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Originally published In Press as doi:10.1074/jbc.M308756200 on October 9, 2003

J. Biol. Chem., Vol. 278, Issue 51, 51395-51407, December 19, 2003
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Initiation of Mucin-type O-Glycosylation in Dictyostelium Is Homologous to the Corresponding Step in Animals and Is Important for Spore Coat Function*

Fei Wang{ddagger}, Talibah Metcalf{ddagger}§, Hanke van der Wel{ddagger}§, and Christopher M. West{ddagger}§

From the {ddagger}Department of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, Florida 32610-0235 and the §Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

Received for publication, August 7, 2003 , and in revised form, October 9, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Like animal cells, many unicellular eukaryotes modify mucin-like domains of secretory proteins with multiple O-linked glycans. Unlike animal mucin-type glycans, those of some microbial eukaryotes are initiated by {alpha}-linked GlcNAc rather than {alpha}-GalNAc. Based on sequence similarity to a recently cloned soluble polypeptide hydroxyproline GlcNAc-transferase that modifies Skp1 in the cytoplasm of the social ameba Dictyostelium, we have identified an enzyme, polypeptide {alpha}-N-acetylglucosaminyltransferase (pp {alpha}-GlcNAc-T2), that attaches GlcNAc to numerous secretory proteins in this organism. Unlike the Skp1 GlcNAc-transferase, pp {alpha}-GlcNAc-T2 is predicted to be a type 2 transmembrane protein. A highly purified, soluble, recombinant fragment of pp {alpha}-GlcNAc-T2 efficiently transfers GlcNAc from UDP-GlcNAc to synthetic peptides corresponding to mucin-like domains in two proteins that traverse the secretory pathway. pp {alpha}-GlcNAc-T2 is required for addition of GlcNAc to peptides in cell extracts and to the proteins in vivo. Mass spectrometry and Edman degradation analyses show that pp {alpha}-GlcNAc-T2 attaches GlcNAc in {alpha}-linkage to the Thr residues of all the synthetic mucin repeats. pp {alpha}-GlcNAc-T2 is encoded by the previously described modB locus defined by chemical mutagenesis, based on sequence analysis and complementation studies. This finding establishes that the many phenotypes of modB mutants, including a permeability defect in the spore coat, can now be ascribed to defects in mucin-type O-glycosylation. A comparison of the sequences of pp {alpha}-GlcNAc-T2 and the animal pp {alpha}-GalNAc-transferases reveals an ancient common ancestry indicating that, despite the different N-acetylhexosamines involved, the enzymes share a common mechanism of action.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Extracellular mucin proteins are employed throughout the eukaryotic kingdom as physical and chemical protective barriers with signaling functions at the cell surface (1-6). Mucin-type domains in these proteins are characterized by multiple repeats of short polypeptide sequences rich in the amino acids Thr, Ser, Pro, Hyp (hydroxyproline), Gly, and/or Val, and the hydroxyamino acids are subject to extensive O-glycosylation. In animals, the O-glycans are anchored via {alpha}-linked GalNAc residues for which additions are catalyzed by members of a well characterized family of UDP-GalNAc:Thr/Ser mucin-type pp {alpha}-GalNAc-Tases1 (7-9). The GalNAc is normally extended by Gal and/or other sugars, which are capped by anionic substituents such as sialic acid or SO4. The O-glycans impose conformational rigidity to the polypeptide (10), protect it from proteolysis (11), and may constitute ligands for receptors or decoys for parasites (1-6). Mucin proteins also typically have N- and/or C-terminal Cys-rich domains, the Cys residues of which participate in intra- and intermolecular disulfide bonds that cross-bridge the proteins into extensive networks (2, 5).

Some microbial eukaryotic mucin-like proteins are O-glycosylated via {alpha}-linked GlcNAc rather than GalNAc. In Trypanosoma cruzi, addition of {alpha}-GlcNAc to Thr residues of glycosylphosphatidylinositol-anchored cell surface mucins has been reconstituted in vitro using a Golgi extract (6, 12), but thus far the gene and protein responsible for this activity have not been identified. Evidence suggests that mucin O-glycosylation is a critical virulence factor for this organism in causing Chagas disease (13). In the model eukaryotic organism Dictyostelium discoideum, the mucin-like repeats of the glycosylphosphatidylinositol-anchored cell surface protein SP29/PsA and other cell surface proteins are similarly modified at Thr and Ser residues by {alpha}-GlcNAc (14, 15, 5), and extended by Fuc-PO4 or PO4-Fuc. Mucin-type O-glycosylation is affected by mutations in the modB locus (16). These mutants were initially identified based on serological changes after chemical mutagenesis, and subsequently SP29/PsA was found to be devoid of its GlcNAc{alpha}1-Thr and -Ser linkages (14). modB mutant strains exhibit many developmental defects including diminished cell-cell adhesion, altered cell sorting, reduced traction in the slug, and defective spore coats (16-22, 5). These intriguing observations suggest that O-glycosylation supports many functions in Dictyostelium, but, until the function of the modB gene is determined, it is not clear whether the phenotypes are caused by the glycosylation defect or whether they are all pleiotropic effects resulting from a separate lesion.

