Glycobiology - current issue

Meeting Announcements

2008-04-14

Erratum

2008-04-14

Differentiating N-linked glycan structural isomers in metastatic and nonmetastatic tumor cells using sequential mass spectrometry

2008-04-14

In an effort to understand the role of molecular glycosylation in cancer a murine model has been used to characterize and fingerprint malignancies in established cell lines that manifest all the hallmarks of metastatic disease: spontaneous development, local invasion, intravasation, immune system survival, extravasation, and secondary tumor formation involving liver, kidney, spleen, lung, and brain. Using astrocyte cell controls, we compared N-linked glycosylation from a nonmetastatic brain tumor cell line and two different metastatic brain tumor cells. Selected ions in each profile were disassembled by ion trap mass spectrometry (MSⁿ) which exhibited multiple structural differences between each tissue. These unique structures were identified within isomeric compositions as pendant nonreducing termini of di- and trisaccharide fragments, probably transparent to a tandem MS approach but distinctively not to sequential ion trap MSⁿ detection.

Deletion of the TbALG3 gene demonstrates site-specific N-glycosylation and N-glycan processing in Trypanosoma brucei

Manthri, S., Guther, M L. S, Izquierdo, L., Acosta-Serrano, A., Ferguson, M. A J — 2008-04-14

We recently suggested a novel site-specific N-glycosylation mechanism in Trypanosoma brucei whereby some protein N-glycosylation sites selectively receive Man₉GlcNAc₂ from Man₉GlcNAc₂-PP-Dol while others receive Man₅GlcNAc₂ from Man₅GlcNAc₂-PP-Dol. In this paper, we test this model by creating procyclic and bloodstream form null mutants of TbALG3, the gene that encodes the -mannosyltransferase that converts Man₅GlcNAc₂-PP-Dol to Man₆GlcNAc₂-PP-Dol. The procyclic and bloodstream form TbALG3 null mutants grow with normal kinetics, remain infectious to mice and tsetse flies, respectively, and have normal morphology. However, both forms display aberrant N-glycosylation of their major surface glycoproteins, procylcin, and variant surface glycoprotein, respectively. Specifically, procyclin and variant surface glycoprotein N-glycosylation sites that are modified with Man₉GlcNAc₂ and processed no further than Man₅GlcNAc₂ in the wild type are glycosylated less efficiently but processed to complex structures in the mutant. These data confirm our model and refine it by demonstrating that the biantennary glycan transferred from Man₅GlcNAc₂-PP-Dol is the only route to complex N-glycans in T. brucei and that Man₉GlcNAc₂-PP-Dol is strictly a precursor for oligomannose structures. The origins of site-specific Man₅GlcNAc₂ or Man₉GlcNAc₂ transfer are discussed and an updated model of N-glycosylation in T. brucei is presented.

Different affinity of galectins for human serum glycoproteins: Galectin-3 binds many protease inhibitors and acute phase proteins

2008-04-14

Here we report the first survey of galectins binding to glycoproteins of human serum. Serum was subjected to affinity chromatography using immobilized galectins, and the bound glycoproteins were analyzed by electrophoresis, Western blotting, and mass spectrometry. Galectins-3, -8, and -9 bound a much broader range of ligands in serum than previously known, galectin-1 bound less, and galectins-2, -4, and -7 bound only traces or no serum ligands. Galectin-3 bound most major glycoproteins, including alpha-2-macroglobulin and acute phase proteins such as haptoglobin. It bound only a selected minor fraction of transferrin, and bound none or little of IgG. Galectins-8 and -9 bound a similar range of glycoproteins as galectin-3, but in lower amounts, and galectin-8 had a relative preference for IgA. Galectin-1 bound mainly a fraction of alpha-2-macroglobulin and only traces of other glycoproteins. The binding of galectin-3 to serum glycoproteins requires affinity for LacNAc, since a mutant (R186S), which has lost this affinity, did not bind any serum glycoproteins. The average affinity of galectin-3 for serum glycoproteins was estimated to correspond to K_d ~1–5 µM by modeling of the affinity chromatography and a fluorescence anisotropy assay. Since galectins are expressed on endothelial cells and other cells exposed to serum components, this report gives new insight into function of galectins and the role of their different fine specificity giving differential binding to the serum glycoproteins.

