RESUMEN
Contact with the urticating setae from the abdomen of adult females of the neo-tropical moth Hylesia metabus gives rise to an urticating dermatitis, characterized by intense pruritus, generalized malaise and occasionally ocular lesions (lepidopterism). The setae contain a pro-inflammatory glycosylated protease homologous to other S1A serine proteases of insects. Deglycosylation with PNGase F in the presence of a buffer prepared with 40% H2 (18)O allowed the assignment of an N-glycosylation site. Five main paucimannosidic N-glycans were identified, three of which were exclusively α(1-6)-fucosylated at the proximal GlcNAc. A considerable portion of these N-glycans are anionic species sulfated on either the 4- or the 6-position of the α(1-6)-mannose residue of the core. The application of chemically and enzymatically modified variants of the toxin in an animal model in guinea pigs showed that the pro-inflammatory and immunological reactions, e.g. disseminated fibrin deposition and activation of neutrophils, are due to the presence of sulfate-linked groups and not on disulfide bonds, as demonstrated by the reduction and S-alkylation of the toxin. On the other hand, the hemorrhagic vascular lesions observed are attributed to the proteolytic activity of the toxin. Thus, N-glycan sulfation may constitute a defense mechanism against predators.
Asunto(s)
Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/química , Polisacáridos/química , Serina Proteasas/química , Animales , Glicosilación , Mariposas Nocturnas/enzimología , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Polisacáridos/metabolismo , Serina Proteasas/metabolismo , Sulfatos/química , Sulfatos/metabolismoRESUMEN
A broad variety of foreign genes can be expressed in transgenic plants, which offer the opportunity for large-scale production of pharmaceutical proteins, such as therapeutic antibodies. Nimotuzumab is a humanized anti-epidermal growth factor receptor (EGFR) recombinant IgG1 antibody approved in different countries for the treatment of head and neck squamous cell carcinoma, paediatric and adult glioma, and nasopharyngeal and oesophageal cancers. Because the antitumour mechanism of nimotuzumab is mainly attributed to its ability to interrupt the signal transduction cascade triggered by EGF/EGFR interaction, we have hypothesized that an aglycosylated form of this antibody, produced by mutating the N(297) position in the IgG(1) Fc region gene, would have similar biochemical and biological properties as the mammalian-cell-produced glycosylated counterpart. In this paper, we report the production and characterization of an aglycosylated form of nimotuzumab in transgenic tobacco plants. The comparison of the plantibody and nimotuzumab in terms of recognition of human EGFR, effect on tyrosine phosphorylation and proliferation in cells in response to EGF, competition with radiolabelled EGF for EGFR, affinity measurements of Fab fragments, pharmacokinetic and biodistribution behaviours in rats and antitumour effects in nude mice bearing human A431 tumours showed that both antibody forms have very similar in vitro and in vivo properties. Our results support the idea that the production of aglycosylated forms of some therapeutic antibodies in transgenic plants is a feasible approach when facing scaling strategies for anticancer immunoglobulins.
