Unique N-Glycan Moieties of the 66-kDa Cell Wall Glycoprotein from the Red Microalga Porphyridium sp.

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 Man8–9Xyl1–2Me3GlcNAc2. 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. 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 Man8–9Xyl1–2Me3GlcNAc2. 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. IntroductionN-Glycosylation is one of the most fundamental protein post-translational modifications in eukaryotes, influencing the physicochemical and biological properties of proteins (1Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4963) Google Scholar, 2Dwek R.A. Chem. Rev. 1996; 96: 683-720Crossref PubMed Scopus (2827) Google Scholar, 3Varki A. Cummings R. Esko J. Freeze H. Hart G. Marth J. Essentials of Glycobiology. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999Google Scholar, 4Wormald M.R. Dwek R.A. Structure. 1999; 7: R155-R160Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 5Taylor M.E. Drickamer K. Introduction to Glycobiology. 2nd Ed. Oxford University Press, Oxford2003Google Scholar). To date, N-glycosylation patterns and N-glycan structures have been studied mainly in mammals, insects, yeasts, and plants (6Weerapana E. Imperiali B. Glycobiology. 2006; 16: 91R-101RCrossref PubMed Scopus (271) Google Scholar), whereas seaweeds and microalgae have received very little attention aside from a few studies conducted on green algae. These studies generally revealed the presence of similar glycans to those found in the other species. For example, a lectin blot analysis in combination with N-glycosidase F (PNGase F) 3The abbreviations used are: PNGase FN-glycosidase F2AB2-aminobenzamideDDWdouble-distilled waterEndo Hendoglycosidase HESIelectrospray ionizationGUglucose unitHexhexosem/zmass/charge ratioNPnormal phaseWAXweak anion-exchangeAraarabinoseXylxyloseFucfucose. or endoglycosidase H (Endo H) treatment on the glycoproteins of the flagellar scale of Scherffelia dubia, a member of the Chlamydomonas family, revealed the presence of both high mannose and processed N-glycans (hybrid and complex types) (7Grunow A. Becker B. Melkonian M. Eur. J. Cell Biol. 1993; 61: 10-20PubMed Google Scholar, 8Becker B. Perasso L. Kammann A. Salzburg M. Melkonian M. Planta. 1996; 199: 503-510Crossref Scopus (20) Google Scholar). Application of a similar protocol to the N-glycan structures of different flagellar strains of Tetraselmis showed that the scale-associated Tetraselmis striata glycoproteins are composed largely of high mannose glycans. On the other hand, those of Tetraselmis chui consist of many unknown complex glycans (9Becker B. Dreschers S. Melkonian M. Eur. J. Phycol. 1995; 30: 307-312Crossref Scopus (7) Google Scholar, 10Becker D. Melkonian M. Eur. J. Cell Biol. 1992; 57: 109-116PubMed Google Scholar, 11Gödel S. Becker B. Melkonian M. Protist. 2000; 151: 147-159Crossref PubMed Scopus (12) Google Scholar). A deeper insight into the glycan structures associated with the glycoprotein pheromone of the chlorophyte Volvox carteri f. nagariensis was obtained by applying a combination of exoglycosidases digestion, gas chromatographic sugar analysis, and two-dimensional HPLC separation (12Balshüsemann D. Jaenicke L. Eur. J. Biochem. 