Recently, the gene that encodes the enzyme that transfers GlcNAc to a hydroxyproline residue in the cytoplasmic/nuclear protein Skp1 of Dictyostelium was identified (23, 24). Although this linkage involves a distinct acceptor hydroxyamino acid and the enzyme resides in the cytoplasm rather than the Golgi apparatus, its sequence exhibits homology with those of the animal pp {alpha}-GalNAc-Tases (25, 26). Here we report the identification and characterization of a related Dictyostelium pp {alpha}-GlcNAc-Tase that is be expressed in the secretory pathway and initiates the O-glycosylation of SP29 and the spore coat protein SP85/PsB. This pp {alpha}-GlcNAc-Tase, referred to as pp {alpha}-GlcNAc-T2, is encoded by the classically defined modB locus demonstrating that modB mutations directly target the first step of mucin-type O-glycosylation. These findings establish a functional role for mucin-type O-glycosylation in Dictyostelium, and suggest that pp {alpha}-GlcNAc-T2, and sequences related to it in other microbes (26) comprise a microbial lineage of Golgi pp {alpha}-GlcNAc-Tases that shares a common evolutionary origin with the animal Golgi pp {alpha}-GalNAc-Tases.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cells
Strain Ax3, an axenic mutant of D. discoideum, was used as the normal reference strain. DL118(H) is an axenic, spontaneously haploidized derivative of the original diploid strain DL118, which carries the chemically mutagenized modB mutant allele from HL220 (16, 17). HU2421 is a non-axenic strain also carrying the HL220 modB allele (28). HW11 is an axenic strain that carries a modB mutant allele, which appears to partially retain function (29). Cis4c is a cisplatin-resistant strain (27) that contains an insertion of the bsr- marker in the cis4c(gntB) gene described in this report. This gene was originally described as having similarity to the kinase domain of a putative phosphatidylinositol 4-phosphate 5-kinase from Arabidopsis. These strains are each haploid.

Cell Extracts
Exponentially growing cells were harvested at <=8 x 106/ml from HL-5 growth medium (30) by centrifugation at 1000 x g for 1 min, washed in 10 mM potassium phosphate buffer, pH 6.5, and frozen as pellets at -80 °C. Stationary phase cells were collected similarly after cells reached maximal density in HL-5 for 1 day. Aggregation, slug, and fruiting body stage cells were obtained by depositing washed growth phase cells on non-nutrient agar plates (30); developing for 8, 12-15, or 30 h, respectively; scraping; and storing at -80 °C. Particulate extracts were prepared by resuspending frozen cell pellets in ice-cold Sucrose Buffer: 0.25 M sucrose, 25 mM Tris-HCl, pH 8.0, 5 mM MnCl2, and protease inhibitors (see below). Suspended cells were sonicated using a probe for two 10-s pulses at 10 watts, and centrifuged at 100,000 x g for 1 h. The pellet was resuspended at 6 x 108 original cells/ml in 0.2% (v/v) Tween 80 in Sucrose Buffer using light probe sonication.

Sequencing the cis4c(gntB) Locus
The ClaI-ClaI fragment of the cis4c locus (see Figs. 1 and 2A) had been cloned previously (27). Nucleotide sequences from this fragment were used to query data bases containing sequences from shotgun-cloned Dictyostelium gDNA fragment for overlapping sequences, accessible via the DictyBase World Wide Web Server (dictybase.org/dicty.html). An alignment of these sequences led to the predicted pp {alpha}-GlcNAc-T2 coding region. This sequence was confirmed by PCR amplification of gDNA using predicted oligonucleotide primers from the data base-derived alignment as described under "Results." Sequences of mutant alleles were determined similarly.



