{"doi":"10.1111/pbi.12602","title":"Reduced paucimannosidic <i>N</i>‐glycan formation by suppression of a specific β‐hexosaminidase from <i>Nicotiana benthamiana</i>","abstract":"<jats:sec><jats:title>Summary</jats:title><jats:p>Plants are attractive hosts for the production of recombinant glycoproteins for therapeutic use. Recent advances in glyco‐engineering facilitate the elimination of nonmammalian‐type glycosylation and introduction of missing pathways for customized <jats:italic>N</jats:italic>‐glycan formation. However, some therapeutically relevant recombinant glycoproteins exhibit unwanted truncated (paucimannosidic) <jats:italic>N</jats:italic>‐glycans that lack Glc<jats:styled-content style=\"fixed-case\">NA</jats:styled-content>c residues at the nonreducing terminal end. These paucimannosidic <jats:italic>N</jats:italic>‐glycans increase product heterogeneity and may affect the biological function of the recombinant drugs. Here, we identified two enzymes, β‐hexosaminidases (<jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>s) that account for the formation of paucimannosidic <jats:italic>N</jats:italic>‐glycans in <jats:italic>Nicotiana benthamiana</jats:italic>, a widely used expression host for recombinant proteins. Subcellular localization studies showed that <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>1 is a vacuolar protein and <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>3 is mainly located at the plasma membrane in <jats:italic>N. benthamiana</jats:italic> leaf epidermal cells. Both enzymes are functional and can complement the corresponding <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>‐deficient <jats:italic>Arabidopsis thaliana</jats:italic> mutants. <jats:italic>In planta</jats:italic> expression of <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>3 demonstrated that core α1,3‐fucose enhances the trimming of Glc<jats:styled-content style=\"fixed-case\">NA</jats:styled-content>c residues from the Fc domain of human IgG. Finally, using <jats:styled-content style=\"fixed-case\">RNA</jats:styled-content> interference, we show that suppression of <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>3 expression can be applied to increase the amounts of complex <jats:italic>N</jats:italic>‐glycans on plant‐produced human α1‐antitrypsin.</jats:p></jats:sec>","journal":"Plant Biotechnology Journal","year":2017,"id":598741,"datarank":0.6090664515819629,"base_score":4.060443010546419,"endowment":4.060443010546419,"self_citation_contribution":0.6090664515819629,"citation_network_contribution":0.0,"self_endowment_contribution":0.6090664515819629,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":57,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":null,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":null,"fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1534461,"name":"Alexandra Castilho","orcid":null,"position":1,"is_corresponding":false},{"id":718642,"name":"Martina Dicker","orcid":null,"position":2,"is_corresponding":false},{"id":1534464,"name":"Flavio Sádio","orcid":null,"position":3,"is_corresponding":false},{"id":1534466,"name":"Ulrike Vavra","orcid":null,"position":4,"is_corresponding":false},{"id":405354,"name":"Clemens Grünwald‐Gruber","orcid":"0000-0002-6097-8348","position":5,"is_corresponding":false},{"id":1534468,"name":"Tae‐Ho Kwon","orcid":null,"position":6,"is_corresponding":false},{"id":1228290,"name":"Friedrich Altmann","orcid":"0000-0002-0112-7877","position":7,"is_corresponding":false},{"id":934701,"name":"Herta Steinkellner","orcid":"0000-0003-4823-1505","position":8,"is_corresponding":false},{"id":1119133,"name":"Richard Strasser","orcid":"0000-0001-8764-6530","position":9,"is_corresponding":false},{"id":1534459,"name":"Yun‐Ji Shin","orcid":null,"position":0,"is_corresponding":false}],"reference_count":0,"raw_metadata":{"has_enrichment":true,"resolved":true,"title":"Reduced paucimannosidic <i>N</i>‐glycan formation by suppression of a specific β‐hexosaminidase from <i>Nicotiana benthamiana</i>","abstract":"<jats:sec><jats:title>Summary</jats:title><jats:p>Plants are attractive hosts for the production of recombinant glycoproteins for therapeutic use. Recent advances in glyco‐engineering facilitate the elimination of nonmammalian‐type glycosylation and introduction of missing pathways for customized <jats:italic>N</jats:italic>‐glycan formation. However, some therapeutically relevant recombinant glycoproteins exhibit unwanted truncated (paucimannosidic) <jats:italic>N</jats:italic>‐glycans that lack Glc<jats:styled-content style=\"fixed-case\">NA</jats:styled-content>c residues at the nonreducing terminal end. These paucimannosidic <jats:italic>N</jats:italic>‐glycans increase product heterogeneity and may affect the biological