title: Structural characterization of recombinant alpha-1-antitrypsin expressed in a human cell line.
abstract: 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 α(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 α(2-3)- and α(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.
authors: Department of BioAnalytics, Grifols Therapeutics, Inc Raleigh, NC 27606, USA. Zihao.Wang@grifols.com
authors: Zihao
authors: Z
authors: Wang
authors: Thomas L
authors: TL
authors: Hilder
authors: Koen
authors: K
authors: van der Drift
authors: Jessica
authors: J
authors: Sloan
authors: Kevin
authors: K
authors: Wee
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002460:Cell Line; D005643:Fucose / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012160:Retina / Q000166:cytology; D013379:Substrate Specificity; D000515:alpha 1-Antitrypsin / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D005643:Fucose; D019158:N-Acetylneuraminic Acid; D005690:Galactose