The spore coat of a fucosylation mutant in Dictyostelium discoideum

doi:10.1016/0012-1606(89)90060-2

Developmental Biology

Volume 133, Issue 2, June 1989, Pages 576-587

https://doi.org/10.1016/0012-1606(89)90060-2 Get rights and content

Abstract

Strain HL250 of Dictyostelium discoideum cannot convert GDP-mannose to GDP-fucose, resulting in an inability to fucosylate protein. This affects a group of proteins which are normally fucosylated intracellularly and then secreted via prespore vesicles to become part of the outer lamina of the spore coat. We have found that strain HL250 nevertheless accumulates typical amounts of these proteins, stores them normally in prespore vesicles, and secretes them normally to become a part of the spore coat. However, affected proteins are proteolyzed after germination, the spore coat is more accessible to penetration by a macromolecular probe, and germination is inefficient in older spores. These findings can be explained by a dependence of the integrity of the outer layer of the spore coat on protein-linked fucose.

References (30)

S. Chang et al.
An epimerase-reductase in l-fucose synthesis
J. Biol. Chem
(1988)
D.N. Cooper et al.
Discoidin-binding polysaccharide from Dictyostelium discoideum
J. Biol. Chem
(1983)
K.M. Devine et al.
Spore coat proteins of Dictyostelium discoideum are packaged in prespore vesicles
Dev. Biol
(1983)
Z. Dische et al.
A specific color reaction of methylpentoses and a spectrophotometric micromethod for their determination
J. Biol. Chem
(1948)
G.W. Erdos et al.
Formation and organization of the spore coat of Dictyostelium discoideum
Exp. Mycol
(1989)
M.R. Hardy et al.
Monosaccharide analysis of glycoconjugates by anion exchange chromatography with pulsed amperometric detection
Anal. Biochem
(1988)
D.E. Hemmes et al.
Structural and enzymatic analysis of the spore wall layers in Dictyostelium discoideum
J. Ultrastruct. Res
(1972)
R.L. Ivatt et al.
Glycoprotein biosynthesis in Dictyostelium discoideum: Developmental regulation of the protein-linked glycans
Cell
(1984)
T.Y. Lam et al.
Synthesis of stage-specific glycoproteins in Dictyostelium discoideum during development
Dev. Biol
(1981)
W.F. Loomis
Genetic tools for Dictyostelium
Methods Cell Biol
(1987)

K. Olden et al.

Carbohydrate moieties of glycoproteins

Biochim. Biophys. Acta

(1982)

M. Orlowski et al.

Plasma membrane proteins of Dictyostelium: The spore coat proteins

Dev. Biol

(1979)

M.L. Reitman et al.

Mouse lymphoma cell lines resistant to pea lectin are defective in fucose metabolism

J. Biol. Chem

(1980)

J. Ripka et al.

Two chinese hamster ovary glycosylation mutants affected in the conversion of GDP-mannose to GDP-fucose

Arch. Biochem. Biophys

(1986)

C.M. West et al.

The expression of glycoproteins in the extracellular matrix of the cellular slime mold Dictyostelium discoideum

Cell Differ

(1988)

Cited by (34)

