Anne-Catherine Fitchette

Plant-specific glycosylation patterns in the context of therapeutic protein production

While N-glycan synthesis in the endoplasmic reticulum (ER) is relatively well conserved in eukaryotes, N-glycan processing and O-glycan biosynthesis in the Golgi apparatus are kingdom specific and result in different oligosaccharide structures attached to glycoproteins in plants and mammals. With the prospect of using plants as alternative hosts to mammalian cell lines for the production of therapeutic glycoproteins, significant progress has been made towards the humanization of protein N-glycosylation in plant cells. To date, successful efforts in this direction have mainly focused on the targeted expression of therapeutic proteins, the knockout of plant-specific N-glycan-processing genes, and/or the introduction of the enzymatic machinery catalyzing the synthesis, transport and addition of human sugars. By contrast, very little attention has been paid until now to the O-glycosylation status of plant-made therapeutic proteins, which is surprising considering that hundreds of human proteins represent good candidates for Hyp-O glycosylation when produced in a plant expression system. This review describes protein N- and O-linked glycosylation in plants and highlights the limitations and advantages of plant-specific glycosylation on plant-made biopharmaceuticals.

Mutations of an α1,6 Mannosyltransferase Inhibit Endoplasmic Reticulum–Associated Degradation of Defective Brassinosteroid Receptors in Arabidopsis

Asn-linked glycans, or the glycan code, carry crucial information for protein folding, transport, sorting, and degradation. The biochemical pathway for generating such a code is highly conserved in eukaryotic organisms and consists of ordered assembly of a lipid-linked tetradeccasaccharide. Most of our current knowledge on glycan biosynthesis was obtained from studies of yeast asparagine-linked glycosylation (alg) mutants. By contrast, little is known about biosynthesis and biological functions of N-glycans in plants. Here, we show that loss-of-function mutations in the Arabidopsis thaliana homolog of the yeast ALG12 result in transfer of incompletely assembled glycans to polypeptides. This metabolic defect significantly compromises the endoplasmic reticulum–associated degradation of bri1-9 and bri1-5, two defective transmembrane receptors for brassinosteroids. Consequently, overaccumulated bri1-9 or bri1-5 proteins saturate the quality control systems that retain the two mutated receptors in the endoplasmic reticulum and can thus leak out of the folding compartment, resulting in phenotypic suppression of the two bri1 mutants. Our results strongly suggest that the complete assembly of the lipid-linked glycans is essential for successful quality control of defective glycoproteins in Arabidopsis.

Protein N-glycosylation is similar in the moss Physcomitrella patens and in higher plants

We have investigated the structure of glycans N-linked to the proteins of the moss Physcomitrella patens. The structural elucidation was carried out by western blotting using antibodies specific for N-glycan epitopes and by analysis of N-linked glycans enzymatically released from a total protein extract by combination of MALDI–TOF and MALDI–PSD mass spectrometry analysis. Nineteen N-linked oligosaccharides were characterised ranging from high-mannose-type and truncated paucimannosidic-type to complex-type N-glycans harbouring core-xylose, core-α(1,3)-fucose and Lewisa, as previously described for proteins from higher plants. This demonstrates that the processing of N-linked glycans, as well as the specificity of glycosidases and glycosyltransferases involved in this processing, are highly conserved between P. patens and higher plants. As a consequence, P. patens appears to be a new promising model organism for the investigation of the biological significance of protein N-glycosylation in the plant kingdom, taking advantage of the potential for gene targeting in this moss.

N-glycan trimming by glucosidase II is essential for Arabidopsis development

Glucosidase II, one of the early N-glycan processing enzymes and a major player in the glycoprotein folding quality control, has been described as a soluble heterodimer composed of α and β subunits. Here we present the first characterization of a plant glucosidase II α subunit at the molecular level. Expression of the Arabidopsis α subunit restored N-glycan maturation capacity in Schizosaccharomyces pombe α− or αβ−deficient mutants, but with a lower efficiency in the last case. Inactivation of the α subunit in a temperature sensitive Arabidopsis mutant blocked N-glycan processing after a first trimming by glucosidase I and strongly affected seedling development.

Plant proteomics and glycosylation.

In plant cells, as in other eucaryotic cells, glycosylation is one of the most studied posttranslational events. It can be of two types, N- or O-glycosylation, depending on the linkage involved between the protein backbone and the oligosaccharide moiety. In this review, we present different methods, commonly used in our laboratory, to study the glycosylation of plant proteins. These approaches rely on blot detection with glycan-specific probes, as well as specific deglycosylation of the glycoproteins, followed by mass spectrometry analysis. Such experiments not only allow determination of whether the protein is a glycoprotein, but also how and where it is glycosylated. The last part of this chapter is dedicated to the specific purification and identification of glycoprotein populations in plant cells, so-called glycoproteomics.

N-glycosylation of plant recombinant pharmaceuticals.

N-glycosylation is a maturation event necessary for the correct function, efficiency, and stability of a high number of biopharmaceuticals. This chapter presented here proposes various methods to determine whether, how, and where a plant pharmaceutical is N-glycosylated. These methods rely on blot detection with glycan-specific probes, specific deglycosylation of glycoproteins followed by mass spectrometry, N-glycan profile analysis, and glycopeptide identification by LC-MS.

Production of high quality and natural-like recombinant allergens needed for high efficiency of allergy treatment

Background Due to a lack of sufficiently accurate and effective tools, allergy is often badly diagnosed. Moreover, its treatment is expensive, long, uncomfortable and of little effectiveness. In this context, less than a quarter of allergic patients receive a treatment currently. Allergies have therefore become an important public health issue, highlighting the many unmet medical needs. ANGANY Genetics has brought together expertise in protein genomics and in therapeutic protein production in plant expression systems, to produce recombinant allergens of an unprecedented quality, offering the following solutions to allergy sufferers: (1) a precise identification of each allergen involved rather than the current approximate diagnosis, (2) pure and certified recombinant allergens rather than imperfect and incomplete allergen extracts and (3) an effective treatment at the best cost to replace symptomatic-only or low-level curative allergy treatments.