1990; 192: 231-237Crossref PubMed Scopus (27) Google Scholar). This experimental protocol showed that the N-glycans contain a chitobiose core with one to four additional Man residues with or without an additional Xyl residue attached to the 2-position of the core branching Man.The current study focuses on the structural characterization of N-glycans in the 66-kDa glycoprotein associated with the cell wall polysaccharide of Porphyridium sp., the most abundant species of red microalga of the division Rhodophyta. Porphyridium sp. has been the subject of intensive study by our group for a number of years (13Lapidot M. Shrestha R.P. Weinstein Y. Arad (Malis) S. Seckbach J. Chapman D.J. Cellular Origin, Life in Extreme Habitats, and Astrobiology: Red Algae in Genomic Age. Vol. 13. Springer, Dordrecht, The Netherlands2010: 205-225Google Scholar, 14Arad (Malis) S. Weinstein Y. Biomedic (Israel). 2003; 1: 32-37Google Scholar, 15Arad S.M. Levy-Ontman O. Curr. Opin. Biotech. 2010; 21: 358-364Crossref PubMed Scopus (176) Google Scholar, 16Arad (Malis) S. Cohen Z. Chemicals from Microalgae. Taylor and Francis, New York1999: 282-287Google Scholar, 17Arad (Malis) S. Richmond A. Richmond A. Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd, Oxford2004: 289-297Google Scholar). The cells of the red microalga are encapsulated within a cell wall complex of a polysaccharide, which includes glycoproteins. This polysaccharide complex was found to possess unique characteristics and bioactivities, which offer a vast range of potential applications (13Lapidot M. Shrestha R.P. Weinstein Y. Arad (Malis) S. Seckbach J. Chapman D.J. Cellular Origin, Life in Extreme Habitats, and Astrobiology: Red Algae in Genomic Age. Vol. 13. Springer, Dordrecht, The Netherlands2010: 205-225Google Scholar, 14Arad (Malis) S. Weinstein Y. Biomedic (Israel). 2003; 1: 32-37Google Scholar, 15Arad S.M. Levy-Ontman O. Curr. Opin. Biotech. 2010; 21: 358-364Crossref PubMed Scopus (176) Google Scholar, 16Arad (Malis) S. Cohen Z. Chemicals from Microalgae. Taylor and Francis, New York1999: 282-287Google Scholar, 17Arad (Malis) S. Richmond A. Richmond A. Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd, Oxford2004: 289-297Google Scholar). Chemical characterization of red microalgae polysaccharide revealed that it is an anionic heteropolymer (molecular mass 3–5 × 106 Da) composed of about 10 different sugars, the main ones being Xyl, Glc, and Gal, with sulfate groups located on the Glc and Gal moieties (16Arad (Malis) S. Cohen Z. Chemicals from Microalgae. Taylor and Francis, New York1999: 282-287Google Scholar, 17Arad (Malis) S. Richmond A. Richmond A. Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd, Oxford2004: 289-297Google Scholar, 18Arad (Malis) S. Stadler T. Mollion J. Verdus M.C. Karamanos Y. Morvan H. Christiaen D. Algal Biotechnology. Elsevier Applied Science, London1988: 65-87Google Scholar, 19Geresh S. Arad (Malis) S. Bioresour. Technol. 1991; 38: 195-201Crossref Scopus (116) Google Scholar, 20Heaney-Kieras J. Chapman D.J. Carbohydr. Res. 1976; 52: 169-177Crossref PubMed Scopus (60) Google Scholar, 21Lupescu N. Arad (Malis) S. Geresh S. Bernstein M. Glazer R. Carbohydr. Res. 1991; 210: 349-352Crossref Scopus (36) Google Scholar). A primary disaccharide building block, 3-O-(α-d-glucopyranosyluronic acid)-l-galactopyranose has been isolated and characterized from the polysaccharides of various red microalgae species (21Lupescu N. Arad (Malis) S. Geresh S. Bernstein M. Glazer R. Carbohydr. Res. 1991; 210: 349-352Crossref Scopus (36) Google Scholar, 22Geresh S. Lupescu N. Arad (Malis) S. Phytochemistry. 