No abstract

We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man 8 -9 Xyl 1-2 Me 3 GlcNAc 2 . The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins.

The investigation of the use of polysaccharides derived from natural sources to support metal catalysis has been the focus of several studies. Even though these molecules seem to be attractive materials, their full potential for use in support of heterogeneous catalysis still needs to be revealed. To that end, we developed a new preparation technique for polysaccharide-based palladium catalysts by immobilizing the palladium phosphine complexes on various renewable polysaccharides. The Suzuki cross-coupling in ethanol, using PdCl 2 (TPPTS) 2 supported by various polysaccharides, was determined by gas chromatography and compared to homogeneous free-catalyst support. The PdCl 2 (TPPTS) 2 , that was immobilized on red algae supports, was successfully used as a heterogeneous catalyst in the Suzuki cross-coupling reaction, yielding high activity, higher than that of the homogeneous complex, without leaching. The FTIR spectrometry of representative heterogeneous polysaccharide-based TPPTS–PdCl 2 catalysts was compared to that of native polysaccharide and polysaccharide-based TPP–PdCl 2 catalysts, indicated on new bands, suggesting that the heterogenization occurs via interactions between the sulfonate group on the TPPTS and the hydroxyl groups on the polysaccharides. EDS and XPS analysis were also performed, confirming that the Pd complex was embedded within the i -carrageenan. A comparison of SEM images of i -carrageenan preparations also shed light on the interaction occurring between the polysaccharides and the TPPTS.

N-glycosylation is one of the most important post-translational modifications that influence protein polymorphism, including protein structures and their functions. Although this important biological process has been extensively studied in mammals, only limited knowledge exists regarding glycosylation in algae. The current research is focused on the red microalga Porphyridium sp., which is a potentially valuable source for various applications, such as skin therapy, food, and pharmaceuticals. The enzymes involved in the biosynthesis and processing of N-glycans remain undefined in this species, and the mechanism(s) of their genetic regulation is completely unknown. In this study, we describe our pioneering attempt to understand the endoplasmic reticulum N-Glycosylation pathway in Porphyridium sp., using a bioinformatic approach. Homology searches, based on sequence similarities with genes encoding proteins involved in the ER N-glycosylation pathway (including their conserved parts) were conducted using the TBLASTN function on the algae DNA scaffold contigs database. This approach led to the identification of 24 encoded-genes implicated with the ER N-glycosylation pathway in Porphyridium sp. Homologs were found for almost all known N-glycosylation protein sequences in the ER pathway of Porphyridium sp.; thus, suggesting that the ER-pathway is conserved; as it is in other organisms (animals, plants, yeasts, etc.).