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  		FIG. 1.
Predicted sequence of the cis4c(gntB) locus. Genomic DNA from position -1364 (relative to the start of the first exon) through 1438 is shown. Nucleotides predicted to be translated are in uppercase, and the putative start codons of the pp {alpha}-GlcNAc-T2 sequence and the nearest upstream, reverse-oriented ORF are in bold uppercase. Nucleotides -105 to 31, and 164 to 1438, are joined in cDNA sequences deposited in the Tsukuba EST data base. Putative polyadenylation motifs (aataaa) are in bold lowercase. Amino acids in the DXD-like DXH motif, and the DX5WGGENXE-like DX6FEGEEXL motif, are in bold. Potential N-glycosylation amino acid sequons are underlined. ClaI sites defining the original fragment cloned from this locus as cis4c (27) are indicated. Sequences corresponding to forward (fp) and reverse (rp) primers used for cloning and sequencing this locus are underlined; additional nt at the 5' end of the primer are given in parentheses. This sequence has been deposited in GenBankTM (accession no. AF509501 [GenBank] ).

 



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  		FIG. 2.
Schematic diagram of pp {alpha}-GlcNAc-T2 DNA and protein expression constructs. A, predicted exons and coding region of cis4c(gntB) DNA. The DpnII site into which the bsr resistance marker was inserted in the original cisplatin-resistant strain (27) and the ClaI sites used to excise the flanking DNA are indicated. B, predicted domains of the expressed protein. The catalytic domain consists of the nucleotide recognition domain 2 (NRD2; Ref. 45), which contains determinants for recognizing UDP-GlcNAc, and the CAT27 domain, including a Gal/GalNAc-like motif previously observed in animal pp {alpha}-GalNAc-Tases and {beta}4-Gal-Tases (7, 9). C, recombinant expression constructs, in which the N terminus including the signal anchor was replaced with a cleavable signal peptide with or without an N-terminal c-Myc epitope tag.

 
Recombinant Expression of pp {alpha}-GlcNAc-T2
DNA was amplified from exon 2 using primers fp3 and rp2 (Figs. 1 and 2), and cloned into the BamHI or BglII sites of pVS4, a previously described plasmid derived from pVEII designed for the expression of secretory proteins under the control of the discoidin 1{gamma} promoter in growing and early developing cells (30). pVS4 contains a start codon followed by the celA cleavable signal peptide, which was used previously to direct the secretion of CelA from growing cells (31) and spore coat proteins from prespore cells (30), and a neophosphotransferase gene enabling growth in the presence of G418. The expression plasmids were electroporated into strain DL118(H) cells, which were then plated onto 10-cm tissue culture dishes in the presence of 7.5 µg/ml G418 in modified HL-5 growth medium (32). Stable transfectants were grown in 120 µg/ml G418 to enrich for higher level expressors, and cloned by plating on Klebsiella aerogenes on SM agar plates. This typically results in selection of clones containing chromosomally integrated tandem arrays of the plasmid (33). Strain HW210, derived from a transfectant using pVS4(gntBEx2) in which the cis4c(gntB) coding region was inserted into the BglII site, was used as a source of recombinant enzyme.

GlcNAc-Tase Activity Assay
Acceptor peptides were synthesized at the UF ICBR Protein Chemistry Core Laboratory on an ABI 430 peptide synthesizer using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Peptide quality was verified by amino acid analysis after acid hydrolysis and MALDI-TOF-MS. Peptides were purified by preparative HPLC on a C18 column using an ascending gradient of acetonitrile in 0.1% trifluoroacetic acid, followed by vacuum drying.

pp {alpha}-GlcNAc-T2 activity was assayed by the transfer of [3H]GlcNAc from UDP-[6-3H]GlcNAc (39.7 Ci/mmol; PerkinElmer Life Sciences) to synthetic peptides corresponding to repeat sequences in SP85 or SP29 (Table I). The reaction mixture typically contained purified enzyme, 25 mM HEPES-NaOH, pH 7.4, 0.1% Tween 80, 5 mM MnCl2, UDP-GlcNAc, and acceptor peptide (typically 0.1-1.0 mM), in a total volume of 24 µl, and was incubated at 22 °C for 30-120 min. UDP-GlcNAc was a mixture of 0.1-0.5 µM UDP-[3H]GlcNAc and unlabeled UDP-GlcNAc (Sigma). The reaction was terminated by dilution to 1 ml with 20 mMsodium pyrophosphate. Incorporation was determined by application onto a 1-ml C18 Sep-Pak cartridge (Millipore) under vacuum, washing six times with 5 ml of water, and elution of the peptide with 5 ml of MeOH. The MeOH eluate was collected into a 20 ml scintillation vial, supplemented with 15 ml of Scintiverse scintillation mixture (Fisher), and counted for radioactivity in a liquid scintillation counter.