1992; 31: 4181-4186Crossref Scopus (43) Google Scholar, 23Geresh S. Dubinsky O. Arad S.M. Christiaen D. Glaser R. Carbohydr. Res. 1990; 208: 301-305Crossref PubMed Scopus (40) Google Scholar), and the chemical structure of different fractions separated from the polysaccharide of Porphyridium sp. have been determined (24Geresh S. Arad S.M. Levy-Ontman O. Zhang W. Tekoah Y. Glaser R. Carbohydr. Res. 2009; 344: 343-349Crossref PubMed Scopus (62) Google Scholar, 25Gloaguen V. Ruiz G. Morvan H. Mouradi-Givernaud A. Maes E Krausz P. Strecker G. Carbohydr. Res. 2004; 339: 97-103Crossref PubMed Scopus (45) Google Scholar).Among the proteins of the Porphyridium sp. cell wall complex that have been detected, the most prominent is a 66-kDa glycoprotein (27Shrestha R.P. Weinstein Y. Bar-Zvi D. Arad S.M. J. Phycol. 2004; 40: 568-580Crossref Scopus (30) Google Scholar, 66Shrestha R.P. A Non-covalenty Bound Cell-wall Glycoprotein of the Red Microalga Porphyridium sp.: Characterization and Functions. Ben-Gurion University of the Negev, Beer-Sheva, Israel1999Google Scholar). This glycoprotein is tightly bound, but not covalently linked, to the cell wall polysaccharide and consists of a polypeptide of ∼58 kDa and sugar moieties of ∼8 kDa. Sequencing of a cDNA clone encoding the 66-kDa glycoprotein revealed that this is a novel protein with four potential N-glycan sites that does not show similarity to any protein in the public domain databases. However, it does show some structural similarities to protein superfamilies within the carbohydrate binding domain, namely, glycosyltransferases, pectin lyase-like, sialidases, and concanavalin A-like lectins/glucanases in the SCOP databases, indicating a possible role of the 66-kDa glycoprotein in synthesis/modification of the cell wall polysaccharide (27Shrestha R.P. Weinstein Y. Bar-Zvi D. Arad S.M. J. Phycol. 2004; 40: 568-580Crossref Scopus (30) Google Scholar). Moreover, this protein was found at the early stages of the cell wall cycle intermediate (28Simon-Bercovitch B. Bar-Zvi D. Arad S.M. J. Phycol. 1999; 35: 78-83Crossref Scopus (19) Google Scholar, 67Simon-Bercovitch B. Cell-wall Formation in the Red Microalga Porphyridium sp. Ben-Gurion University of the Negev, Eilat, Israel1997Google Scholar) and in all cell wall-modified mutants. In addition, the glycoprotein was shown to play a role in biorecognition (30Ucko M. Shrestha R.P. Mesika P. Bar-Zvi D. Arad (Malis) S. J. Phycol. 1999; 35: 1276-1281Crossref Scopus (18) Google Scholar). Initial characterization of the sugar moieties by lectin blot analysis and PNGase F treatment suggested the presence of terminal Man (27Shrestha R.P. Weinstein Y. Bar-Zvi D. Arad S.M. J. Phycol. 2004; 40: 568-580Crossref Scopus (30) Google Scholar).4 The glycoprotein was also detected by a xylose-specific antibody, indicating the presence of Xyl.In the current study we characterized for the first time the N-linked glycans of the 66-kDa sugar moiety within the cell wall polysaccharide of Porphyridium sp. This study of the N-linked glycosylation patterns in Porphyridium sp. constitutes the first step in the search for enzymes involved in glycosylation-related pathways in this microalga. Because the cell wall glycoprotein was suggested to be involved in polysaccharide biosynthesis, unraveling its structure may contribute to our understanding of the biosynthesis of this polysaccharide. In addition, knowledge of the N-linked glycosylation might be of value for biotechnological applications, especially for production of therapeutic proteins, using the algae as a "cell factory," as well as for studying the evolution of glycosylation processes.DISCUSSIONThis study constitutes the first investigation of the N-glycan structures of a glycoprotein from within the cell wall polysaccharide of the red microalga Porphyridium sp. Glycan analysis showed four prominent N-linked