[PMID]: | 28452462 |
[Au] Autor: | Yang Q; An Y; Zhu S; Zhang R; Loke CM; Cipollo JF; Wang LX |
[Ad] Endereço: | Department of Chemistry and Biochemistry, University of Maryland , College Park, Maryland 20742, United States. |
[Ti] Título: | Glycan Remodeling of Human Erythropoietin (EPO) Through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation. |
[So] Source: | ACS Chem Biol;12(6):1665-1673, 2017 06 16. |
[Is] ISSN: | 1554-8937 |
[Cp] País de publicação: | United States |
[La] Idioma: | eng |
[Ab] Resumo: | The tremendous structural heterogeneity of N-glycosylation of glycoproteins poses a great challenge for deciphering the biological functions of specific glycoforms and for developing protein-based therapeutics. We have previously reported a chemoenzymatic glycan remodeling method for producing homogeneous glycoforms of N-glycoproteins including intact antibodies, which consist of endoglycosidase-catalyzed deglycosylation and novel glycosynthase-catalyzed transglycosylation, but its application to complex glycoproteins carrying multiple N-glycans remains to be examined. We report here site-selective chemoenzymatic glycosylation remodeling of recombinant human erythropoietin (EPO) that contains three N-glycans. We found that the generation of a HEK293S GnT I knockout FUT8 overexpressing cell line enabled the production of an unusual Man GlcNAc Fuc glycoform, which could be converted to the core-fucosylated GlcNAc-EPO intermediate acceptor for enzymatic transglycosylation. With this acceptor, homogeneous sialylated glycoform or azide-tagged glycoform were produced using the glycosynthase (EndoF3-D165A) catalyzed transglycosylation. Interestingly, a remarkable site-selectivity was observed in the transglycosylation reactions, leading to the introduction of two N-glycans selectively at the Asn-38 and Asn-83 sites, which was confirmed by a detailed MS/MS analysis of the transglycosylation product. Finally, a different N-glycan was attached at the third (Asn-24) site by pushing the enzymatic transglycosylation with a distinct glycan oxazoline, achieving the site-selective glycosylation modification of the protein. This study represents the first example of site-selective chemoenzymatic glycan engineering of complex glycoproteins carrying multiple N-glycans. |
[Mh] Termos MeSH primário: |
Engenharia Celular/métodos Eritropoetina/metabolismo Polissacarídeos/metabolismo Engenharia de Proteínas/métodos
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[Mh] Termos MeSH secundário: |
Asparagina/metabolismo Glicosilação Células HEK293 Seres Humanos Espectrometria de Massas em Tandem
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[Pt] Tipo de publicação: | JOURNAL ARTICLE; RESEARCH SUPPORT, N.I.H., EXTRAMURAL |
[Nm] Nome de substância:
| 0 (Polysaccharides); 11096-26-7 (Erythropoietin); 7006-34-0 (Asparagine) |
[Em] Mês de entrada: | 1709 |
[Cu] Atualização por classe: | 180201 |
[Lr] Data última revisão:
| 180201 |
[Sb] Subgrupo de revista: | IM |
[Da] Data de entrada para processamento: | 170429 |
[St] Status: | MEDLINE |
[do] DOI: | 10.1021/acschembio.7b00282 |
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