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  		TABLE I
Synthetic peptides employed as acceptor substrates

 
The assay of the particulate cell extract was similar, except that Tris buffer was used in place of HEPES, and the reaction solution was supplemented with 0.25 M sucrose and 0.2% (v/v) Tween 80. The reaction was terminated by dilution with 1 ml of 10 mM NaEDTA, pH 8.0, in Sucrose Buffer and centrifugation at 100,000 x g for 90 min. The supernatant was applied to the Sep-Pak as above to measure incorporation into peptide.

pH dependence of activity was determined after dilution of the purified enzyme in 20 mM HEPES, 20 mM Trizma base, and 20 mM MES (in place of HEPES alone), which had been adjusted to pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0 with HCl or NaOH.

Purification of Expressed pp {alpha}-GlcNAc-T2
Buffers were adjusted to their pH values at 22 °C, filtered, degassed, and stored at 4 °C. Buffer A was composed of 20% (NH4)2SO4 in 50 mM Tris-HCl, pH 7.5. Buffer B was 5 mM Tris-HCl, pH 7.5. Buffer C was 50 mM HEPES-NaOH, 5 mM MgCl2, pH 7.4. All procedures were carried out at 4 °C or on ice unless otherwise indicated. 1500 ml of an HL-5 culture of strain HW210, grown to 2 x 107 cells/ml, was centrifuged at 1600 g for 5 min to remove cells, and the supernatant was further clarified at 7000 x g for 10 min. (NH4)2SO4 (ultrapure, ICN) was added to a final concentration of 30% saturation with stirring, and centrifuged at 27,000 x g for 10 min. This process was sequentially repeated by bringing the (NH4)2SO4 concentration of the supernatant to 50, 70, and 90%. The pellets from each step were dissolved in 10 ml of 50 mM Tris-HCl, pH 7.5, containing 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride.

The 50-70% (NH4)2SO4 cut was adjusted to 20% (w/v) (NH4)2SO4, and centrifuged at 12,000 x g for 30 min. The supernatant was loaded onto a 2.6 x 20 cm phenyl-Sepharose Fast-Flow (high sub) column (Amersham Biosciences) equilibrated in Buffer A at 4 °C. The column was washed with Buffer A until the A280 returned to base-line level, and eluted with a descending linear 750-ml gradient of Buffer A to Buffer B. The column was washed with Buffer B until the A280 returned to near base-line level, and a 750-ml ascending linear gradient from 0-70% ethylene glycol in Buffer B, followed by 200 ml of 70% ethylene glycol in Buffer B was then applied.

Fractions from the main pp GlcNAc-Tase activity peak from the phenyl-Sepharose column were pooled, dialyzed against Buffer C for 6 h, and load onto a DEAE-5PW column (8 mm ID x 7.5 cm, TSK-GEL, 10 µm) equilibrated with Buffer C. The column was eluted in an ascending gradient of 0-0.5 M NaCl in the same buffer at 22 °C at 1 ml/min. The enzymatically active fractions were pooled and concentrated in a Centriplus-30 centrifugal concentrator device (Amicon) at 4 °C, and aliquots were stored at -80 °C.

Antibody against pp {alpha}-GlcNAc-T2
A peptide, LEEDPNQDPSFPNSYC, predicted from the coding region of pp {alpha}-GlcNAc-T2 to be surface-exposed, was synthesized, coupled to keyhole limpet hemocyanin, and used to induce an antiserum in rabbits against the full-length protein (Sigma-Genosys). Preimmune serum and serum from the third bleed were used at 1:50 dilution in Western blotting.

SDS-PAGE and Western Blotting
Samples were suspended or diluted in SDS sample buffer, separated on a freshly prepared 7-20% linear gradient polyacrylamide slab gel, and electrotransferred to nitrocellulose using a Bio-Rad semidry blotting apparatus (30). Blots in Figs. 3 and 4 were blocked in 5% nonfat dried milk in TBS (100 mM NaCl, 50 mM Tris-HCl, pH 7.5), and the blot in Fig. 9 was blocked using a 1:1 dilution with TBS of Blocking Buffer from Li-Cor (Lincoln, NE). Primary and secondary antibodies were diluted in the respective blocking buffers, and blots were washed using TBS. Secondary antibodies in Figs. 3 and 4 were alkaline phosphatase-conjugated (Jackson Laboratories), diluted 1:2000, and detected colorimetrically (30). The secondary antibody in Fig. 9 was Alexa-Fluor 680-conjugated goat anti-mouse IgG (Li-Cor) diluted 1:10,000, and imaged at 700 nm in a Li-Cor Odyssey infrared imager. For Fig. 4, the low Mr standard kit from Sigma was used; for Fig. 9, the BenchMark prestained protein ladder from Invitrogen was used.