glycans, based on the oligomannose topology, that were different from structures found so far in other organisms. These novel structures contain the same four monosaccharides: GlcNAc, Man, 6-O-MeMan, and Xyl. All have the same core structure with the characteristic structure [Man]4[O-MeMan]3[Xyl]1[GlcNAc]2, containing methylated Man residues (Fig. 7A).Several studies have reported methylated sugars as constituents of glycoproteins in other organisms, including nematodes, yeasts, snails, algae, and planarians (51Vliegenthart J.F.G. Montreuil J. Montreuil J. Schachter H. Vliegenthart J.F.G. Glycoproteins. Elsevier Science B.V., Amsterdam1995: 13-28Google Scholar, 52Hall R.L Wood E.J. Kamberling J.P. Gerwig G.J. Vliegenthart F.G. Biochem. J. 1977; 165: 173-176Crossref PubMed Scopus (49) Google Scholar, 53Kamerling J.P. Vliegenthart J.F.G. Montreuil J. Vliegenthart J.F.G. Schachter H. Glycoproteins II. Elsevier Science B.V., Amsterdam1997: 123-161Google Scholar, 54Lommerse J.P. Thomas-Oates J.E. Gielens C. Préaux G. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1997; 249: 195-222Crossref PubMed Scopus (69) Google Scholar, 55Stoeva S. Rachev R. Severov S. Voelter W. Genov N. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 1995; 110: 761-765Crossref PubMed Scopus (24) Google Scholar, 56Stoeva S. Schütz J. Gebauer W. Hundsdörfer T. Manz C. Markl J. Voelter W. Biochim. Biophys. Acta. 1999; 1435: 94-109Crossref PubMed Scopus (31) Google Scholar, 57Khoo K.H. Maizels R.M. Page A.P. Taylor G.W. Rendell N.B. Dell A. Glycobiology. 1991; 1: 163-171Crossref PubMed Scopus (88) Google Scholar, 58Guérardel Y. Balanzino L. Maes E. Leroy Y. Coddeville B. Oriol R. Strecker G. Biochem. J. 2001; 357: 167-182Crossref PubMed Scopus (99) Google Scholar, 59Puanglarp N. Oxley D. Currie G.J. Bacic A. Craik D.J. Yellowlees D. Eur. J. Biochem. 1995; 232: 873-880Crossref PubMed Google Scholar, 60Dolashka-Angelova P. Beck A. Dolashki A. Beltramini M. Stevanovic S. Salvato B. Voelter W. Biochem. J. 2003; 374: 185-192Crossref PubMed Google Scholar, 61Gutternigg M. Ahrer K. Grabher-Meier H. Bürgmayr S. Staudacher E. Eur. J. Biochem. 2004; 271: 1348-1356Crossref PubMed Scopus (35) Google Scholar, 62Natsuka S. Hirohata Y. Nakakita S. Sumiyoshi W. Hase S. FEBS J. 2011; 278: 452-460Crossref PubMed Scopus (33) Google Scholar). For example, in the study by Hall et al. (52Hall R.L Wood E.J. Kamberling J.P. Gerwig G.J. Vliegenthart F.G. Biochem. J. 1977; 165: 173-176Crossref PubMed Scopus (49) Google Scholar), 3-O-MeGal was found in hemocyanin from Helix pomatia (Roman snail), and both 3-O-MeGal and 3-O-MeMan were identified in hemocyanin from Lymnaea stagnalis (a freshwater snail). In mollusks, 3-O-MeMan and/or 3-O-MeGal were found in some hemocyanins (53Kamerling J.P. Vliegenthart J.F.G. Montreuil J. Vliegenthart J.F.G. Schachter H. Glycoproteins II. Elsevier Science B.V., Amsterdam1997: 123-161Google Scholar, 54Lommerse J.P. Thomas-Oates J.E. Gielens C. Préaux G. Kamerling J.P. Vliegenthart J.F.G. Eur. J. Biochem. 1997; 249: 195-222Crossref PubMed Scopus (69) Google Scholar, 55Stoeva S. Rachev R. Severov S. Voelter W. Genov N. Comp. Biochem. Physiol. B. Biochem. Mol. Biol. 1995; 110: 761-765Crossref PubMed Scopus (24) Google Scholar, 56Stoeva S. Schütz J. Gebauer W. Hundsdörfer T. Manz C. Markl J. Voelter W. Biochim. Biophys. Acta. 1999; 1435: 94-109Crossref PubMed Scopus (31) Google Scholar), and in nematodes, O-linked glycans were shown to contain 2-O-methylated fucose (57Khoo K.H. Maizels R.M. Page A.P. Taylor G.W. Rendell N.B. Dell A. Glycobiology. 