Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1 - 6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2 - 3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2 - 6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.

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Peptide:N-glycanase (PNGase) A is used preferentially to cleave the glycans from plant and insect glycopeptides. Although many putative PNGase A homologous genes have been found in the plant and fungus kingdoms through sequence similarity analyses, only several PNGases from plants and one from a filamentous fungus have been characterized. In this study, we identified and characterized a PNGase A-like enzyme, PNGase Yl, in the dimorphic yeast Yarrowia lipolytica. The corresponding gene was cloned and recombinantly expressed in Pichia pastoris. The purified enzyme cleaved glycans from glycopeptides with the maximum activity at pH 5. No metal ions were required for full activity, and rather it was repressed by three metal ions (Fe(3+), Cu(2+) and Zn(2+)). Using glycopeptide substrates, PNGase Yl was shown to release various types of N-glycans including high-mannose and complex-type glycans as well as glycans containing core-linked α(1,3)-fucose that are frequently found in plants and insects. Moreover, in comparison with PNGase A, PNGase Yl was able to cleave with higher efficiency the glycans from some denatured glycoproteins. Taken together, our results suggest that PNGase Yl, the first biochemically characterized yeast PNGase A homologue, can be developed through protein engineering as a useful deglycosylation tool for N-glycosylation study.