Dominique Michaud

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.

Preventing unintended proteolysis in plant protein biofactories.

Summary Numerous reports have been published over the last decade assessing the potential of plants as useful hosts for the heterologous expression of clinically useful proteins. Significant progress has been made, in particular, in optimizing transgene transcription and translation in plants, and in elucidating the complex post‐translational modifications of proteins typical of the plant cell machinery. In this article, we address the important issue of recombinant protein degradation in plant expression platforms, which directly impacts on the final yield, homogeneity and overall quality of the resulting protein product. Unlike several more stable and structurally less complex pharmaceuticals, recombinant proteins present a natural tendency to structural heterogeneity, resulting in part from the inherent instability of polypeptide chains expressed in heterologous environments. Proteolytic processing, notably, may dramatically alter the structural integrity and overall accumulation of recombinant proteins in plant expression systems, both in planta during expression and ex planta after extraction. In this article, we describe the current strategies proposed to minimize protein hydrolysis in plant protein factories, including organ‐specific transgene expression, organelle‐specific protein targeting, the grafting of stabilizing protein domains to labile proteins, protein secretion in natural fluids and the co‐expression of companion protease inhibitors.

Impact of environmental stress on aphid clonal resistance to parasitoids: Role of Hamiltonella defensa bacterial symbiosis in association with a new facultative symbiont of the pea aphid.

Resistance to endoparasitoids in aphids involves complex interactions between insect and microbial players. It is now generally accepted that the facultative bacterial symbiont Hamiltonella defensa of the pea aphid Acyrthosiphon pisum is implicated in its resistance to the parasitoid Aphidius ervi. It has also been shown that heat negatively affects pea aphid resistance, suggesting the thermosensitivity of its defensive symbiosis. Here we examined the effects of heat and UV-B on the resistance of A. pisum to A. ervi and we relate its stability under heat stress to different facultative bacterial symbionts hosted by the aphid. For six A. pisum clones harboring four different facultative symbiont associations, the impact of heat and UV-B was measured on their ability to resist A. ervi parasitism under controlled conditions. The results revealed that temperature strongly affected resistance, while UV-B did not. As previously shown, highly resistant A. pisum clones singly infected with H. defensa became more susceptible to parasitism after exposure to heat. Interestingly, clones that were superinfected with H. defensa in association with a newly discovered facultative symbiont, referred to as PAXS (pea aphid X-type symbiont), not only remained highly resistant under heat stress, but also expressed previously unknown, very precocious resistance to A. ervi compared to clones with H. defensa alone. The prevalence of dual symbiosis involving PAXS and H. defensa in local aphid populations suggests its importance in protecting aphid immunity to parasitoids under abiotic stress.

Constitutive expression of digestive cysteine proteinase forms during development of the colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae)

Changes in digestive proteinase activities were monitored throughout development of the Colorado potato beetle (CPB), Leptinotarsa decemlineata Say. Consistent with high growth rates generally observed for larval stages, larva midgut extracts exhibited high specific proteinase activity as compared with adult extracts. Apart from these quantitative variations, no qualitative difference was noted between developmental stages. Specific activity was maximal at mildly acidic pH, and was activated by reducing agents and phenylmethylsulfonyl fluoride. The activity was significantly decreased by trans-epoxysuccinyl-l-leucylamido (4-guanidino) butane and pepstatin, indicating the presence of cysteine and aspartate proteinase activity in the extracts, respectively. As demonstrated by the use of class-specific activity gels, this activity was principally due to the constitutive expression of at least 9 cysteine proteinase forms active over a wide pH range. In accordance with the apparent existence of a unique digestive proteolytic system throughout CPB development, the rice cysteine proteinase inhibitor oryzacystatin I had the same inhibitory effect on extracts of each developmental stage, but in contrast to early stages, growth of 3rd and 4th instars was not significantly affected when the inhibitor was fed to the insects. These observations indicate the potential of the rice cystatin for inhibition of CPB growth but also suggest a stage-related efficiency for this control strategy, independent of the nature of proteinase forms used by the insect.