1991; 1: 163-171Crossref PubMed Scopus (88) Google Scholar, 58Guérardel Y. Balanzino L. Maes E. Leroy Y. Coddeville B. Oriol R. Strecker G. Biochem. J. 2001; 357: 167-182Crossref PubMed Scopus (99) Google Scholar). However, only a few studies have investigated the overall glycan structure (composition and monosaccharide sequence) of molecules that contain methylated sugars. For example, the structures of N-linked glycans from a lectin of the giant clam Hippopus hippopus were found to be primarily of the oligomannose type, but in addition, some contained a 6-O-methylated group on the terminal Man residue of the chain (59Puanglarp N. Oxley D. Currie G.J. Bacic A. Craik D.J. Yellowlees D. Eur. J. Biochem. 1995; 232: 873-880Crossref PubMed Google Scholar). Another study investigating the primary structures of two biantennary N-glycans of the glycoprotein from Rapana venosa (marine snail) hemocyanin showed that the glycans contain a 3-O-methyl group on the terminal Gal and/or GlcNAc (58Guérardel Y. Balanzino L. Maes E. Leroy Y. Coddeville B. Oriol R. Strecker G. Biochem. J. 2001; 357: 167-182Crossref PubMed Scopus (99) Google Scholar). A terminal 3-O-MeMan was also found in N-glycans from the gastropod Arion lusitanicus (61Gutternigg M. Ahrer K. Grabher-Meier H. Bürgmayr S. Staudacher E. Eur. J. Biochem. 2004; 271: 1348-1356Crossref PubMed Scopus (35) Google Scholar) and from the planarian Dugesia japonica (62Natsuka S. Hirohata Y. Nakakita S. Sumiyoshi W. Hase S. FEBS J. 2011; 278: 452-460Crossref PubMed Scopus (33) Google Scholar).Although the organisms described above are able to methylate terminal monosaccharides (Man and Gal) in glycans, the red microalga Porphyridium sp. is the first organism to be described that is able to methylate internal Man residues, i.e. residues other than those directly at the non-reducing terminus. The presence of three O-MeMan groups and a Xyl on the antennae in the glycans is probably also the reason that the terminal Man residues were not removed by mannosidase digestion.Xyl residues are found in N-glycans from plants (63Lerouge P. Cabanes-Macheteau M. Rayon C. Fischette-Lainé A.C. Gomord V. Faye L. Plant Mol. Biol. 1998; 38: 31-48Crossref PubMed Google Scholar), insects (64Altmann F. Staudacher E. Wilson I.B. März L. Glycoconj. J. 1999; 16: 109-123Crossref PubMed Scopus (280) Google Scholar), molluscs (53Kamerling J.P. Vliegenthart J.F.G. Montreuil J. Vliegenthart J.F.G. Schachter H. Glycoproteins II. Elsevier Science B.V., Amsterdam1997: 123-161Google Scholar), and in rare examples of parasitic helminths (65Khoo K.H. Chatterjee D. Caulfield J.P. Morris H.R. Dell A. Glycobiology. 1997; 7: 663-677Crossref PubMed Scopus (117) Google Scholar) but not normally in mammals. In addition, the position and linkage of Xyl (attached to the 2-position of the core branching Man) is the same in all the organisms mentioned above. In this study we found for the first time a Xyl residue attached to the Man of the 6-antenna and linked 1 → 3- to the penultimate GlcNAc of the core). These findings indicate the existence of special glycosyltransferases and glycosylation pathways, unique to the red microalgae. It is not known whether the Xyl residues reported here have a similar allergenic nature to the Xyl residues found in other glycans (26van Ree R. Cabanes-Macheteau M. Akkerdaas J. Milazzo J.P. Loutelier-Bourhis C. Rayon C. Villalba M. Koppelman S. Aalberse R. Rodriguez R. Faye L. Lerouge P. J. Biol. Chem. 2000; 275: 11451-11458Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar, 29Garcia-Casado G. Sanchez-Monge R. Chrispeels M.J. Armentia A. Salcedo G. Gomez L. Glycobiology. 