Expression and assembly of a fully active antibody in algae

Mayfield, Stephen P.; Franklin, Scott; Lerner, Richard A.

doi:10.1073/pnas.0237108100

Cited by 311 publications

(173 citation statements)

References 20 publications

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“…Several studies have already demonstrated that the green alga C. reinhardtii is a convenient platform for producing recombinant proteins, including those of human origin (23). For example, a large single-chain antibody directed against glycoprotein D of the herpes simplex virus (24) and full-length IgG1 monoclonal antibodies directed against anthrax protective antigen 83 (25) have been successfully expressed in the chloroplast of transgenic C. reinhardtii cells. The production of secreted therapeutic proteins such as erythropoietin has also been evaluated (26).…”

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confidence: 99%

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

Mathieu‐Rivet

Scholz

Arias

et al. 2013

Molecular & Cellular Proteomics

142

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Chlamydomonas reinhardtii is a green unicellular eukaryotic model organism for studying relevant biological and biotechnological questions. The availability of genomic resources and the growing interest in C. reinhardtii as an emerging cell factory for the industrial production of biopharmaceuticals require an in-depth analysis of protein N-glycosylation in this organism. Accordingly, we used a comprehensive approach including genomic, glycomic, and glycoproteomic techniques to unravel the N-glycosylation pathway of C. reinhardtii. Using mass-spectrometry-based approaches, we found that both endogenous soluble and membrane-bound proteins carry predominantly oligomannosides ranging from Man-2 to Man-5. In addition, minor complex N-linked glycans were identified as being composed of partially 6-Omethylated Man-3 to Man-5 carrying one or two xylose residues. These findings were supported by results from a glycoproteomic approach that led to the identification of 86 glycoproteins. Here, a combination of in-source collision-induced dissodiation (CID) for glycan fragmentation followed by mass tag-triggered CID for peptide sequencing and PNGase F treatment of glycopeptides in the presence of 18 O-labeled water in conjunction with CID mass spectrometric analyses were employed. In conclusion, our data support the notion that the biosynthesis and maturation of N-linked glycans in the endoplasmic reticulum and Golgi apparatus occur via a GnT I-independent pathway yielding novel complex N-linked glycans that maturate differently from their counterparts in land plants. Molecular & Cellular

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confidence: 99%

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

Mathieu‐Rivet

Scholz

Arias

et al. 2013

Molecular & Cellular Proteomics

142

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“…We showed that a gD-Dendra2 fusion protein localized primarily to the cell surface in the presence or absence of an anti-gD antibody under non-ABB conditions (8); thus, we could use this protein to investigate the fate of a cell surface antigen under ABB conditions. We used an anti-gD IgG antibody (20) with a human Fc (anti-gD hFc ) that can bind to gE-gI and to gD to create ABB complexes and two types of control IgGs to create non-ABB complexes: the anti-gD antibody fused with a mouse Fc (antigD mFc ), which binds gD, but not gE-gI; and a human IgG against an unrelated antigen (IgG hFc ), which binds gE-gI, but not gD (Fig. 1).…”

mentioning

confidence: 99%

Characterization of Antibody Bipolar Bridging Mediated by the Human Cytomegalovirus Fc Receptor gp68

Ndjamen

Joshi

Fraser

et al. 2016

J Virol

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The human cytomegalovirus glycoprotein gp68 functions as an Fc receptor for host IgGs and can form antibody bipolar bridging (ABB) complexes in which gp68 binds the Fc region of an antigen-bound IgG. Here we show that gp68-mediated endocytosis transports ABB complexes into endosomes, after which the complex is routed to lysosomes, presumably for degradation. These results suggest gp68 contributes to evasion of IgG-mediated immune responses by mediating destruction of host IgG and viral antigens. The betaherpesvirus human cytomegalovirus (HCMV) affects 50 to 98% of people, causing severe symptoms in newborns and a lifetime latent infection that can be lethal in immunocompromised individuals (1). HCMV can also establish recurrent secondary infections after reactivation from latency (2). Immune evasion strategies of herpesviruses include expression of viral Fc receptors (Fc␥Rs) that bind host IgG to evade immune responses mediated by host Fc␥Rs (3-6). Viral Fc␥Rs can participate in antibody bipolar bridging (ABB), whereby an antibody simultaneously binds antigen via its fragment antigen-binding (Fab) arms and an Fc receptor using its Fc (7-9). While there is likely a large excess of nonviral IgG compared with antiviral IgG, the proximity of viral Fc␥Rs to Fc regions from IgGs bound to viral antigens on an infected cell could allow viral Fc␥Rs to preferentially bind antiviral IgGs. ABB protects virally infected cells from antibodyand complement-dependent neutralization (10), antibody-dependent cell-mediated cytotoxicity (11), and granulocyte attachment (12). The HCMV glycoproteins gp68, gp34, Toll-like receptor 12 (TLR12), and TLR13 act as Fc␥Rs to bind human IgG (3,6,13,14). Recent studies reported formation of ABB complexes with gp68 and with gp34 and demonstrated their functional importance by showing that cells infected with HCMV lacking gp68 and/or gp34 triggered stronger activation of the host Fc␥Rs and NK cells than cells infected with wildtype HCMV (15).In previous studies of ABB, we used cells expressing gE-gI, a herpes simplex virus 1 (HSV-1) Fc␥R, and gD, an HSV-1 cell surface antigen, to show that anti-gD IgGs formed ABB complexes with gE-gI and gD and that anti-gD IgG and gD were internalized in a gE-gI-dependent process, resulting in lysosomal localization of IgG and gD, but not gE-gI (8) (Fig. 1). Since gE-gI binds Fc at neutral/basic, but not acidic, pH (8, 16), these results were consistent with dissociation of IgG-antigen complexes from gE-gI upon trafficking to acidic intracellular vesicles. In contrast, the gp68-Fc interaction is broadly stable across acidic and basic pHs (17), suggesting a potentially different intracellular trafficking pathway if gp68, like gE-gI, can internalize ABB complexes.To investigate ABB mediated by HCMV gp68, we adapted the model system used to characterize gE-gI-mediated ABB (8). In the gE-gI studies, we transiently expressed gE-gI and gD in HeLa cells and then investigated the trafficking of gE-gI and gD under ABB and non-ABB conditions (8). We chose gD as the model ...

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“…Successful expression and assembly of a recombinant human monoclonal IgA antibody has already been demonstrated for Chlamydomonas reinhardtii [6], while stable expression of the hepatitis B surface antigen gene has been shown in Dunaliella salina [7]. Since Dunaliella is otherwise used for nutrition, there is no need for purification of the antigen, so the intact algae could be used to deliver a vaccine.…”

Section: Antibody and Vaccine Productionmentioning

confidence: 99%

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2018

JAPLR

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Expression and assembly of a fully active antibody in algae

Cited by 311 publications

References 20 publications

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

Exploring the N-glycosylation Pathway in Chlamydomonas reinhardtii Unravels Novel Complex Structures

Characterization of Antibody Bipolar Bridging Mediated by the Human Cytomegalovirus Fc Receptor gp68

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