Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana
| Autor: | Vanesa Soledad Marin Viegas, Silvana Petruccelli, Herta Steinkellner, Silvina Mangano, Carolina Gabriela Ocampo, Jorge Fabricio Lareu, Andreas Loos |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0106 biological sciences
0301 basic medicine Glycosylation Biología Nicotiana benthamiana N-glycosylation molecular farming Plant Science Vacuole 01 natural sciences MOLECULAR FARMING purl.org/becyt/ford/1 [https] chemistry.chemical_compound N-linked glycosylation Research Articles biology N‐glycosylation Antibodies Monoclonal Química N-GLYCOSYLATION vacuolar transport Recombinant Proteins Cell biology vacuolar sorting signals Biochemistry symbols CIENCIAS NATURALES Y EXACTAS Research Article Biotechnology VACUOLAR TRANSPORT Glycan Otras Ciencias Biológicas Immunoglobulins Ciencias Biológicas 03 medical and health sciences symbols.namesake Polysaccharides Tobacco purl.org/becyt/ford/1.6 [https] Secretory pathway fungi SECRETORY PATHWAY Golgi apparatus biology.organism_classification secretory pathway VACUOLAR SORTING SIGNALS 030104 developmental biology chemistry Vacuolar transport Vacuoles IMMUNOGLOBULIN biology.protein immunoglobulin Agronomy and Crop Science 010606 plant biology & botany |
| Zdroj: | CONICET Digital (CONICET) Consejo Nacional de Investigaciones Científicas y Técnicas instacron:CONICET SEDICI (UNLP) Universidad Nacional de La Plata instacron:UNLP Plant Biotechnology Journal |
| DOI: | 10.1111/pbi.12580 |
| Popis: | Plant-based platforms are extensively used for the expression of recombinant proteins, including monoclonal antibodies. However, to harness the approach effectively and leverage it to its full potential, a better understanding of intracellular processes that affect protein properties is required. In this work, we examined vacuolar (vac) targeting and deposition of the monoclonal antibody (Ab) 14D9 in Nicotiana benthamiana leaves. Two distinct vacuolar targeting signals (KISIA and NIFRGF) were C-terminal fused to the heavy chain of 14D9 (vac-Abs) and compared with secreted and ER-retained variants (sec-Ab, ER-Ab, respectively). Accumulation of ER- and vac-Abs was 10- to 15-fold higher than sec-Ab. N-glycan profiling revealed the predominant presence of plant typical complex fucosylated and xylosylated GnGnXF structures on sec-Ab while vac-Abs carried mainly oligomannosidic (Man 7-9) next to GnGnXF forms. Paucimannosidic glycans (commonly assigned as typical vacuolar) were not detected. Confocal microscopy analysis using RFP fusions showed that sec-Ab-RFP localized in the apoplast while vac-Abs-RFP were exclusively detected in the central vacuole. The data suggest that vac-Abs reached the vacuole by two different pathways: direct transport from the ER bypassing the Golgi (Ab molecules containing Man structures) and trafficking through the Golgi (for Ab molecules containing complex N-glycans). Importantly, vac-Abs were correctly assembled and functionally active. Collectively, we show that the central vacuole is an appropriate compartment for the efficient production of Abs with appropriate post-translational modifications, but also point to a reconsideration of current concepts in plant glycan processing. Centro de Investigación y Desarrollo en Criotecnología de Alimentos |
| Databáze: | OpenAIRE |
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