1996; 6: 471-477Crossref PubMed Scopus (179) Google Scholar). The glycosylation pattern might be important when using the red microalgae as cell factories for biopharmaceuticals.The overall structures of the N-glycans in Porphyridium sp. are novel, but the glycans appear to exhibit a core oligomannose topology that is common in plant and yeast N-glycans. Thus, the N-glycans of the Porphyridium sp. glycoprotein combine structural features from plants and those from the giant clam H. hippopus (which also contains 6-O-Man). IntroductionN-Glycosylation is one of the most fundamental protein post-translational modifications in eukaryotes, influencing the physicochemical and biological properties of proteins (1Varki A. Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4963) Google Scholar, 2Dwek R.A. Chem. Rev. 1996; 96: 683-720Crossref PubMed Scopus (2827) Google Scholar, 3Varki A. Cummings R. Esko J. Freeze H. Hart G. Marth J. Essentials of Glycobiology. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999Google Scholar, 4Wormald M.R. Dwek R.A. Structure. 1999; 7: R155-R160Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 5Taylor M.E. Drickamer K. Introduction to Glycobiology. 2nd Ed. Oxford University Press, Oxford2003Google Scholar). To date, N-glycosylation patterns and N-glycan structures have been studied mainly in mammals, insects, yeasts, and plants (6Weerapana E. Imperiali B. Glycobiology. 2006; 16: 91R-101RCrossref PubMed Scopus (271) Google Scholar), whereas seaweeds and microalgae have received very little attention aside from a few studies conducted on green algae. These studies generally revealed the presence of similar glycans to those found in the other species. For example, a lectin blot analysis in combination with N-glycosidase F (PNGase F) 3The abbreviations used are: PNGase FN-glycosidase F2AB2-aminobenzamideDDWdouble-distilled waterEndo Hendoglycosidase HESIelectrospray ionizationGUglucose unitHexhexosem/zmass/charge ratioNPnormal phaseWAXweak anion-exchangeAraarabinoseXylxyloseFucfucose. or endoglycosidase H (Endo H) treatment on the glycoproteins of the flagellar scale of Scherffelia dubia, a member of the Chlamydomonas family, revealed the presence of both high mannose and processed N-glycans (hybrid and complex types) (7Grunow A. Becker B. Melkonian M. Eur. J. Cell Biol. 1993; 61: 10-20PubMed Google Scholar, 8Becker B. Perasso L. Kammann A. Salzburg M. Melkonian M. Planta. 1996; 199: 503-510Crossref Scopus (20) Google Scholar). Application of a similar protocol to the N-glycan structures of different flagellar strains of Tetraselmis showed that the scale-associated Tetraselmis striata glycoproteins are composed largely of high mannose glycans. On the other hand, those of Tetraselmis chui consist of many unknown complex glycans (9Becker B. Dreschers S. Melkonian M. Eur. J. Phycol. 1995; 30: 307-312Crossref Scopus (7) Google Scholar, 10Becker D. Melkonian M. Eur. J. Cell Biol. 1992; 57: 109-116PubMed Google Scholar, 11Gödel S. Becker B. Melkonian M. Protist. 2000; 151: 147-159Crossref PubMed Scopus (12) Google Scholar). A deeper insight into the glycan structures associated with the glycoprotein pheromone of the chlorophyte Volvox carteri f. nagariensis was obtained by applying a combination of exoglycosidases digestion, gas chromatographic sugar analysis, and two-dimensional HPLC separation (12Balshüsemann D. Jaenicke L. Eur. J. Biochem. 