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  		FIG. 3.
O-glycosylation of cell surface proteins in mutant cells. Cells from strain Ax3 (normal or wild type (wt)), the cis4c(gntB) mutant, or strain DL118(H) (modB mutant) were developed to the slug stage, harvested, and subjected to SDS-PAGE and Western blot analysis using the mAbs listed below each panel. A, the anti-carbohydrate mAb 16.1 binds SP85 (SP85+) in wild-type cells but not in either cis4c(gntB) or modB mutant cells. B, probing with mAb 5F5, specific for the polypeptide backbone of SP85, shows that SP85 exhibits a lower apparent Mr in both mutant strains (SP85-) compared with normal cells (SP85+). C, probed with a mixture of mAbs 54.2 and 83.5. The anti-carbohydrate mAb 54.2, which binds both SP85 and SP29 (SP29+) in wild-type cells, fails to bind these proteins in the mutant strains. In contrast, the binding of the anti-fucose mAb 83.5, which recognizes many proteins including SP96 and SP75, is not affected in the mutant strains. D, probing with mAb MUD1, specific for the polypeptide backbone of SP29, shows that this protein exhibits the same lower apparent Mr in both mutant strains (SP29-).

 



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  		FIG. 4.
Purified recombinant fragment of pp {alpha}-GlcNAc-T2. The recombinant fragment of pp {alpha}-GlcNAc-T2 described in Fig. 2C (lacking the Myc tag) was purified from the growth medium of strain HW210 as described in the text and analyzed by SDS-PAGE followed by staining with Coomassie Blue (CB) or Western blotting and probing with immune serum (Imm) from a rabbit challenged with a pp {alpha}-GlcNAc-T2 synthetic peptide, or preimmune serum (PI) from the same animal. Mr markers are to the right.

 



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  		FIG. 9.
Complementation of the modB-glycosylation defect by cis4c(gntB). Whole cell pellets from strains DL118(H) (modB mutant), HW210 (DL118(H) transfected with the non-Myc-tagged cis4c(gntB) construct in Fig. 2C), or Ax3 (normal), from three stages of the life cycle (late growth phase, 106 cells/lane; late aggregation stage, 2 x 106 cells/lane; or fruiting body stage, 2 x 106 cells/lane) were subjected to SDS-PAGE and Western blotted with mAb 54.2, which detects GA-XX dependent on {alpha}-linked GlcNAc. Mr standards are shown at the right with values in kilodaltons.

 
Characterization of the Reaction Product
Radiochemical Incorporation—Dried SP85 peptide-2 was dissolved at 4 mM in 50 mM Tris-HCl, pH 7.4, and the concentration was confirmed spectroscopically at A280 based on the expected absorbance of the two Tyr residues. 0.1 mM peptide and 1.2 mM UDP-GlcNAc (labeled or unlabeled in parallel reactions) were added to a standard enzyme reaction buffered in a volume of 0.5 ml. Reaction progress was monitored on 5-µl aliquots that were periodically withdrawn and analyzed for incorporation of radiolabel into peptide on a C18 Sep-Pak, and the reaction was concluded when incorporation plateaued. Molar incorporation of GlcNAc was calculated based on its specific radioactivity.

HPLC Purification—The reaction mixture containing unlabeled UDP-GlcNAc, or the unmodified peptide alone, was loaded on a C18 reverse phase column (4.6 x 250 mm, LKB TSK ODS-120T, 5 µm) equilibrated in 5% acetonitrile, 0.1% trifluoroacetic acid in water. The column was eluted in an ascending linear gradient up to 95% acetonitrile, 0.08% trifluoroacetic acid in water, at 1 ml/min for 40 min. Elution was monitored at 215 and 280 nm, and radioactivity of fractions was determined by liquid scintillation counting after dilution into 5 ml of Scintiverse LC (Fisher).

Matrix-assisted Laser Adsorption Time-of-Flight (MALDI-TOF) Mass Spectrometry—HPLC fractions containing purified (glyco)peptides were mixed with equal volumes of saturated re-crystallized {alpha}-cyano-4-hydroxycinnamic acid (Aldrich) in 50% acetonitrile. One µl (~10 pmol) was deposited on a sample plate and allowed to air-dry. Spectra were collected on a PerSeptive Biosystem Voyager DE MALDI-TOF-MS operated in the positive ion and reflectron modes. A mixture of known peptides were used as an external standard.

