proteolytic destruction by enzymes from inflammatory cells. A1PI deficiency is an inherited disorder associated with pulmonary emphysema and a higher risk of chronic obstructive pulmonary disease (COPD). Here we present the structural characterization of a recombinant form of human A1PI (Hu-recA1PI) expressed in the human PER.C6 cell line using an array of analytical and biochemical techniques. Hu-recA1PI had the same primary structure as plasma-derived A1PI (pd-A1PI) except reduced N-terminal heterogeneity. The secondary and tertiary structures were indistinguishable from pd-A1PI. Like pd-A1PI, Hu-recA1PI was modified by N-linked glycosylation on N46, N83, and N246. Unlike pd-A1PI, most glycans on recA1P1 were core fucosylated via ¦Á(1-6) linkage. In addition, significantly higher amounts of tri- and tetraantennary glycans were observed. These differences in glycosylation contributed to the apparent higher molecular weight and lower isoelectric point (pI) of Hu-recA1PI than pd-A1PI. Hu-recA1PI contained both ¦Á(2-3)- and ¦Á(2-6)-linked sialic acids and had very similar sialylation levels as pd-A1PI. Hu-recA1PI glycans were differentially distributed, with N46 containing mostly biantennary glycans, N83 containing primarily tri- and tetraantennary glycans, and N247 containing exclusively biantennary glycans.