Marie-Christine Kiefer-Meyer

Down-regulated expression of plant-specific glycoepitopes in alfalfa.

SUMMARY Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N-glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N-glycosylation in alfalfa. The first was a knock-down of two plant-specific N-glycan maturation enzymes, beta1,2-xylosyltransferase and alpha1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human beta1,4-galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N-acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum. Both knock-down and knock-in strategies strongly, but not completely, inhibited the biosynthesis of alpha1,3-fucose- and beta1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human beta1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N-glycans.

Cloning and sequence analysis of laccase-encoding cDNA clones from tobacco

Three laccase-encoding cDNAs were cloned from a tobacco stem cDNA library. One of them contains a full length sequence coding for a cationic laccase. The predicted polypeptide sequence shows 48% identity with sycamore laccase. Amino acid comparisons with other laccases and ascorbate oxidases have shown that this new plant laccase sequence also contains four potential copper binding regions which are highly conserved among the blue copper oxidases.

N-Glycans of Phaeodactylum tricornutum Diatom and Functional Characterization of Its N-Acetylglucosaminyltransferase I Enzyme

N-Glycosylation, a major co- and post-translational event in the synthesis of proteins in eukaryotes, is unknown in aquatic photosynthetic microalgae. In this paper, we describe the N-glycosylation pathway in the diatom Phaeodactylum tricornutum. Bio-informatic analysis of its genome revealed the presence of a complete set of sequences potentially encoding for proteins involved in the synthesis of the lipid-linked Glc3Man9GlcNAc2-PP-dolichol N-glycan, some subunits of the oligosaccharyltransferase complex, as well as endoplasmic reticulum glucosidases and chaperones required for protein quality control and, finally, the α-mannosidase I involved in the trimming of the N-glycan precursor into Man-5 N-glycan. Moreover, one N-acetylglucosaminyltransferase I, a Golgi glycosyltransferase that initiates the synthesis of complex type N-glycans, was predicted in the P. tricornutum genome. We demonstrated that this gene encodes for an active N-acetylglucosaminyltransferase I, which is able to restore complex type N-glycans maturation in the Chinese hamster ovary Lec1 mutant, defective in its endogeneous N-acetylglucosaminyltransferase I. Consistent with these data, the structural analyses of N-linked glycans demonstrated that P. tricornutum proteins carry mainly high mannose type N-glycans ranging from Man-5 to Man-9. Although representing a minor glycan population, paucimannose N-glycans were also detected, suggesting the occurrence of an N-acetylglucosaminyltransferase I-dependent maturation of N-glycans in this diatom.

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

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-O-methylated 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 18O-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.

Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals

Microalgae are currently used for the production of food compounds. Recently, few microalgae species have been investigated as potential biofactories for the production of biopharmaceuticals. Indeed in this context, microalgae are cheap, classified as Generally Recognized As Safe (GRAS) organisms and can be grown easily. However, problems remain to be solved before any industrial production of microalgae-made biopharmaceuticals. Among them, post-translational modifications of the proteins need to be considered. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. Therefore, the evaluation of microalgae as alternative cell factory for biopharmaceutical productions thus requires to investigate their N-glycosylation capability in order to determine to what extend it differs from their human counterpart and to determine appropriate strategies for remodeling the microalgae glycosylation into human-compatible oligosaccharides. Here, we review the secreted recombinant proteins which have been successfully produced in microalgae. We also report on recent bioinformatics and biochemical data concerning the structure of glycans N-linked to proteins from various microalgae phyla and comment the consequences on the glycan engineering strategies that may be necessary to render those microalgae-made biopharmaceuticals compatible with human therapy.

PECTIN METHYLESTERASE48 is involved in Arabidopsis pollen grain germination

Modifying homogalacturonans in the intine cell wall during maturation of the pollen grain is central for proper germination. Germination of pollen grains is a crucial step in plant reproduction. However, the molecular mechanisms involved remain unclear. We investigated the role of PECTIN METHYLESTERASE48 (PME48), an enzyme implicated in the remodeling of pectins in Arabidopsis (Arabidopsis thaliana) pollen. A combination of functional genomics, gene expression, in vivo and in vitro pollen germination, immunolabeling, and biochemical analyses was used on wild-type and Atpme48 mutant plants. We showed that AtPME48 is specifically expressed in the male gametophyte and is the second most expressed PME in dry and imbibed pollen grains. Pollen grains from homozygous mutant lines displayed a significant delay in imbibition and germination in vitro and in vivo. Moreover, numerous pollen grains showed two tips emerging instead of one in the wild type. Immunolabeling and Fourier transform infrared analyses showed that the degree of methylesterification of the homogalacturonan was higher in pme48−/− pollen grains. In contrast, the PME activity was lower in pme48−/−, partly due to a reduction of PME48 activity revealed by zymogram. Interestingly, the wild-type phenotype was restored in pme48−/− with the optimum germination medium supplemented with 2.5 mm calcium chloride, suggesting that in the wild-type pollen, the weakly methylesterified homogalacturonan is a source of Ca2+ necessary for pollen germination. Although pollen-specific PMEs are traditionally associated with pollen tube elongation, this study provides strong evidence that PME48 impacts the mechanical properties of the intine wall during maturation of the pollen grain, which, in turn, influences pollen grain germination.

Comparative in depth RNA sequencing of P. tricornutum’s morphotypes reveals specific features of the oval morphotype

Phaeodactylum tricornutum is the most studied diatom encountered principally in coastal unstable environments. It has been hypothesized that the great adaptability of P. tricornutum is probably due to its pleomorphism. Indeed, P. tricornutum is an atypical diatom since it can display three morphotypes: fusiform, triradiate and oval. Currently, little information is available regarding the physiological significance of this morphogenesis. In this study, we adapted P. tricornutum Pt3 strain to obtain algal culture particularly enriched in one dominant morphotype: fusiform, triradiate or oval. These cultures were used to run high-throughput RNA-Sequencing. The whole mRNA transcriptome of each morphotype was determined. Pairwise comparisons highlighted biological processes and molecular functions which are up- and down-regulated. Finally, intersection analysis allowed us to identify the specific features from the oval morphotype which is of particular interest as it is often described to be more resistant to stresses. This study represent the first transcriptome wide characterization of the three morphotypes from P. tricornutum performed on cultures specifically enriched issued from the same Pt3 strain. This work represents an important step for the understanding of the morphogenesis in P. tricornutum and highlights the particular features of the oval morphotype.

Endoplasmic Reticulum Body-Related Gene Expression in Different Root Zones of Arabidopsis Isolated by Laser-Assisted Microdissection.

ER body–related genes are specifically regulated in different root zones Methyl jasmonate induces ER body formation in Arabidopsis root Methyl jasmonate induces the overexpression of ER body–related genes in the root Primary root zones can be easily microdissected without any chemical treatment