Fluorescent protein fusions to a human immunodeficiency virus monoclonal antibody reveal its intracellular transport through the plant endomembrane system

Irons, Sarah L.; Nuttall, James; Floß, Doreen M.; Frigerio, Lorenzo; Kotzer, Amanda M.; Hawes, Chris

doi:10.1111/j.1467-7652.2008.00348.x

Cited by 7 publications

(8 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In plants, little is known about the impact of FP fusions. Fusions of monomeric RFP and yellow Venus fluorescent protein to mAb 2G12 were not able to reach the apoplast but being retained in prevacuolar compartments (Irons et al ., ). At difference of such observations, we showed that the mAb14D9‐RFP fusions sorted to the assigned final destinations.…”

Section: Discussionmentioning

confidence: 97%

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Ocampo

Lareu

Viegas

et al. 2016

Plant Biotechnology Journal

View full text Add to dashboard Cite

SummaryPlant‐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.

show abstract

Section: Discussionmentioning

confidence: 97%

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Ocampo

Lareu

Viegas

et al. 2016

Plant Biotechnology Journal

View full text Add to dashboard Cite

show abstract

“…Subcellular localization of antibodies in plants have been studied in a few cases and with various materials (De Wilde et al 1996;During et al 1990), making it difficult to draw a general conclusion. Using reporter fluorescent proteins fused to the light and heavy chains, Irons et al (2008) showed that an antibody transiently expressed in tobacco leaf epidermal cells is located in punctuate structures in close association with the ER and partly overlapping with a pre-vacuolar compartment marker. Whether these punctuate structures correspond to those observed with LO-BM2 expressed in tobacco plants will require a more detailed investigation using various markers of the secretory pathway.…”

Section: Discussionmentioning

confidence: 99%

Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells

et al. 2009

View full text Add to dashboard Cite

Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment.

show abstract

“…In addition, the use of a broad acting cocktail of protease inhibitors at high concentration had little effect on the IgG fragment pattern for MAb 2G12. These findings suggest that degradation of MAb 2G12 is not related to the process of extraction, more likely, degradation occurs before extraction, and either along the secretory pathway, in the apoplastic space, or in subcellular storage compartments [ 28 ]. These observations support the findings of Hassan et al [ 29 ] who showed little benefit from using protease inhibitors during the extraction of a murine IgG 1 κ at neutral pH to prevent the loss of functional MAb.…”

Section: Discussionmentioning

confidence: 99%

Antibody degradation in tobacco plants: a predominantly apoplastic process

et al. 2011

View full text Add to dashboard Cite

BackgroundInterest in using plants for production of recombinant proteins such as monoclonal antibodies is growing, but proteolytic degradation, leading to a loss of functionality and complications in downstream purification, is still a serious problem.ResultsIn this study, we investigated the dynamics of the assembly and breakdown of a human IgG1κ antibody expressed in plants. Initial studies in a human IgG transgenic plant line suggested that IgG fragments were present prior to extraction. Indeed, when the proteolytic activity of non-transgenic Nicotiana tabacum leaf extracts was tested against a human IgG1 substrate, little activity was detectable in extraction buffers with pH > 5. Significant degradation was only observed when the plant extract was buffered below pH 5, but this proteolysis could be abrogated by addition of protease inhibitors. Pulse-chase analysis of IgG MAb transgenic plants also demonstrated that IgG assembly intermediates are present intracellularly and are not secreted, and indicates that the majority of proteolytic degradation occurs following secretion into the apoplastic space.ConclusionsThe results provide evidence that proteolytic fragments derived from antibodies of the IgG subtype expressed in tobacco plants do not accumulate within the cell, and are instead likely to occur in the apoplastic space. Furthermore, any proteolytic activity due to the release of proteases from subcellular compartments during tissue disruption and extraction is not a major consideration under most commonly used extraction conditions.

show abstract

Fluorescent protein fusions to a human immunodeficiency virus monoclonal antibody reveal its intracellular transport through the plant endomembrane system

Cited by 7 publications

References 64 publications

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Vacuolar targeting of recombinant antibodies in Nicotiana benthamiana

Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells

Antibody degradation in tobacco plants: a predominantly apoplastic process

Contact Info

Product

Resources

About