Development of Dictyostelium discoideum is associated with alteration of fucosylated N-glycan structures (original) (raw)
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Cell surface oligosaccharides on Dictyostelium during development
Journal of Cell Science, 1991
Developmental studies of the changes in protein glycosylation are useful in elucidating the role of oligosaccharides in biological events. We have used the chemical technique, hydrazinolysis, to release oligosaccharides from cell surface glycoproteins of Dictyostelium discoideum. Oligomannose type, xylose- and fucose-containing oligosaccharides were found to be present. The charged oligosaccharides contained sulphate and mannose 6-phosphate residues; no sialic acid was detected. The charged oligosaccharides also contained significant amounts of xylose, arabinose, fucose and galactose, as well as mannose and N-acetylglucosamine, which were the main constituents of the neutral glycans. By monitoring the chemical characteristics of the liberated oligosaccharides, dramatic changes in both the charge and size distribution of cell surface oligosaccharides were observed throughout the 24 h period of cell development. A comparison, however, between the neutral glycan structures of prestalk ...
Journal of Biological Chemistry, 2000
A decreased level of fucosylation on certain spore coat proteins of Dictyostelium discoideum alters the permeability of the spore coat. Here the post-translational modifications of a major spore coat protein, SP96, are studied in a wild type strain (X22) and a fucosylationdefective mutant (HU2470). A novel phosphoglycan structure on SP96 of the wild type strain, consisting of Fuc(␣1-3)GlcNAc-␣-1-P-Ser , was identified by electrospray ionization mass spectrometry and NMR. It was shown using monosaccharide and gas chromatography mass spectrometry analysis that SP96 in the mutant HU2470 contained approximately 20% of wild type levels of fucose, as a result of a missing terminal fucose on the novel glycan structure. The results support previous predictions, based on inhibition studies on different fucose-deficient strains, about the nature of monoclonal antibody epitopes identified by monoclonal antibodies MUD62 and MUD166, which are known to identify Olinked glycans (Champion, A., Griffiths, K., Gooley, A. A., Gonzalez, B. Y., Gritzali, M., West, C. M., and Williams, K. L. (1995) Microbiology 141, 785-797). Quantitative studies on wild type SP96 indicated that there were approximately 60 sites with phosphodiester-linked Nacetylglucosamine-fucose disaccharide units and a further approximately 20 sites with fucose directly linked to the protein.
The Journal of biological chemistry, 1987
Previous studies have shown that the majority of the asparagine-linked high mannose oligosaccharide units present on the lysosomal enzymes of the slime mold Dictyostelium discoideum contain mannose 6-sulfate and mannose 6-phosphate residues, the latter being diesterified to methyl groups. Using a variety of techniques, including NMR spectroscopy and fast atom bombardment mass spectrometry, we have found that many of these oligosaccharides also contain an N-acetylglucosamine residue linked beta 1-4 to the mannose residue linked alpha 1-6 to the beta-linked core mannose. (Formula: see text) As far as we are aware, this is the first description of an intersecting N-acetylglucosamine residue in this position on a high mannose oligosaccharide.
The Journal of biological chemistry, 1987
Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cell...
The glycoproteins of Dictyostelium discoideum
Experimental Cell Research, 1979
The glycoproteins ofD. discoideum have been analyzed by direct binding of radio-iodinated lectins to SDS gels of the successive developmental stages. Compared with the total pattern of proteins, many changes are found in the glycoproteins during development. WGA reacts with few gel bands from the vegetative cells and most of these, including a very intense band at the top of the gel, are lost during the first few hours of development. Approximately halfway through the developmental cycle at least 14 new glycoproteins reacting with WGA begin to appear and progressively accumulate. In contrast, ConA labels many glycoproteins over the complete molecular weight range and most are unaffected during development. Lectins which bind fucose label a single component at the top of the gel of vegetative cells and this decreases rapidly as development begins. No other reactive gel bands are revealed by fucose-binding lectins until the final stages of spore and stalk formation, when four high molecular weight glycoproteins are detected. Lectins specific for terminal galactose residues and for N-acetyl-galactosamine, including the intrinsic lectins produced by D. discoideum during its development, failed to reveal any reactive glycoproteins.
Journal of Proteome Research, 2013
The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the Nglycomic potential of this fascinating model organism.
European Journal of Biochemistry, 1998
One class of O-glycosylation in the simple eukaryote Dictyostelium discoideum involves the addition of a single N-acetylglucosamine residue to Ser and Thr residues on secreted or membrane-bound proteins at an early stage of development. A previously developed in vivo approach for the identification of acceptor sites for O-glycosylation was used to further characterise the specificity of the UDP-GlcNAc:polypeptide N-acetylglucosaminyltransferase(s). Glutathione S-transferase fusion proteins were constructed to express and secrete the mucin peptide repeat for MUC1 (PDT 1 RPAPGS 1 T 2 APPAHGVT 3 S 2 A) and a MUC2-like peptide (PT 1 T 2 T 3 PIT 4 T 5 T 6 T 7 T 8 T 9 VT 10 PT 11 PT 12 PT 13 GT 14 QT 15 ), respectively (superscript numbers indicate residues with the potential to be glycosylated). Monosaccharide analysis, electrospray-ionisation mass spectrometry and protein sequencing showed that the modification is a single N-acetylglucosamine attached to certain Thr residues. The MUC1 repeat was glycosylated on T 2 and T 3 and there were no modifications on T 1 or on S 1 and S 2 . The MUC2 glycopeptide was glycosylated on T 1 , T 3 , T 5 , T 7 , T 9 , T 10 , T 11 , T 12 , T 13 and T 14 . Our results show that the D. discoideum glycosylation apparatus incorporates GlcNAc residues into peptide sequences similar to those reported for the addition of GalNAc residues in mammalian tissues. The anomeric linkage of the GlcNAc residues to the polypeptide chain was shown to be in A configuration as determined by NMR studies.
The EMBO journal, 1987
The functions of type 1 and 2 carbohydrates of the contact site A (csA) glycoprotein of Dictyostelium discoideum have been investigated using mutants lacking type 2 carbohydrate. In two mutant strains, HG220 and HG701, a 68-kd glycoprotein was synthesized as the final product of csA biosynthesis. This glycoprotein accumulated to a much lower extent on the surfaces of mutant cells than the mature 80-kd glycoprotein did in wild-type cells. There was also no accumulation of the 68-kd glycoprotein observed within the mutant cells nor was a precursor of lower molecular mass detected, in accordance with previous findings that indicated cotranslational linkage of type 1 carbohydrate by N-glycosylation. Pulse-chase labelling showed that a 50-kd glycopeptide was cleaved off from the mutant 68-kd glycoprotein and released into the medium, while the fully glycosylated 80-kd glycoprotein of the wild type was stable. These results assign a function to type 2 carbohydrate in protecting the cell-s...