Structural analysis of N‐ glycans of the planarian Dugesia japonica

N-Acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of the bisecting GlcNAc branch on N-glycans, is usually described as a metastasis suppressor. Overexpression of GnT-III inhibited migration in multiple types of tumor cells. However, these results seem controversial to the clinical observations for the increased expression of GnT-III in human hepatomas, glioma, and ovarian cancers. Here, we present evidence that these inconsistencies are mainly attributed to the different expression pattern of cell sialylation. In detail, we show that overexpression of GnT-III significantly inhibits ␣2,3-sialylation but not ␣2,6-sialylation. The migratory ability of cells without or with a low level of ␣2,6-sialylation is consistently suppressed after GnT-III overexpression. In contrast, the effects of GnT-III overexpression are variable in tumor cells that are highly ␣2,6-sialylated. Overexpression of GnT-III promotes the cell migration in glioma cells U-251 and hepatoma cells HepG2, although it has little influence in human breast cancer cell MDA-MB-231 and gastric cancer cell MKN-45. Interestingly, up-regulation of ␣2,6-sialylation by overexpressing ␤-galactoside ␣2,6-sialyltranferase 1 in the ␣2,6-hyposialylated HeLa-S3 cells abolishes the anti-migratory effects of GnT-III. Conversely, depletion of ␣2,6-sialylation by knock-out of ␤-galactoside ␣2,6-sialyltranferase 1 in ␣2,6-hypersialylated HepG2 cells endows GnT-III with the anti-migratory ability. Taken together, our data clearly demonstrate that high expression of ␣2,6-sialylation on the cell surface could affect the anti-migratory role of GnT-III, which provides an insight into the mechanistic roles of GnT-III in tumor metastasis.Alterations in N-linked glycosylation have been observed in various malignant diseases such as cancer. Certain glycan structures resulting from the dysregulated glycosyltransferases and glycosidases are well known tumor markers and closely associated with the malignant progression (1). As examples, the ␣1,6-fucosylated ␣-fetoprotein catalyzed by ␣1,6-fucosyltransferase has been developed as a biomarker for primary hepatoma by Abelev (2). An increase in ␤1,6-GlcNAc branching of N-glycans as a consequence of enhanced expression of N-acetylglucosaminyltransferase V (GnT-V) 2 is convincingly correlated with the increased frequency of tumor metastasis (3-6). N-Acetylglucosaminyltransferase III (GnT-III) is one more important glycosyltransferase that has been receiving much attention for its involvement in the biology of tumor (7,8). It catalyzes the transfer of N-acetylglucosamine (GlcNAc) in a ␤1,4 linkage to mannose on N-glycans forming a bisecting GlcNAc structure. This step plays critical roles in determining the structure of N-glycans because introduction of the bisecting GlcNAc residue could preclude further processing and elongation of N-glycans, such as the formation of ␤1,6-GlcNAc branching structures (9). Considering also that overexpression of GnT-III inhibited the metastasis of multiple types of carcinomas (10, 1...

Section: Methodsmentioning

confidence: 99%

Expression of N-Acetylglucosaminyltransferase III Suppresses α2,3-Sialylation, and Its Distinctive Functions in Cell Migration Are Attributed to α2,6-Sialylation Levels

Isaji

et al. 2016

“…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 (58). A terminal 3-O-MeMan was also found in N-glycans from the gastropod Arion lusitanicus (61) and from the planarian Dugesia japonica (62).…”

Section: Discussionmentioning

confidence: 99%

“…Several studies have reported methylated sugars as constituents of glycoproteins in other organisms, including nematodes, yeasts, snails, algae, and planarians (51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61)(62). For example, in the study by Hall et al (52), 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).…”

Section: Discussionmentioning

confidence: 99%

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

Levy‐Ontman

Arad

Harvey

et al. 2011

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.

“…Samples were run on 12.6% gels. The separated proteins were transferred to PVDF membranes, followed by immunodetection with antibodies against phospho-p44/42 MAPK (Thr Purification and Structural Analysis of N-Glycans-Preparation and purification of pyridylaminated (PA-)N-glycans from glycopeptides derived from TIB-141 were carried out as described previously (25,26), except that the PA-glycans were first subjected to affinity chromatography on an hG9Null-immobilized column as described above. PA-glycans dissolved in TBS were applied to the column.…”

Section: Methodsmentioning

confidence: 99%

“…S3). Structural analysis of the PA-N-glycans was carried out according to the method of Natsuka et al (25) (i.e. consecutive glycosidase digestion and RP-HPLC analysis).…”

Section: Methodsmentioning

confidence: 99%

Cooperative Interactions of Oligosaccharide and Peptide Moieties of a Glycopeptide Derived from IgE with Galectin-9

Nakakita¹,

Itoh²,

Nakakita³

et al. 2016

Self Cite

We previously showed that galectin-9 suppresses degranulation of mast cells through protein-glycan interaction with IgE. To elucidate the mechanism of the interaction in detail, we focused on identification and structural analysis of IgE glycans responsible for the galectin-9-induced suppression using mouse monoclonal IgE (TIB-141). TIB-141 in combination with the antigen induced degranulation of RBL-2H3 cells, which was almost completely inhibited by human and mouse galectin-9. Sequential digestion of TIB-141 with lysyl endopeptidase and trypsin resulted in the identification of a glycopeptide (H-Lys13-Try3; 48 amino acid residues) with a single N-linked oligosaccharide near the N terminus capable of neutralizing the effect of galectin-9 and another glycopeptide with two N-linked oligosaccharides (H-Lys13-Try1; 16 amino acid residues) having lower activity. Enzymatic elimination of the oligosaccharide chain from H-Lys13-Try3 and H-Lys13-Try1 completely abolished the activity. Removal of the C-terminal 38 amino acid residues of H-Lys13-Try3 with glutamyl endopeptidase, however, also resulted in loss of the activity. We determined the structures of N-linked oligosaccharides of H-Lys13-Try1. The galectin-9-binding fraction of pyridylaminated oligosaccharides contained asialo-and monosialylated bi/tri-antennary complex type oligosaccharides with a core fucose residue. The structures of the oligosaccharides were consistent with the sugar-binding specificity of galectin-9, whereas the nonbinding fraction contained monosialylated and disialylated biantennary complex type oligosaccharides with a core fucose residue. Although the oligosaccharides linked to H-Lys13-Try3 could not be fully characterized, these results indicate the possibility that cooperative binding of oligosaccharide and neighboring polypeptide structures of TIB-141 to galectin-9 affects the overall affinity and specificity of the IgElectin interaction.