Role of a cytoplasmic dual-function glycosyltransferase in O<inf>2</inf> regulation of development in Dictyostelium
2009, Journal of Biological Chemistry
Citation Excerpt :
Strains carrying disruptions of phyA/P4H1 (3), pgtA (5), or agtA (7) have been described, and these strains accumulate unmodified Skp1 or Skp1 bearing a glycan consisting of one or three sugars, respectively, compared with the pentasaccharide that accumulates in the parental normal strain (Fig. 2A). In addition, a gmd− strain (HL250) with a deficiency in conversion of GDP-Man to GDP-Fuc accumulates Skp1 with a disaccharide (13, 14, 16). Because this strain is defective in all fucosylation, we reexamined a previously constructed strain in which the N-terminal domain of the PgtA diglycosyltransferase (PgtA-N) is constitutively expressed in a pgtA-null background, and whose extracts exhibit β3-GalT but not α2FucT activity as expected (5).
In the social amoeba Dictyostelium, a terminal step in development is regulated by environmental O₂. Prolyl 4-hydroxylase-1 (P4H1) was previously implicated in mediating the O₂ signal, and P4H1-null cells require elevated O₂ to culminate. The E3-ubiquitin ligase adaptor Skp1 is a P4H1 substrate, and here we investigate the function of PgtA, a dual function β3-galactosyltransferase/α2-fucosyltransferase that contributes the 2nd and 3rd sugars of the pentasaccharide cap formed on Skp1 hydroxyproline. Although pgtA-null cells, whose Skp1 contains only a single sugar (N-acetylglucosamine or GlcNAc), show wild-type O₂ dependence of culmination, cells lacking AgtA, an α3-galactosyltransferase required to extend the trisaccharide, require elevated O₂ as for P4H1-null cells. Skp1 is the only detectable protein modified by purified PgtA added to pgtA-null extracts. The basis for specificity of PgtA was investigated using native Skp1 acceptor glycoforms and a novel synthetic peptide containing GlcNAcα1,4-hydroxy(trans)proline. Cysteine-alkylation of Skp1 strongly inhibited modification by the PgtA galactosyltransferase but not the fucosyltransferase. Furthermore, native and synthetic Skp1 glycopeptides were poorly galactosylated, not processively fucosylated, and negligibly inhibitory, whereas the fucosyltransferase was active toward small substrates. In addition, the galactosyltransferase exhibited an atypical concentration dependence on UDP-galactose. The results provide the first evidence that Skp1 is the functional target of P4H1 in O₂ regulation, indicate a gatekeeper function for the β3-galactosyltransferase in the PgtA dual reaction, and identify an unexpected P4H1-dependent yet antagonistic function for PgtA that is reversed by AgtA.
Comparative analysis of spore coat formation, structure, and function in Dictyostelium
2003, International Review of Cytology
Dictyostelium produces spores at the end of its developmental cycle to propagate the lineage. The spore coat is an essential feature of spore biology contributing a semipermeable chemical and physical barrier to protect the enclosed amoeba. The coat is assembled from secreted proteins and a polysaccharide, and from cellulose produced at the cell surface. They are organized into a polarized molecular sandwich with proteins forming layers surrounding the microfibrillar cellulose core. Genetic and biochemical studies are beginning to provide insight into how the deliveries of protein and cellulose to the cell surface are coordinated and how cysteine-rich domains of the proteins interact to form the layers. A multidomain inner layer protein, SP85/PsB, seems to have a central role in regulating coat assembly and contributing to a core structural module that bridges proteins to cellulose. Coat formation and structure have many parallels in walls from plant, algal, yeast, protist, and animal cells.
A bifunctional diglycosyltransferase forms the Fucα1,2Galβ1,3-disaccharide on Skp1 in the cytoplasm of Dictyostelium
2002, Journal of Biological Chemistry
Citation Excerpt :
The modified plasmids were transfected into E. coli strain ER2566, expression was induced, and cells were lysed also as before (7). α1,2-Fuc-Tase activity was assayed as described previously (7) using GDP-[2-3H]Fuc (17.3 Ci/mmol, diluted 5–20-fold with unlabeled GDP-Fuc; New England Nuclear) as the donor and either 0.36 mmGalβ1,3GlcNAcα1-pNP (pNP-GlcNAc-Gal) or Skp1-GlcNAc-Gal purified to near homogeneity from Dictyostelium strain HL250, which is unable to synthesize GDP-Fuc (14). Reactions were conducted in a 50 μl volume containing 500 μg/ml bovine serum albumin, 50 mm HEPES-NaOH (pH 7.5), 100 mm NaCl, 10 mm MgCl2, 2 mm MnCl2, 0.1 mm EDTA, 5 mm dithiothreitol, and 0.05% Tween 20 at 30 °C for the time indicated.
Skp1 is a subunit of the Skp1 cullin-1 F-box protein (SCF) family of E3 ubiquitin ligases and of other regulatory complexes in the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by a pentasaccharide with the type I blood group H antigen (Fucα1,2Galβ1,3GlcNAc-) at its core. Addition of the Fuc is catalyzed by FT85, a 768-amino acid protein whose fucosyltransferase activity maps to the C-terminal half of the protein. A strain whose FT85 gene is interrupted by a genetic insertion produces a truncated, GlcNAc-terminated glycan on Skp1, suggesting that FT85 may also have β-galactosyltransferase activity. In support of this model, highly purified native and recombinant FT85 are each able to galactosylate Skp1 from FT85 mutant cells. Site-directed mutagenesis of predicted key amino acids in the N-terminal region of FT85 abolishes Skp1 β-galactosyltransferase activity with minimal effects on the fucosyltransferase. In addition, a recombinant form of the N-terminal region exhibits β-galactosyltransferase but not fucosyltransferase activity. Kinetic analysis of FT85 suggests that its two glycosyltransferase activities normally modify Skp1 processively but can have partial function individually. In conclusion, FT85 is a bifunctional diglycosyltransferase that appears to be designed to efficiently extend the Skp1 glycan in vivo.
A Non-Golgi α1,2-Fucosyltransferase That Modifies Skp1 in the Cytoplasm of Dictyostelium