Edman Degradation
Peptide or glycopeptide fractions from HPLC separations (500 pmol based on A280) were loaded onto a Polybrene-pretreated glass fiber filter and subjected to standard Edman chemistry cycles in a model 494-HT (Procise) sequencer (Applied Biosystems, Foster City, CA), using pre-derivatized phenylthiohydantoin-derivatives as standards. For the GlcNAc{alpha}1-Thr standard, a GlcNAc-rich peptide preparation was derived from a sialoglycoprotein fraction from T. cruzi by periodate oxidation and partial acid hydrolysis, as described (6). This fraction contained almost exclusively GlcNAc{alpha}1-Thr as determined by NMR, and was provided by J. Previato (Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil). A synthetic Myc peptide containing GlcNAc{beta}1-Thr corresponding to the region surrounding Thr-58 of the intact protein (34) was provided by N. Zachara and G. Hart (Johns Hopkins University, Baltimore, MD).

Lectin Labeling of Spores
Cells were allowed to develop for 48 h on non-nutrient agar plates. Spores were transferred by hand-picking with a loop into a solution of 0.2% (v/v) Nonidet P-40 in KP Buffer (17 mM potassium phosphate, pH 6.5) on ice. Spores were centrifuged at 10,000 x g for 15 s and resuspended in KP Buffer or 6 M urea, 1% (v/v) 2-mercaptoethanol, in TBS (150 mM NaCl, 10 mM Tris-HCl, pH 7.4). The urea-suspended spores were incubated in a boiling water bath for 3 min and diluted 5-fold with KP. All spores were collected by centrifugation, resuspended in TBS, centrifuged again, and then resuspended in 10 µg/ml fluorescein isothiocyanate-conjugated Ricinus communis agglutinin-I (120) (Vector Laboratories, Ignacio, CA), 5 mg/ml crystalline hemoglobin (Sigma) in TBS, for 1 h at 22 °C, essentially as previously described (22). Spores were recovered and washed twice in TBS by centrifugation and deposited on a glass slide for fluorescence and phase contrast microscopy. Images from representative samples were collected through a 40x objective using a Kodak DC-290 camera (1.5 s for fluorescence images), and processed identically in Adobe Photoshop software.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Architecture of the Predicted pp {alpha}-GlcNAc-T2 Gene and Protein Product—To search for new proteins related to the Dictyostelium Skp1 GlcNAc-Tase (GnT51 or pp {alpha}-GlcNAc-T1), publicly accessible data bases containing Dictyostelium genomic and cDNA sequences were queried using the tBLASTn algorithm. A cloned ClaI-ClaI fragment of Dictyostelium gDNA, known as cis4c, was found that had been isolated in a screen for genes that when disrupted resulted in increased resistance to the drug cisplatin (27). The sequence comprised a single long ORF, which conceptually encodes a polypeptide sequence that includes a 284-amino acid stretch with 29% identity and 52% similarity to that of the apparent catalytic domain of pp {alpha}-GlcNAc-T1 (Fig. 1, nt 220-1224). Inspection of the flanking gDNA sequences suggested the existence of a second, short ORF upstream of the main ORF, which appeared to represent an additional exon. This two-exon model (Fig. 2B) was confirmed by comparison with cDNA sequences deposited in the Tsukuba EST data base, including clones SLJ654 (GenBankTM AU060413 [GenBank] ) and SLC558 (GenBankTM AU060997 [GenBank] ), which extend from nt -105 to 1438 (Fig. 1), and lack an intervening 132-nt DNA fragment that is A/T-enriched and terminated with consensus intron splice donor (GT) and splice acceptor motifs (AG). The predicted start codon was in a typical sequence context including the presence of an A at position -3, and the predicted stop codon (TAA) was followed by multiple putative polyadenylation signals (Fig. 1). The deduced spliced coding sequence is predicted to encode a 403-amino acid protein with a theoretical Mr of 46,532 and a predicted isoelectric point of 6.3. It is separated from an upstream ORF in reverse orientation by 1331 nt of G/C-poor sequence, typical of non-coding Dictyostelium DNA (Fig. 1), and from a downstream reverse-oriented coding region of 2368 G/C-poor nt. The sequence of the putative coding region with intervening intron was verified by direct sequencing of a PCR product produced using fp2 and rp2 (Fig. 1). The upstream region, presumably containing expression regulatory elements, was confirmed by PCR amplification using fp1 and rp1 and partial sequencing. cis4c was previously shown by Northern blot analysis to be expressed throughout the life cycle (27).

