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N‐glycosylation and biological activity of recombinant human alpha1‐antitrypsin expressed in a novel human neuronal cell line
Author(s) -
Blanchard Véronique,
Liu Xi,
Eigel Susann,
Kaup Matthias,
Rieck Silke,
Janciauskiene Sabina,
Sandig Volker,
Marx Uwe,
Walden Peter,
Tauber Rudolf,
Berger Markus
Publication year - 2011
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.23158
Subject(s) - glycan , pngase f , exoglycosidase , glycosylation , glycoprotein , biochemistry , endoglycosidase , chemistry , recombinant dna , epitope , fucosylation , microbiology and biotechnology , n linked glycosylation , biology , antibody , gene , immunology
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. Biotechnol. Bioeng. 2011;108:2118–2128. © 2011 Wiley Periodicals, Inc.