1990; 192: 231-237Crossref PubMed Scopus (27) Google Scholar). This experimental protocol showed that the N-glycans contain a chitobiose core with one to four additional Man residues with or without an additional Xyl residue attached to the 2-position of the core branching Man.The current study focuses on the structural characterization of N-glycans in the 66-kDa glycoprotein associated with the cell wall polysaccharide of Porphyridium sp., the most abundant species of red microalga of the division Rhodophyta. Porphyridium sp. has been the subject of intensive study by our group for a number of years (13Lapidot M. Shrestha R.P. Weinstein Y. Arad (Malis) S. Seckbach J. Chapman D.J. Cellular Origin, Life in Extreme Habitats, and Astrobiology: Red Algae in Genomic Age. Vol. 13. Springer, Dordrecht, The Netherlands2010: 205-225Google Scholar, 14Arad (Malis) S. Weinstein Y. Biomedic (Israel). 2003; 1: 32-37Google Scholar, 15Arad S.M. Levy-Ontman O. Curr. Opin. Biotech. 2010; 21: 358-364Crossref PubMed Scopus (176) Google Scholar, 16Arad (Malis) S. Cohen Z. Chemicals from Microalgae. Taylor and Francis, New York1999: 282-287Google Scholar, 17Arad (Malis) S. Richmond A. Richmond A. Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd, Oxford2004: 289-297Google Scholar). The cells of the red microalga are encapsulated within a cell wall complex of a polysaccharide, which includes glycoproteins. This polysaccharide complex was found to possess unique characteristics and bioactivities, which offer a vast range of potential applications (13Lapidot M. Shrestha R.P. Weinstein Y. Arad (Malis) S. Seckbach J. Chapman D.J. Cellular Origin, Life in Extreme Habitats, and Astrobiology: Red Algae in Genomic Age. Vol. 13. Springer, Dordrecht, The Netherlands2010: 205-225Google Scholar, 14Arad (Malis) S. Weinstein Y. Biomedic (Israel). 2003; 1: 32-37Google Scholar, 15Arad S.M. Levy-Ontman O. Curr. Opin. Biotech. 2010; 21: 358-364Crossref PubMed Scopus (176) Google Scholar, 16Arad (Malis) S. Cohen Z. Chemicals from Microalgae. Taylor and Francis, New York1999: 282-287Google Scholar, 17Arad (Malis) S. Richmond A. Richmond A. Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd, Oxford2004: 289-297Google Scholar). Chemical characterization of red microalgae polysaccharide revealed that it is an anionic heteropolymer (molecular mass 3–5 × 106 Da) composed of about 10 different sugars, the main ones being Xyl, Glc, and Gal, with sulfate groups located on the Glc and Gal moieties (16Arad (Malis) S. Cohen Z. Chemicals from Microalgae. Taylor and Francis, New York1999: 282-287Google Scholar, 17Arad (Malis) S. Richmond A. Richmond A. Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing Ltd, Oxford2004: 289-297Google Scholar, 18Arad (Malis) S. Stadler T. Mollion J. Verdus M.C. Karamanos Y. Morvan H. Christiaen D. Algal Biotechnology. Elsevier Applied Science, London1988: 65-87Google Scholar, 19Geresh S. Arad (Malis) S. Bioresour. Technol. 1991; 38: 195-201Crossref Scopus (116) Google Scholar, 20Heaney-Kieras J. Chapman D.J. Carbohydr. Res. 1976; 52: 169-177Crossref PubMed Scopus (60) Google Scholar, 21Lupescu N. Arad (Malis) S. Geresh S. Bernstein M. Glazer R. Carbohydr. Res. 1991; 210: 349-352Crossref Scopus (36) Google Scholar). A primary disaccharide building block, 3-O-(α-d-glucopyranosyluronic acid)-l-galactopyranose has been isolated and characterized from the polysaccharides of various red microalgae species (21Lupescu N. Arad (Malis) S. Geresh S. Bernstein M. Glazer R. Carbohydr. Res. 1991; 210: 349-352Crossref Scopus (36) Google Scholar, 22Geresh S. Lupescu N. Arad (Malis) S. Phytochemistry. 