Selective inhibition of Colorado potato beetle cathepsin H by oryzacystatins I and II

The use of oryzacystatins I and II, two cysteine proteinase inhibitors naturally produced in rice grains, represents an attractive way for the control of Coleoptera insect pests. The present study was done to analyze the inhibitory effect of recombinant oryzacystatins produced in Escherichia coli as fusion proteins against digestive proteinases of the major pest Colorado potato beetle (Leptinotarsa decemlineata Say). Both inhibitors had a significant effect on total proteolytic activity, but maximal inhibitions ranged from 20 to 80% for pHs varying from 5.0 to 7.0, respectively. This pH‐dependent efficiency of plant cystatins was due to the selective inactivation of potato beetle cathepsin H, as demonstrated by the use of inhibitors with different specificities against cathepsins B and H. These results demonstrate the importance of having an adequate knowledge of insect proteinases specifically recognized by the inhibitors to be used in pest control strategies.

Oryzacystatin I expressed in transgenic potato induces digestive compensation in an insect natural predator via its herbivorous prey feeding on the plant.

We observed recently that the rice cysteine proteinase inhibitor, oryzacystatin I (OCI) expressed in transgenic potato does not affect growth and development of the two‐spotted stinkbug predator (Perillus bioculatus) via its herbivorous prey feeding on the plant. Here we monitored the inhibitory activity of recombinant OCI along this potato → herbivore → predator continuum, to determine if the absence of effect was associated with a digestive compensatory response of the predator following inhibition of its proteinases by the recombinant cystatin. After confirming that OCI is present in the plant, and ingested in an active form by potato beetle larvae, quantitative and electrophoretic assays allowed us to determine that the recombinant cystatin (representing about 0.8% of total soluble proteins in leaves) was entirely bound to a ∼30‐kDa target proteinase in the preys midgut, forming a sodium dodecyl sulphate (SDS)‐stable complex detected on immunoblots with an anti‐OCI polyclonal antibody. Despite the apparent absence of free, residual OCI in the beetles midgut, digestive protease activity in the predator, known to include OCI‐sensitive activity, was altered negatively when the prey was fed the modified plant. This inhibitory process at the third trophic level was accompanied by a compensatory response in the predator, by which serine‐type proteinases were synthesized de novo. Overall, our data suggest that the affinity between OCI and the predators OCI‐sensitive proteinases is: (i) as strong as (or stronger than) the affinity between OCI and the potato beetle 30‐kDa‐sensitive proteinase; and (ii) stronger than the affinity between these enzymes and the plant endogenous homologue of OCI, potato multicystatin, induced in the plant by potato beetle feeding. Our results also show that predatory organisms can adapt their digestive metabolism to the presence of plant antidigestive proteins ingested by their herbivorous preys. In a broader context, this study stresses the need to monitor the inhibitory effects of PI‐expressing plants not only on the herbivorous insects targeted, but also on the organisms likely to consume these pests in the environment.

GROWTH COMPENSATION AND FASTER DEVELOPMENT OF COLORADO POTATO BEETLE (COLEOPTERA : CHRYSOMELIDAE) FEEDING ON POTATO FOLIAGE EXPRESSING ORYZACYSTATIN I

Feeding, growth, development, and food conversion efficiency of Colorado potato beetle larvae reared on foliage from a “Kennebec” potato line expressing oryzacystatin I (OCI) at about 1% of its total soluble proteins were compared to those of larvae feeding on untransformed foliage from the same line. During stages L1 to L3, larvae feeding on OCI consumed leaf material 14% faster, gained weight 28% faster, and weighed 20% more at the end of the L3 stage, compared to controls. Continued exceptional performance on OCI during the final L4 stage was expressed as faster development than controls, an effect that persisted during pupal development and resulted in emergence of similar weight adults 1 day earlier than controls. Larvae initially maintained on control foliage and switched to OCI foliage during L4 did not overcompensate as those on OCI foliage throughout development, but performed similarly to larvae on control foliage throughout. Total azocaseinase activity in midgut extracts from these 4th instars 1 d after switching to OCI foliage was sensitive to inhibition by a recombinant form of OCI expressed in Escherichia coli, but was no longer sensitive 4 d after switching, indicating a gradual adaptation of the insect digestive protease system, based on the production of OCI insensitive proteases. Despite OCI potato foliage being consumed faster by small larvae using it for food, there was no indication that it was less efficient than untransformed foliage as food protein. Arch. Insect Biochem. Physiol. 40:69–79, 1999.

