title: N-glycosylation and biological activity of recombinant human alpha1-antitrypsin expressed in a novel human neuronal cell line.
abstract: Human alpha-1-antitrypsin (A1AT) is a protease inhibitor that is involved in the protection of lungs from neutrophil elastase enzyme that drastically modifies tissue functioning. The glycoprotein consists of 394 amino acids and is N-glycosylated at Asn-46, Asn-83, and Asn-247. A1AT deficiency is currently treated with A1AT that is purified from human serum. In view of therapeutic applications, rA1AT was produced using a novel human neuronal cell line (AGE1.HN®) and we investigated the N-glycosylation pattern as well as the in vitro anti-inflammatory activity of the recombinant glycoprotein. rA1AT (300 mg/L) was biologically active as analyzed using elastase assay. The N-glycan pool, released by PNGase F digestion, was characterized using 2D-HPLC, MALDI-TOF mass spectrometry, and by exoglycosidase digestions. A total of 28 N-glycan structures were identified, ranging from diantennary to tetraantennary complex-type N-glycans. Most of the N-glycans were found to be (α1-6) core-fucosylated and part of them contain the Lewis X epitope. The two major compounds are a monosialylated diantennary difucosylated glycan and a disialylated diantennary core-fucosylated glycan, representing 25% and 18% of the total N-glycan pool, respectively. Analysis of the site-specificity revealed that Asn-247 was mainly occupied by diantennary N-glycans whereas Asn-46 was occupied by di-, and triantennary N-glycans. Asn-83 was exclusively occupied by sialylated tri- and tetraantennary N-glycans. Next, we evaluated the anti-inflammatory activity of rA1AT using A1AT purified from human serum as a reference. rA1AT was found to inhibit the production of TNF-α in neutrophils and monocytes as commercial A1AT does.
authors.affiliation: Glycodesign and Glycoanalytics, Central Institute of Laboratory Medicine and Pathobiochemistry, Charité Medical University, Charité Platz 1, 10117 Berlin, Germany. veronique.blanchard@charite.de
authors.forename: Véronique
authors.initials: V
authors.lastname: Blanchard
authors.forename: Xi
authors.initials: X
authors.lastname: Liu
authors.forename: Susann
authors.initials: S
authors.lastname: Eigel
authors.forename: Matthias
authors.initials: M
authors.lastname: Kaup
authors.forename: Silke
authors.initials: S
authors.lastname: Rieck
authors.forename: Sabina
authors.initials: S
authors.lastname: Janciauskiene
authors.forename: Volker
authors.initials: V
authors.lastname: Sandig
authors.forename: Uwe
authors.initials: U
authors.lastname: Marx
authors.forename: Peter
authors.initials: P
authors.lastname: Walden
authors.forename: Rudolf
authors.initials: R
authors.lastname: Tauber
authors.forename: Markus
authors.initials: M
authors.lastname: Berger
mesh_terms: D000893:Anti-Inflammatory Agents / Q000378:metabolism / Q000494:pharmacology; D001709:Biotechnology / Q000379:methods*; D002460:Cell Line; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012794:Sialic Acids / Q000032:analysis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D000893:Anti-Inflammatory Agents; D011994:Recombinant Proteins; D012794:Sialic Acids; D014409:Tumor Necrosis Factor-alpha; D000515:alpha 1-Antitrypsin; D019272:Leukocyte Elastase