Asunto(s)
Anticuerpos Monoclonales Humanizados/biosíntesis , Anticuerpos Monoclonales Humanizados/farmacología , Receptores ErbB/antagonistas & inhibidores , Inmunoglobulinas/biosíntesis , Nicotiana/genética , Nicotiana/metabolismo , Planticuerpos/farmacología , Animales , Antineoplásicos/metabolismo , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Desnudos , Agricultura Molecular/métodos , Fosforilación/efectos de los fármacos , Planticuerpos/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Ratas , Ratas Wistar , Proteínas Recombinantes , Tirosina/metabolismoRESUMEN
Plant cells are able to perform most of the post-translational modifications that are required by recombinant proteins to achieve adequate bioactivity and pharmacokinetics. However, regarding N-glycosylation the processing of plant N-glycans in the Golgi apparatus displays major differences when compared with that of mammalian cells. These differences in N-glycosylation are expected to influence serum clearance rate of plant-derived monoclonal antibodies. The monoclonal antibody against the hepatitis B virus surface antigen expressed in Nicotiana tabacum leaves without KDEL endoplasmic reticulum (ER) retention signal (CB.Hep1(-)KDEL) and with a KDEL (Lys-Asp-Glu-Leu) fused to both IgG light and heavy chains (CB.Hep1(+)KDEL) were tested for in vivo stability in mice. Full characterization of N-glycosylation and aggregate formation in each monoclonal antibody batch was determined. The mouse counterpart (CB.Hep1) was used as control. Both (CB.Hep1(-)KDEL) and (CB.Hep1(+)KDEL) showed a faster initial clearance rate (first 24 h) compared with the analogous murine antibody while the terminal phase was similar in the three antibodies. Despite the differences between CB.Hep1(+)KDEL and CB.Hep1(-)KDEL N-glycans, the in vivo elimination in mice was indistinguishable from each other and higher than the murine monoclonal antibody. Molecular modelling confirmed that N-glycans linked to plantibodies were oriented away from the interdomain region, increasing the accessibility of the potential glycan epitopes by glycoprotein receptors that might be responsible for the difference in stability of these molecules.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Retículo Endoplásmico/metabolismo , Anticuerpos contra la Hepatitis B/biosíntesis , Nicotiana/inmunología , Planticuerpos/metabolismo , Señales de Clasificación de Proteína , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Clonación Molecular , Epítopos/inmunología , Epítopos/metabolismo , Femenino , Regulación de la Expresión Génica de las Plantas , Glicosilación , Anticuerpos contra la Hepatitis B/inmunología , Antígenos de la Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Hojas de la Planta/inmunología , Hojas de la Planta/metabolismo , Planticuerpos/inmunología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/inmunología , Plantas Modificadas Genéticamente/metabolismo , Polisacáridos/inmunología , Polisacáridos/aislamiento & purificación , Polisacáridos/metabolismo , Estabilidad Proteica , Conejos , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Plants synthesize N-glycans containing the antigenic sugars alpha(1,3)-fucose and beta(1,2)-xylose. Therefore it is important to monitor these N-glycans in monoclonal antibodies produced in plants (plantibodies). We evaluated several techniques to characterize the N-glycosylation of a plantibody produced in tobacco plants with and without the KDEL tetrapeptide endoplasmic reticulum retention signal which should inhibit or drastically reduce the addition of alpha(1,3)-fucose and beta(1,2)-xylose. Ammonium hydroxide/carbonate-based chemical deglycosylation and PNGase A enzymatic release were investigated giving similar 2-aminobenzamide-labeled N-glycan HPLC profiles. The chemical release does not generate peptides which is convenient for MS analysis of unlabeled pool but its main drawback is that it induces degradation of alpha1,3-fucosylated N-glycan reducing terminal sugar. Three analytical methods for N-glycan characterization were evaluated: (i) MALDI-MS of glycopeptides from tryptic digestion; (ii) negative-ion ESI-MS/MS of released N-glycans; (iii) normal-phase HPLC of fluorescently labeled glycans in combination with exoglycosidase sequencing. The MS methods identified the major glycans, but the HPLC method was best for identification and relative quantitation of N-glycans. Negative-mode ESI-MS/MS permitted also the correct identification of the linkage position of the fucose residue linked to the inner core N-acteylglucosamine (GlcNAc) in complex N-glycans.
Asunto(s)
Anticuerpos Monoclonales/química , Cromatografía Líquida de Alta Presión/métodos , Nicotiana/metabolismo , Planticuerpos/química , Polisacáridos/química , Hidróxido de Amonio , Anticuerpos Monoclonales/metabolismo , Glicosilación , Hidróxidos/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Hojas de la Planta/metabolismo , Planticuerpos/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , ortoaminobenzoatos/químicaRESUMEN
Development of convenient strategies for identification of plant N-glycan profiles has been driven by the emergence of plants as an expression system for therapeutic proteins. In this article, we reinvestigated qualitative and quantitative aspects of plant N-glycan profiling. The extraction of plant proteins through a phenol/ammonium acetate procedure followed by deglycosylation with peptide N-glycosidase A (PNGase A) and coupling to 2-aminobenzamide provides an oligosaccharide preparation containing reduced amounts of contaminants from plant cell wall polysaccharides. Such a preparation was also suitable for accurate qualitative and quantitative evaluation of the N-glycan content by mass spectrometry. Combining these approaches allows the profiling to be carried out from as low as 500 mg of fresh leaf material. We also demonstrated that collision-induced dissociation (CID) mass spectrometry in negative mode of N-glycans harboring alpha(1,3)- or alpha(1,6)-fucose residue on the proximal GlcNAc leads to specific fragmentation patterns, thereby allowing the discrimination of plant N-glycans from those arising from mammalian contamination.
