期刊
MOLECULAR PHARMACEUTICS
卷 10, 期 7, 页码 2616-2629出版社
AMER CHEMICAL SOC
DOI: 10.1021/mp400043r
关键词
alpha 1-antitrypsin; clearance; mass spectrometry; N-glycan; serum half-life
资金
- BMBF within the program InnoProfile [03IP5111]
- Sonnenfeld Foundation
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 a-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.
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