The AP-2{alpha} transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of a PTEN gene

2008-04-14

Ganglioside GM3 inhibits the growth of several cancer cells and induces cell cycle arrest by regulating cellular signal pathways. Our previous results have shown that GM3 suppresses tumor suppressor PTEN-mediated cancer cell proliferation. However, the precise molecular mechanism(s) for the transcriptional regulation of a PTEN gene induced by GM3 remains unclear. Here, we show, for the first time, that GM3 induces transcription factor AP-2-mediated PTEN expression in colon cancer cells. The enhanced expression of PTEN by GM3 in both HCT116 and p53-null HCT116 cells has been shown to be not associated with p53 function. Thus, to further determine the mechanism underlying the regulation of PTEN gene expression by GM3, we characterized the promoter region of the PTEN gene. Promoter analysis of the 5'-flanking region of the PTEN gene showed that the region between –1175 and –1077 from the translational initiation site, which contains the AP-2 binding site, functions as the GM3-inducible promoter in colon cancer cells. Furthermore, gel shift assays, site-directed mutagenesis, and chromatin immunoprecipitation assay obviously indicated that the AP-2 is essential for the expression of PTEN in GM3-stimulated colon cancer cells. Moreover, siRNA against AP-2 diminished the enhancement of AP-2 and PTEN expressions in GM3-induced colon cancer cells. The transient expression of AP-2 also results in the induction of PTEN transcription in AP-2-negative colon cancer cells. Additionally, GM3 induced AP-2-mediated PTEN expression through the inhibition of autocrine-ligand-mediated EGFR activation. These results suggest that the AP-2 transcription factor is required for the ganglioside GM3-stimulated transcriptional regulation of the PTEN gene.

Roles of complex gangliosides in the development of experimental autoimmune encephalomyelitis

Miyamoto, K., Takada, K., Furukawa, K., Furukawa, K., Kusunoki, S. — 2008-04-14

We induced experimental autoimmune encephalomyelitis (EAE) in GM2/GD2 synthase knockout mice (GM2/GD2^–/–), which cannot synthesize complex gangliosides, such as GM1, GD1a, GD1b, GT1b, and GQ1b, to investigate the roles of complex gangliosides in the pathogenesis of this disease. We used myelin-oligodendrocyte glycoprotein (MOG) as an immunogen. In active immunization EAE, the severity of clinical score was not different but the disease onset was significantly delayed in GM2/GD2^–/– compared with those in wild-type mice. When we transferred MOG-reactive T cells from GM2/GD2^–/– or wild-type mice to wild-type mice, no significant differences were observed between the two groups. In contrast, when we transferred MOG-reactive T cells from wild-type mice to GM2/GD2^–/– or to wild-type mice, the onset of EAE in GM2/GD2^–/– mice was delayed. The recall response of MOG-specific T cells, the function of antigen presenting cells, or the expression of several adhesion molecules in the endothelium were not significantly different between GM2/GD2^–/– and wild-type mice. On the other hand, quantitative analysis of cellular infiltration in the central nervous system (CNS) on day 9 of active immunization EAE showed that the CD4⁺ cell number in the CNS isolated from GM2/GD2^–/– mice was significantly less than that from wild-type mice. It indicated that the delayed onset of EAE in GM2/GD2^–/– mice was due to the delay of the migration of pathogenic T cells into the CNS. Thus, the complex gangliosides may be involved in the T cell–endothelial cell interaction in the pathogenetic process of EAE.

A new type of plant chitinase containing LysM domains from a fern (Pteris ryukyuensis): Roles of LysM domains in chitin binding and antifungal activity

Onaga, S., Taira, T. — 2008-04-14

Chitinase-A (PrChi-A), of molecular mass 42 kDa, was purified from the leaves of a fern (P. ryukyuensis) using several column chromatographies. The N-terminal amino acid sequence of PrChi-A was similar to the lysin motif (LysM). A cDNA encoding PrChi-A was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1459 nucleotides and encoded an open-reading frame of 423-amino-acid residues. The deduced amino acid sequence indicated that PrChi-A is composed of two N-terminal LysM domains and a C-terminal catalytic domain, belonging to the group of plant class IIIb chitinases, linked by proline, serine, and threonine-rich regions. Wild-type PrChi-A had chitin-binding and antifungal activities, but a mutant without LysM domains had lost both activities. These results suggest that the LysM domains contribute significantly to the antifungal activity of PrChi-A through their binding activity to chitin in the cell wall of fungi. This is the first report of the presence in plants of a family-18 chitinase containing LysM domains.