An amino acid stretch near the N terminus of the ORF, from position 6 to 23, is predicted to encode a non-cleaved signal anchor that would retain a very short N-terminal region in the cytoplasm and direct most of the polypeptide into the lumen of the rER, resulting in a type 2 membrane protein (Fig. 2B). This is followed by 34 residues that may correspond to a spacer or stem seen in other type 2 membrane protein glycosyltransferases that separates the catalytic domain from the membrane surface (35). The rest of the ORF consists of 341 amino acids, which can be aligned to nearly the entire length of sequence of pp {alpha}-GlcNAc-T1 except for a poly-Asn tract in the latter (Fig. 2 of Ref. 25; Fig. 3 of Ref. 26). The first 259 residues (residues 63-321, Fig. 1) can also be aligned with the catalytic domain of the pp {alpha}-GalNAc-Tases (see "Discussion"). The catalytic domain of most pp {alpha}-GalNAc-Tases is followed by a 150-residue C-terminal domain, which resembles the tripartite lectins ricin and abrin and is thought to be involved in acceptor substrate recognition (9), although this domain is absent from the putative pp {alpha}-GalNAc-Tase Gly-8 from Caenorhabditis elegans. The ORF appears also to lack this domain; however, a sequence corresponding to the triply repeated signature motif QXWX#X2(D/E/N/Q) (# = hydrophobic side chain) of the ricin-like domain is present once near the C terminus at residue 379 (Fig. 1). This locus is referred to as the cis4c(gntB) gene and is predicted to encode a Golgi-associated UDP-GlcNAc:polypeptide {alpha}-GlcNAc-Tase referred to as pp {alpha}-GlcNAc-T2.

Dependence of Cell Surface Protein O-glycosylation on the cis4c(gntB) Gene—GlcNAc{alpha}1-Thr and -Ser linkages have been previously described on the cell surface glycosphingolipid-linked protein SP29/PsA (14, 15). This linkage is also thought to be present on the spore coat protein SP85/PsB based on cross-reactivity with the anti-carbohydrate mAbs 54.2 (36) and MUD50 (37), which bind the previously described glycoantigen GA-XX associated with O-linked GlcNAc on SP29. Binding of mAb 54.2 is reduced or eliminated in strains containing mutant alleles of the modB locus, resulting in increased mobility of SP29 and SP85 during SDS-PAGE (17, 30). In the cisplatin-resistant strain that originally defined the cis4c(gntB) locus (27), the coding region for the predicted catalytic domain is interrupted by a bsr resistance cassette (Fig. 2A). If this locus encodes a pp {alpha}-GlcNAc-T2 that forms GlcNAc{alpha}1-Thr and -Ser linkages on SP29 and SP85, then their glycosylation is predicted to be blocked in the cis4c mutant strain.

To assess the glycosylation status of SP29 and SP85, whole cell extracts from the slug stage, during which these proteins are expressed, were analyzed by Western blotting using mAbs that recognize carbohydrate and peptide determinants on these glycoproteins. The normal strain Ax3 and the modB mutant strain DL118(H) were compared with the cis4c(gntB) mutant strain as positive and negative controls for O-glycosylation. As shown in panels A and B of Fig. 3, SP85 was recognized by the anti-carbohydrate mAb 16.1 in wild-type cells but not in either of the two mutants, and migrated more rapidly in the mutants. The absence of the carbohydrate epitope and the lower apparent Mr indicate that O-glycosylation of SP85 is inhibited in the cis4c(gntB) mutant strain in the same way that it is affected by the modB mutation (17, 36). A similar effect was observed for the glycosylation of SP29, which is shown in the lower Mr region of panel C (Fig. 3). The effects of the mutations on O-glycosylation was specific, because fucosylation of other proteins such as SP96 and SP75, detected with mAb 83.5 which recognizes GA-X and is equivalent to MUD62 (37), was not affected in these strains (higher Mr region of panel C). These results are consistent with the model that cis4c(gntB) encodes the pp {alpha}-GlcNAc-T2 enzyme that initiates O-glycosylation on SP29 and SP85.

Recombinant Expression of the pp {alpha}-GlcNAc-Tase-like Protein—To determine whether the cis4c(gntB) sequence encodes a glycosyltransferase, DNA encoding most of exon 2, from near the beginning of the predicted stem region (position of fp3, Fig. 1) to the C terminus (position of rp2) and including the putative catalytic domain (Fig. 2C), was inserted into an integrating Dictyostelium vector (pVS4) containing promoter sequences that direct stable expression in high density growing and aggregating cells, and a cleavable signal peptide to allow processing through the secretory pathway and secretion into the HL-5 growth medium (pVS4(gntBEx2)). A second construct inserted the same DNA sequence into an alternative restriction site, resulting in the presence of a c-Myc tag at the N terminus after cleavage of the signal peptide (pVS4(mycgntBEx2)). These vectors were transformed into strain DL118(H), a modB mutant strain shown below to possess an inactive allele of the pp {alpha}-GlcNAc-T2 gene.

