title: Production of homogeneous glycoprotein with multisite modifications by an engineeredN-glycosyltransferase mutant.
abstract: Naturally occurringN-glycoproteins exhibit glycoform heterogeneity with respect toN-glycan sequon occupancy (macroheterogeneity) and glycan structure (microheterogeneity). However, access to well-defined glycoproteins is always important for both basic research and therapeutic purposes. As a result, there has been a substantial effort to identify and understand the catalytic properties ofN-glycosyltransferases, enzymes that install the first glycan on the protein chain. In this study we found that ApNGT, a newly discovered cytoplasmicN-glycosyltransferase fromActinobacillus pleuropneumoniae, has strict selectivity toward the residues around the Asn ofN-glycosylation sequon by screening a small library of synthetic peptides. The inherent stringency was subsequently demonstrated to be closely associated with a critical residue (Gln-469) of ApNGT which we propose hinders the access of bulky residues surrounding the occupied Asn into the active site. Site-saturated mutagenesis revealed that the introduction of small hydrophobic residues at the site cannot only weaken the stringency of ApNGT but can also contribute to enormous improvement of glycosylation efficiency against both short peptides and proteins. We then employed the most efficient mutant (Q469A) other than the wild-type ApNGT to produce a homogeneous glycoprotein carrying multiple (up to 10)N-glycans, demonstrating that this construct is a promising biocatalyst for potentially addressing the issue of macroheterogeneity in glycoprotein preparation.
authors.affiliation: From the State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, China and.
authors.forename: Qitao
authors.initials: Q
authors.lastname: Song
authors.affiliation: the Department of Chemistry, Georgia State University, Atlanta, Georgia 30303.
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Wu
authors.affiliation: From the State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, China and.
authors.forename: Yueyuan
authors.initials: Y
authors.lastname: Fan
authors.affiliation: From the State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, China and.
authors.forename: Woran
authors.initials: W
authors.lastname: Song
authors.affiliation: From the State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, China and.
authors.forename: Peiru
authors.initials: P
authors.lastname: Zhang
authors.affiliation: From the State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, China and.
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.affiliation: From the State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, China and.
authors.forename: Faxing
authors.initials: F
authors.lastname: Wang
authors.affiliation: From the State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, China and.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: From the State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, China and pwang@nankai.edu.cn.
authors.forename: Peng G
authors.initials: PG
authors.lastname: Wang
authors.affiliation: From the State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin 300353, China and jiansongcheng@nankai.edu.cn.
authors.forename: Jiansong
authors.initials: J
authors.lastname: Cheng
mesh_terms: D000188:Actinobacillus / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense
chemical_list: D001426:Bacterial Proteins; D006023:Glycoproteins; D016695:Glycosyltransferases
keywords: N-linked glycosylation; glycoprotein; glycosyltransferase; post-translational modification (PTM); protein engineering