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Additional N-glycosylation in the N-terminal region of recombinant human alpha-1 antitrypsin enhances the circulatory half-life in Sprague-Dawley rats

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Abstract

Glycosylation affects the circulatory half-lives of therapeutic proteins. However, the effects of an additional N-glycosylation in the unstructured region or the loop region of alpha-1 antitrypsin (A1AT) on the circulatory half-life of the protein are largely unknown. In this study, we investigated the role of an additional N-glycosylation site (Q4N/D6T, Q9N, D12N/S14T, A70N, G148T, R178N, or V212N) to the three naturally occurring N-glycosylation sites in human A1AT. A single-dose (445 μg/kg) pharmacokinetic study using male Sprague-Dawley rats showed that, among the seven recombinant A1AT (rA1AT) mutants, Q9N and D12N/S14T showed the highest serum concentration and area under the curve values, as well as similar circulatory half-lives that were 2.2-fold higher than plasma-derived A1AT and 1.7-fold higher than wild-type rA1AT. We further characterized the Q9N mutant regarding the N-glycan profile, sialic acid content, protease inhibitory activity, and protein stability. The results indicate that an additional N-glycosylation in the flexible N-terminal region increases the circulatory half-life of rA1AT without altering its protease inhibitory activity. Our study provides novel insight into the use of rA1AT for the treatment of emphysema with an increased injection interval relative to the clinically used plasma-derived A1AT.

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Abbreviations

A1AT:

Alpha-1 antitrypsin

nA1AT:

Plasma-derived native A1AT

rA1AT:

Recombinant A1AT

Neu5Ac:

N-acetylneuraminic acid

Neu5Gc:

N-glycolylneuramic acid

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Authors and Affiliations

  1. Alteogen Inc., 62, Yuseong-daero 1628 beon-gil, Yuseong-gu, Daejeon, 34054, South Korea

    Hye-Shin Chung, Ji-Sun Kim, Sang Mee Lee & Soon Jae Park

  2. Department of Biological Sciences and Biotechnology, Hannam University, Daejeon, 34054, South Korea

    Hye-Shin Chung

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  1. Hye-Shin Chung
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  2. Ji-Sun Kim
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Correspondence to Soon Jae Park.

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Chung, HS., Kim, JS., Lee, S.M. et al. Additional N-glycosylation in the N-terminal region of recombinant human alpha-1 antitrypsin enhances the circulatory half-life in Sprague-Dawley rats. Glycoconj J 33, 201–208 (2016). https://doi.org/10.1007/s10719-016-9657-3

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  • DOI: https://doi.org/10.1007/s10719-016-9657-3

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