Unexpected Effects of Different Potato Resistance Factors to the Colorado Potato Beetle (Coleoptera: Chrysomelidae) on the Potato Aphid (Homoptera: Aphididae)

Abstract Improving the use of bioctechnological and classical plant resistance for herbivore pest control with less reliance on chemicals critically depends on predictable interactions with secondary pests. Performance of the potato aphid Macrosiphum euphorbiae (Thomas), a secondary pest of potato in eastern North America, was studied on potato, Solanum tuberosum L., lines with traits of potential resistance to primary pests. The lines tested were ‘Newleaf’, a transgenic ‘Superior’ cultivar expressing the Bacillus thuringiensis Berliner CryIIIA toxin, which is highly resistant to the Colorado potato beetle, Leptinotarsa decemlineata (Say); a transgenic ‘Kennebec’ cultivar expressing rice cystatin I, a protease inhibitor previously shown to inhibit cathepsin like digestive enzymes in the Colorado potato beetle; NYL 235–4, a potato derived by selective breeding following hybridization with S. berthaulthii, with a moderate density of glandular trichomes providing resistance to small insects by contact; and the commercial cultivars Superior and Kennebec used as controls. Transgenic Superior potatoes negatively affected M. euphorbiae’s growth and fecundity, in contrast with the OCI potato, which improved aphid performance. The flight incidence of young alatae of M. euphorbiae that completed development on transgenic Superior was significantly higher than in aphids from other potato lines. Aphid resistance in the ‘NYL 235–4’ line was complex and depended on aphid access being limited to leaves, which reduced survival and fecundity. However, when aphids had access to whole NYL 235–4 plants, aphid performance was restored, as they preferentially fed and reproduced on NYL 235–4 stems and apical buds of unfolding leaflets. The results illustrate that the performance of a secondary pest of potato can vary unpredictably, depending on the nature of the resistance factors involved in developing specific resistance to a primary pest.

A proteomic analysis of the aphid Macrosiphum euphorbiae under heat and radiation stress

Temperature and solar radiation can be important sources of abiotic stress for small herbivorous insects living in close association with plants. We examined the effects of daily fluctuations of heat and UV radiation on the proteome and performance of winged and wingless morphs of the aphid Macrosiphum euphorbiae. A daily regime of 4h of heat stress at 35 degrees C had more negative effects on the aphids fitness than a similar period of UV-B stress (11.6kJm(-2) per day), and these effects were most pronounced on wingless aphids. Aphid proteomes as detected on 2-D gels revealed approximately 470 protein spots, with the fluctuating heat stress leading to many more changes than exposure to UV-B. The reduced performance of aphids under heat stress correlated with lower abundance of several enzymes in central pathways of energy metabolism, including the TCA cycle and the respiratory chain. Several exoskeletal proteins were induced or their abundance was increased under high temperature stress, suggesting that cuticle barrier enhancement at molting in response to heat stress is an aphid adaptation to stressful thermal conditions. The proteome of winged aphids was more broadly modulated under stress than that of wingless aphids. Greater homeostatic capabilities as revealed at the proteomic level could explain the higher tolerance of the alate aphid morph to environmental stress and its more stable performance and fitness.

Synthesis of active oryzacystatin I in transgenic potato plants

SummaryTransformation of potato (Solanum tuberosum L.) with cysteine proteinase inhibitor (PI) genes represents a potential way of controlling the major insect pest Colorado potato beetle (CPB; Leptinotarsa decemlineata Say). The present study describes the Agrobacterium-mediated transformation of potato (cv. Kennebec) with an oryzacystatin I (OCI) cDNA clone linked to a CaMV 35S promoter. The transgenic plants accumulated active OCI in potato leaves, as demonstrated by the papain-inhibitory activity of transgenic plant leaf extracts. In addition to their anti-papain activity, the extracts also caused a partial but significant inhibition of CPB digestive proteinases, similar to that observed with pure inhibitors. Recombinant OCI did not alter the activity of the major potato leaf endogenous proteinases, which seemed to be of the serine-type. Therefore we suggest that the OCI cDNA can be used for the production of CPB-resistant transgenic potato plants without interfering with endogenous proteinases of these plants.