Asunto(s)
Plantas/química , Polisacáridos/análisis , Polisacáridos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Glicopéptidos/química , Glicopéptidos/metabolismo , Medicago sativa/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa/metabolismo , Hojas de la Planta/química , Polisacáridos/química , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Nicotiana/química , ortoaminobenzoatos/químicaRESUMEN
Plants are potential hosts for the expression of recombinant glycoproteins intended for therapeutic purposes. However, N-glycans of mammalian glycoproteins produced in transgenic plants differ from their natural counterparts. The use of the endoplasmic reticulum (ER)-retention signal has been proposed to restrict glycosylation of plantibodies to only high-mannose-type N-glycans. Furthermore, little is known about the influence of plant development and growth conditions on N-linked glycosylation. Here, we report a detailed N-glycosylation profiling study of CB.Hep1, a mouse IgG2b monoclonal antibody (mAb) against hepatitis B surface antigen (HBsAg) currently expressed in tobacco plants (Nicotiana tabacum L.). The KDEL ER-retention signal was fused to the C-terminal of both light and heavy chains. The structures of the N-linked glycans of this mAb produced in transgenic tobacco plants at various growth stages were analysed by high-performance liquid chromatography (HPLC) profiling techniques and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and compared with those of murine origin. The high-mannose-type oligosaccharides accounted for more than 80% of the total N-glycans, with Man7GlcNAc2 being the most abundant species. Some complex N-glycans bearing xylose and small amounts of oligosaccharides with both xylose and fucose were identified. No appreciable differences were detected when comparing glycosylation at different leaf ages, e.g. from seedling leaves up to 8 weeks old and top or basal leaves of mature plants, or between leaves, stems and whole plants. A strict retention of glycoproteins to ER by the use of the tetrapeptide KDEL was not sufficient, even though the majority of the resulting N-glycosylation was of the high-mannose type. It is highly likely to be dependent on other factors, which are most probably protein specific.
RESUMEN
When generating stably transformed transgenic plants, transient gene expression experiments are especially useful to rapidly confirm that the foreign molecule of interest is correctly assembled and retains its biological activity. TheraCIM(R) (CIMAB S.A., Havana) is a recombinant humanized antibody against the Epidermal Growth Factor receptor (EGF-R), now in clinical trials for cancer therapy in Cuba and other countries. An aglycosylated version (Asn 297 was mutated for Gln 297) of this antibody was transiently expressed in tobacco leaves after vacuum-mediated infiltration of recombinant Agrobacterium tumefaciens that carried a binary plasmid bearing the antibody heavy and light chain genes and plant regulation signals. Protein extracts from "agroinfiltrated" leaves were tested by ELISA and Western blot, showing that the fully assembled antibody was accumulated in plant tissues. The absence of plant specific glycans did not interfere in the assembling or in the activity of the plantibody, as demonstrated in this work. Indirect immunofluorescence demonstrated that the aglycosylated antibody expressed in plants recognizes the EGF-R expressed on the surface of A431 human tumor culture cells.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Receptores ErbB/inmunología , Nicotiana/genética , Nicotiana/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Ingeniería de Proteínas/métodos , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados , Células Cultivadas , Regulación de la Expresión Génica de las Plantas/fisiología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Factores de TiempoRESUMEN
Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein, which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS. The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N-glycans, from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) of which, Man(8-9)GlcNAc(2) were the most abundant. O-Glycopeptides are distributed mostly towards the protein N-terminus. While the first N-glycosylated site (Asn(122)) is located between EGF-like domains 2 and 3, where the O-glycopeptides were found, two other N-glycosylated sites (Asn(329) and Asn(363)) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr.