The cell-wall active mannuronan C5-epimerases in the model brown alga Ectocarpus: From gene context to recombinant protein.

Mannuronan C5-epimerases (ManC5-Es) catalyze in brown algae the remodeling of alginate, a major cell-wall component which is involved in many biological functions in these organisms. ManC5-Es are present as large multigenic families in brown algae, likely indicating functional specificities and specializations. ManC5-Es control the distribution pattern of (1-4) linked ß-d-mannuronic acid (M) and α-l-guluronic acid (G) residues in alginates, giving rise to widely different polysaccharide compositions and sequences, depending on tissue, season, age, or algal species. As such they are also a source of powerful new tools for the biotechnological and enzymatic processing of alginates, to match the growing interest for food hydrocolloids and in biomedical and nanotechnological applications. We report here the first heterologous production of a ManC5-E of brown algal origin that is successfully refolded in an active form. The activity was measured by 1H NMR and by an indirect enzymatic assay using a known bacterial alginate lyase. The transcript expression as a function of the developmental program of the brown alga Ectocarpus, together with the bioinformatic analyses of the corresponding gene context of this multigenic family, is also presented.

Rhamnogalacturonan II structure shows variation in the side chains monosaccharide composition and methylation status within and across different plant species.

A paradigm regarding rhamnogalacturonans II (RGII) is their strictly conserved structure within a given plant. We developed and employed a fast structural characterization method based on chromatography and mass spectrometry, allowing analysis of RGII side chains from microgram amounts of cell wall. We found that RGII structures are much more diverse than so far described. In chain A of wild-type plants, up to 45% of the l-fucose is substituted by l-galactose, a state that is seemingly uncorrelated with RGII dimerization capacity. This led us to completely reinvestigate RGII structures of the Arabidopsis thaliana fucose-deficient mutant mur1, which provided insights into RGII chain A biosynthesis, and suggested that chain A truncation, rather than l-fucose to l-galactose substitution, is responsible for the mur1 dwarf phenotype. Mass spectrometry data for chain A coupled with NMR analysis revealed a high degree of methyl esterification of its glucuronic acid, providing a plausible explanation for the puzzling RGII antibody recognition. The ß-galacturonic acid of chain A exhibits up to two methyl etherifications in an organ-specific manner. Combined with variation in the length of side chain B, this gives rise to a family of RGII structures instead of the unique structure described up to now. These findings pave the way for studies on the physiological roles of modulation of RGII composition.

The two endo-ß-N-acetylglucosaminidase genes from Arabidopsis thaliana encode cytoplasmic enzymes controlling free N-glycan levels.

Endo-ß-N-acetylglucosaminidases (ENGases) cleave N-glycans from proteins and/or peptides by hydrolyzing the O-glycosidic linkage between the two core-N-acetylglucosamine (GlcNAc) residues. Although, two homologous genes potentially encoding ENGases have been identified in Arabidopsis thaliana, their respective substrate specificity, their subcellular and their organ specific localization was hitherto unknown. In order to investigate the role of ENGases in this model plant species, we transiently expressed the two A. thaliana genes in Nicotiana benthamiana and determined the substrate specificities, as well as the Km values, of the purified recombinant enzymes. The assumed predominantly cytosolic localisation of both enzymes, here referred to as AtENGase85A and AtENGase85B, was determined by confocal microscopy of plant leaves expressing the respective GFP-fusion constructs. For the individual characterization of the two enzymes expression patterns in planta, single knock-out plants were selected for both genes. Although both enzymes are present in most organs, only AtENGase85A (At5g05460) was expressed in stems and no ENGase activity was detected in siliques. A double knock-out was generated by crossing but-like single knock-out plants-no apparent phenotype was observed. In contrast, in this double knock-out, free N-glycans carrying a single GlcNAc at the reducing end are completely absent and their counterparts with two GlcNAc-visible only at a trace level in wild type-accumulated dramatically.

Nucleotide and nucleotide sugar analysis by liquid chromatography-electrospray ionization-mass spectrometry on surface-conditioned porous graphitic carbon.

We examined the analysis of nucleotides and nucleotide sugars by chromatography on porous graphitic carbon with mass spectrometric detection, a method that evades contamination of the MS instrument with ion pairing reagent. At first, adenosine triphosphate (ATP) and other triphosphate nucleotides exhibited very poor chromatographic behavior on new columns and could hardly be eluted from columns previously cleaned with trifluoroacetic acid. Satisfactory performance of both new and older columns could, however, be achieved by treatment with reducing agent and, unexpectedly, hydrochloric acid. Over 40 nucleotides could be detected in cell extracts including many isobaric compounds such as ATP, deoxyguanosine diphosphate (dGTP), and phospho-adenosine-5'-phosphosulfate or 3',5'-cyclic adenosine 5'-monophosphate (AMP) and its much more abundant isomer 2',3'-cyclic AMP. A fast sample preparation procedure based on solid-phase extraction on carbon allowed detection of very short-lived analytes such as cytidine 5'-monophosphate (CMP)-2-keto-deoxy-octulosonic acid. In animal cells and plant tissues, about 35 nucleotide sugars were detected, among them rarely considered metabolites such as uridine 5'-diphosphate (UDP)-l-arabinopyranose, UDP-L-arabinofuranose, guanosine 5'-diphosphate (GDP)-L-galactofuranose, UDP-L-rhamnose, and adenosine diphosphate (ADP)-sugars. Surprisingly, UDP-arabinopyranose was also found in Chinese hamster ovary (CHO) cells. Due to the unique structural selectivity of graphitic carbon, the method described herein distinguishes more nucleotides and nucleotide sugars than previously reported approaches.

Class I alpha-mannosidases are required for N-glycan processing and root development in Arabidopsis thaliana.

In eukaryotes, class I alpha-mannosidases are involved in early N-glycan processing reactions and in N-glycan-dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ER-type alpha-mannosidase I (MNS3) and the two Golgi-alpha-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected alpha1,2-mannose residue from Man(9)GlcNAc(2), MNS1/2 readily removed three alpha1,2-mannose residues from Man(8)GlcNAc(2). Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man(8)GlcNAc(2) accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man(9)GlcNAc(2), demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I alpha-mannosidases in wild-type seedlings resulted in a similar root phenotype. These findings show that class I alpha-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis.