title: Development and analysis of alpha 1-antitrypsin neoglycoproteins: the impact of additional N-glycosylation sites on serum half-life.
abstract: Therapeutic efficacy of glycoproteins is affected by many factors, including molecular size and net charge; both are influenced by the presence and composition of glycan structures. Human alpha 1-antitrypsin (A1AT) was cloned and expressed in human embryonic kidney cells (HEK293) that are capable of mammalian glycosylation. Utilizing PCR-based site-directed mutagenesis, new A1AT variants were created with single, double, or triple additional N-glycosylation sites to the three naturally occurring N-glycosylation sites. Because of the supplementary N-glycans, the A1AT variants exhibited an increased molecular weight. Retention of inhibitory activity was shown via trypsin inhibitory assay. The A1AT variants were treated with PNGase F, and the resulting N-glycans were analyzed by MALDI-TOF mass spectrometry. The N-glycan profile of the recombinant A1AT variants was mostly composed of monofucosylated bi-, tri-, and tetraantennary complex-type N-glycans, with a tendency toward higher antennary structures compared to the wild-type. The relevance of N-glycosylation in A1AT for the circulatory serum half-life was demonstrated in CD1 mice. The A1AT neoglycoprotein with an additional N-glycosylation site at position N123 exhibited a 62% increase in serum half-life. Additionally, using a two-compartment model, the A1AT variants exhibited increased α-phase values, especially N123 (223%) and N201 (255%). The results suggest the recombinant A1AT neoglycoprotein as a serious alternative to A1AT derived from human plasma.
authors.affiliation: Institute of Laboratory Medicine, Clinical Chemistry and Pathobiochemistry, Berlin, Germany.
authors.forename: Astrid
authors.initials: A
authors.lastname: Lusch
authors.forename: Matthias
authors.initials: M
authors.lastname: Kaup
authors.forename: Uwe
authors.initials: U
authors.lastname: Marx
authors.forename: Rudolf
authors.initials: R
authors.lastname: Tauber
authors.forename: Véronique
authors.initials: V
authors.lastname: Blanchard
authors.forename: Markus
authors.initials: M
authors.lastname: Berger
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006023:Glycoproteins / Q000097:blood* / Q000737:chemistry* / Q000493:pharmacokinetics; D006031:Glycosylation; D006801:Humans; D051379:Mice; D011134:Polysaccharides / Q000097:blood / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000515:alpha 1-Antitrypsin / Q000097:blood* / Q000737:chemistry* / Q000493:pharmacokinetics
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides; D000515:alpha 1-Antitrypsin