function of the recombinant drugs. Here, we identified two enzymes, β‐hexosaminidases (<jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>s) that account for the formation of paucimannosidic <jats:italic>N</jats:italic>‐glycans in <jats:italic>Nicotiana benthamiana</jats:italic>, a widely used expression host for recombinant proteins. Subcellular localization studies showed that <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>1 is a vacuolar protein and <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>3 is mainly located at the plasma membrane in <jats:italic>N. benthamiana</jats:italic> leaf epidermal cells. Both enzymes are functional and can complement the corresponding <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>‐deficient <jats:italic>Arabidopsis thaliana</jats:italic> mutants. <jats:italic>In planta</jats:italic> expression of <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>3 demonstrated that core α1,3‐fucose enhances the trimming of Glc<jats:styled-content style=\"fixed-case\">NA</jats:styled-content>c residues from the Fc domain of human IgG. Finally, using <jats:styled-content style=\"fixed-case\">RNA</jats:styled-content> interference, we show that suppression of <jats:styled-content style=\"fixed-case\">HEXO</jats:styled-content>3 expression can be applied to increase the amounts of complex <jats:italic>N</jats:italic>‐glycans on plant‐produced human α1‐antitrypsin.</jats:p></jats:sec>","is_dataset_classified":null,"base_score":4.060443010546419,"endowment":4.060443010546419,"datacite_reuse_total":0,"file_count":0,"downloads":0,"views":0,"has_version_chain":false,"is_dataset":false,"is_oa":false,"pmid":"27421111","pmcid":"PMC5259580","openalex_id":"https://openalex.org/W2464790038","authors":[],"funders":[{"funder_name":"Austrian Science Fund","grant_id":"TRP 242‐B20","title":null},{"funder_name":"Österreichische Forschungsförderungsgesellschaft","grant_id":"822757","title":null},{"funder_name":"Austrian Science Fund FWF","grant_id":"TRP 242","title":"Production of O-glycan modified biopharmaceuticals in plants"},{"funder_name":"Bundesministerium für Verkehr, Innovation und Technologie","grant_id":"","title":null}],"total_grants":4,"fwci":5.0087,"citation_percentile":0.94814415,"influential_citations":0,"citation_trend":[{"year":2017,"count":5},{"year":2018,"count":6},{"year":2019,"count":5},{"year":2020,"count":4},{"year":2021,"count":11},{"year":2022,"count":8},{"year":2023,"count":6},{"year":2024,"count":7},{"year":2025,"count":5}],"oa_status":"gold","license":"cc-by","oa_locations":[{"url":"https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/pbi.12602","host_type":"journal"},{"url":"https://onlinelibrary.wiley.com/doi/pdfdirect/10.1111/pbi.12602","host_type":"publisher"},{"url":"https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fpbi.12602","host_type":"publisher"},{"url":"https://onlinelibrary.wiley.com/doi/pdf/10.1111/pbi.12602","host_type":"publisher"},{"url":"https://doi.org/10.1111/pbi.12602","host_type":"journal"},{"url":"https://pubmed.ncbi.nlm.nih.gov/27421111","host_type":"repository"},{"url":"https://www.ncbi.nlm.nih.gov/pmc/articles/5259580","host_type":"repository"},{"url":"https://europepmc.org/articles/PMC5259580","host_type":"Europe_PMC"},{"url":"https://europepmc.org/articles/PMC5259580?pdf=render","host_type":"Europe_PMC"},{"url":"http://dx.doi.org/10.1111/pbi.12602","host_type":""},{"url":"https://dx.doi.org/10.1111/pbi.12602","host_type":""}],"fields_of_study":["Transgenic Plants and Applications","Glycosylation and Glycoproteins Research","Plant Virus Research Studies","0301 basic medicine","0303 health sciences","03 medical and health sciences","Arabidopsis","Base Sequence","Cell Membrane","Genes, Plant","Glycosylation","Plant Leaves","Plant Proteins","Plants, Genetically Modified","Polysaccharides","Recombinant Proteins","Nicotiana","Vacuoles","beta-N-Acetylhexosaminidases"],"mesh_terms":["Base Sequence","beta-N-Acetylhexosaminidases","Cell Membrane","Glycosylation","Plant Proteins","Polysaccharides","Recombinant Proteins","Nicotiana","Vacuoles","Genes, Plant","Arabidopsis","Plant Leaves","Plants, Genetically Modified"],"keywords":["Nicotiana benthamiana","Biology","Recombinant DNA","Glycosylation","Glycan","Glycoprotein","Biochemistry","Arabidopsis thaliana","Cell biology","N-linked glycosylation","Mutant","Gene","N-Glycosylation","Α1-antitrypsin","Glyco-engineering","Plant-made Pharmaceuticals","Nicotiana","Base Sequence","Cell Membrane","Arabidopsis","Genes, Plant","Plants, Genetically Modified","Recombinant Proteins","beta-N-Acetylhexosaminidases","Plant Leaves","Polysaccharides","Vacuoles","Research Articles","Plant Proteins"],"sdg_mappings":[{"sdg_number":2,"sdg_label":"2. 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