2001, Journal of Biological Chemistry
Citation Excerpt :
Clonal strain HW260 was analyzed by PCR using FT85-specific primers P9 and P10 that flanked the bsrinsertion site and was found to contain an insertion in theFT85 gene whose length corresponded to that of thebsr locus (Fig.7 A). Skp1 produced by mutant strain HW260 migrated slightly ahead of normal Skp1 on a SDS-PAGE gel (Fig. 7 B), close to the position of Skp1 produced by a strain (HL250) unable to fucosylate Skp1 (3) because of a mutation in GDP-Fuc synthesis (26). To verify that Skp1(HW260) was not fucosylated, extracts of HW260 and normal strain Ax3 cells were compared after metabolic-labeling with [3H]Fuc during growth in HL-5 or differentiation in KP buffer.
Skp1 is a subunit of the SCF-E3 ubiquitin ligase that targets cell cycle and other regulatory factors for degradation. In Dictyostelium, Skp1 is modified by a pentasaccharide containing the type 1 blood group H trisaccharide at its core. To address how the third sugar, fucose α1,2-linked to galactose, is attached, a proteomics strategy was applied to determine the primary structure of FT85, previously shown to copurify with the GDP-Fuc:Skp1 α1,2-fucosyltransferase. Tryptic-generated peptides of FT85 were sequenced de novo using Q-TOF tandem mass spectrometry. Degenerate primers were used to amplify FT85 genomic DNA, which was further extended by a novel linker polymerase chain reaction method to yield an intronless open reading frame of 768 amino acids. Disruption of the FT85 gene by homologous recombination resulted in viable cells, which had altered light scattering properties as revealed by flow cytometry. FT85 was necessary and sufficient for Skp1 fucosylation, based on biochemical analysis of FT85 mutant cells and Escherichia coli that express FT85 recombinantly. FT85 lacks sequence motifs that characterize all other known α1,2-fucosyltransferases and lacks the signal-anchor sequence that targets them to the secretory pathway. The C-terminal region of FT85 harbors motifs found in inverting Family 2 glycosyltransferase domains, and its expression in FT85 mutant cells restores fucosyltransferase activity toward a simple disaccharide substrate. Whereas most prokaryote and eukaryote Family 2 glycosyltransferases are membrane-bound and oriented toward the cytoplasm where they glycosylate lipid-linked or polysaccharide precursors prior to membrane translocation, the soluble, eukaryotic Skp1-fucosyltransferase modifies a protein that resides in the cytoplasm and nucleus.
Spore coat formation and timely sporulation depend on cellulose in Dictyostelium
2001, Differentiation
Cellulose is a major component of the extracellular coat that surrounds the terminally-differentiated spore of Dictyostelium. It is sandwiched between two layers of proteins that derive from prespore vesicles by exocytosis. Strains unable to synthesize cellulose due to null mutations in the gene encoding the catalytic subunit of cellulose synthase (dcsA) failed to make detergent-resistant spores but produced small, highly refractile, round spore-like cells up to a day late. Although these cells resembled spores in appearance, they were unstable, only transiently ellipsoid in shape, and sensitive to hypo-osmotic shock, drying, or detergents. Differentiation of these pseudo-spores was induced in the normal time frame by activation of the cAMP-dependent protein kinase or co-development with wild type cells, and coat proteins were secreted by the dcsA-null cells at the same time as wild type cells. A substantial fraction of secreted coat proteins was loosely associated with the surface of the mutant cells, resembling the precoat posited to form early during normal sporulation. Transmission electron microscopy revealed that the precoat had little ultrastructural organization in the absence of cellulose. Thus, cellulose in the coat appears to be required for the organization of the precoat precursors as well as the stability, dormancy, and shape of the spore.
Multiple O-glycoforms on the spore coat protein sp96 in Dictyostelium discoideum. Fuc(α1-3)GlcNAc-α-1-P-Ser is the major modification
2000, Journal of Biological Chemistry
Citation Excerpt :
Spores are surrounded by three different layers, in which proteins are embedded, which build up a shield and protect the dormant amoeba from environmental stress. Some of the proteins carry post-translational modifications, and previous studies have shown that a lack in fucosylation on these results in increased permeability of the spore coat, which makes the spores less viable with time (17, 46). There is emerging a picture of the assembly of the spore coat, involving a complex of proteins (7) and cellulose binding (9,10).
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 fucosylation-defective mutant (HU2470). A novel phosphoglycan structure on SP96 of the wild type strain, consisting of Fuc(α1–3)GlcNAc-α-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 identifyO-linked 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-linkedN-acetylglucosamine-fucose disaccharide units and a further approximately 20 sites with fucose directly linked to the protein. Over 70% of the serine sites are modified, with less than 1% of these sites as phosphoserine. Threonine and tyrosine residues were not found to be modified.

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^☆: This work was supported by NIH Grants GM-33015 and GM-37539.

³: M. Girard was supported by NIH training Grant HL-07489-08.

⁴: M. Gritzali and R. D. Brown received support from a program cofunded by the Gas Research Institute and the Institute of Food and Agricultural Sciences.

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Full paperThe spore coat of a fucosylation mutant in Dictyostelium discoideum☆

Abstract

J. Biol. Chem

J. Biol. Chem

Dev. Biol

J. Biol. Chem

Exp. Mycol

Anal. Biochem

J. Ultrastruct. Res

Cell

Dev. Biol

Methods Cell Biol

Biochim. Biophys. Acta

Dev. Biol

J. Biol. Chem

Arch. Biochem. Biophys

Cell Differ

Full paper
The spore coat of a fucosylation mutant in Dictyostelium discoideum☆