1992; 31: 4181-4186Crossref Scopus (43) Google Scholar, 23Geresh S. Dubinsky O. Arad S.M. Christiaen D. Glaser R. Carbohydr. Res. 1990; 208: 301-305Crossref PubMed Scopus (40) Google Scholar), and the chemical structure of different fractions separated from the polysaccharide of Porphyridium sp. have been determined (24Geresh S. Arad S.M. Levy-Ontman O. Zhang W. Tekoah Y. Glaser R. Carbohydr. Res. 2009; 344: 343-349Crossref PubMed Scopus (62) Google Scholar, 25Gloaguen V. Ruiz G. Morvan H. Mouradi-Givernaud A. Maes E Krausz P. Strecker G. Carbohydr. Res. 2004; 339: 97-103Crossref PubMed Scopus (45) Google Scholar).Among the proteins of the Porphyridium sp. cell wall complex that have been detected, the most prominent is a 66-kDa glycoprotein (27Shrestha R.P. Weinstein Y. Bar-Zvi D. Arad S.M. J. Phycol. 2004; 40: 568-580Crossref Scopus (30) Google Scholar, 66Shrestha R.P. A Non-covalenty Bound Cell-wall Glycoprotein of the Red Microalga Porphyridium sp.: Characterization and Functions. Ben-Gurion University of the Negev, Beer-Sheva, Israel1999Google Scholar). This glycoprotein is tightly bound, but not covalently linked, to the cell wall polysaccharide and consists of a polypeptide of ∼58 kDa and sugar moieties of ∼8 kDa. Sequencing of a cDNA clone encoding the 66-kDa glycoprotein revealed that this is a novel protein with four potential N-glycan sites that does not show similarity to any protein in the public domain databases. However, it does show some structural similarities to protein superfamilies within the carbohydrate binding domain, namely, glycosyltransferases, pectin lyase-like, sialidases, and concanavalin A-like lectins/glucanases in the SCOP databases, indicating a possible role of the 66-kDa glycoprotein in synthesis/modification of the cell wall polysaccharide (27Shrestha R.P. Weinstein Y. Bar-Zvi D. Arad S.M. J. Phycol. 2004; 40: 568-580Crossref Scopus (30) Google Scholar). Moreover, this protein was found at the early stages of the cell wall cycle intermediate (28Simon-Bercovitch B. Bar-Zvi D. Arad S.M. J. Phycol. 1999; 35: 78-83Crossref Scopus (19) Google Scholar, 67Simon-Bercovitch B. Cell-wall Formation in the Red Microalga Porphyridium sp. Ben-Gurion University of the Negev, Eilat, Israel1997Google Scholar) and in all cell wall-modified mutants. In addition, the glycoprotein was shown to play a role in biorecognition (30Ucko M. Shrestha R.P. Mesika P. Bar-Zvi D. Arad (Malis) S. J. Phycol. 1999; 35: 1276-1281Crossref Scopus (18) Google Scholar). Initial characterization of the sugar moieties by lectin blot analysis and PNGase F treatment suggested the presence of terminal Man (27Shrestha R.P. Weinstein Y. Bar-Zvi D. Arad S.M. J. Phycol. 2004; 40: 568-580Crossref Scopus (30) Google Scholar).4 The glycoprotein was also detected by a xylose-specific antibody, indicating the presence of Xyl.In the current study we characterized for the first time the N-linked glycans of the 66-kDa sugar moiety within the cell wall polysaccharide of Porphyridium sp. This study of the N-linked glycosylation patterns in Porphyridium sp. constitutes the first step in the search for enzymes involved in glycosylation-related pathways in this microalga. Because the cell wall glycoprotein was suggested to be involved in polysaccharide biosynthesis, unraveling its structure may contribute to our understanding of the biosynthesis of this polysaccharide. In addition, knowledge of the N-linked glycosylation might be of value for biotechnological applications, especially for production of therapeutic proteins, using the algae as a "cell factory," as well as for studying the evolution of glycosylation processes.