Several clones from a cell population transfected with pvs4(mycgntBEx2) were found to secrete a protein with an apparent Mr of 48,000 that could be detected in Western blots with Myc-specific mAb 9E10 (data not shown). This band had a larger apparent Mr than expected after cleavage of the signal peptide, 43,585. This might be the result of utilization of potential N-glycosylation sites in the polypeptide (Fig. 1). The band was not detected in parental cell growth medium, suggesting that it corresponded to the expected truncated pp {alpha}-GlcNAc-T2 protein. Ammonium sulfate fractionation of HL-5 growth medium from one of the clones revealed that the majority of expressed protein was precipitated between 50 and 70% saturated ammonium sulfate, based on Western blot analysis (data not shown).

Purification and Activity of the pp {alpha}-GlcNAc-Tase-like Protein—The 50-70% ammonium sulfate fraction from stable clones transfected with pVS4(mycgntBEx2) or pVS4(gntBEx2), lacking the Myc tag, were analyzed for pp {alpha}-GlcNAc-Tase activity. Aliquots were incubated with UDP-[3H]GlcNAc, 5 mM MgCl2, and a synthetic peptide, SP85 peptide-1, representing three iterations of a tetrapeptide repeat that occurs in SP85 (Table I). Transfer of 3H from UDP-[3H]GlcNAc to the peptide was assayed based on binding of dpm to a C18 Sep-Pak cartridge and release with MeOH. The 50-70% ammonium sulfate fraction from several clones was found to incorporate dpm into the peptide in a time-dependent fashion, whereas no incorporation above background was observed in a corresponding fraction from the parental strain DL118(H), and specific activity was enriched over 3-fold relative to the 30-50 and 70-90% ammonium sulfate cuts (data not shown).

The 50-70% ammonium sulfate fraction from the conditioned growth medium of strain HW210 (from pVS4(gntBEx2) lacking the Myc tag) exhibited maximal activity and was used for further purification. Activity was found to adsorb to a phenyl-Sepharose column in the presence of ammonium sulfate and could be eluted with an ascending gradient of ethylene glycol after washout of the ammonium sulfate as described under "Experimental Procedures." This activity pool adsorbed to DEAE-Sepharose at pH 7.4, consistent with the predicted acidic pI, and could be eluted in an ascending gradient of NaCl. The resulting activity pool was concentrated by centrifugal ultrafiltration and analyzed by SDS-PAGE. Staining of the gel revealed a major band of protein with an apparent Mr of 47,000 (Fig. 4), compared with the expected value of 42,070. A band at the same position was labeled with a rabbit antiserum raised against a synthetic peptide corresponding to a conceptually translated region of cis4c(gntB), as described under "Experimental Procedures," but not with an identical dilution of preimmune antiserum from the same animal. This protein, purified to near homogeneity from the growth medium, therefore represents a soluble recombinant partial length product of the cis4c(gntB) gene, lacking its putative transmembrane domain.

Catalytic Properties of Purified Recombinant pp {alpha}-GlcNAc-T2—The highly purified recombinant pp {alpha}-GlcNAc-T2 exhibited time-dependent transfer of 3H from UDP-[3H]GlcNAc to SP85 peptide-1 (Fig. 5A), and this activity was abolished in the presence of EDTA as for pp {alpha}-GlcNAc-T1 (23). Dilution of the enzyme in 5 mM MnCl2 sustained activity better than dilution in MgCl2 or CoCl2 (data not shown), suggesting that Mn2+ is a natural cofactor. Activity was inhibited when NaCl or KCl was increased above 10 mM, or if pH was greater than 8.5 or less than 6.5 (data not shown).



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  		FIG. 5.
Kinetic analysis of pp {alpha}-GlcNAc-T2 activity. A, time course of the GlcNAc-Tase reaction in the presence of 4.5 µM UDP-[3H]GlcNAc and 2 mM SP85 peptide-1, as described under "Experimental Procedures" in the absence or presence of 20 mM EDTA. B, reactions were performed in the presence of 50 µM UDP-[3H]GlcNAc using varying concentrations of SP85-peptide-2, SP29-peptide-1, or T. cruzi mucin-peptide 1, for 1 h. C, double-reciprocal plot of the data shown for SP29-peptide-1 in panel B, which indicates an apparent Km for the SP29-peptide-1 of 84 µM. D, the concentration of UDP-GlcNAc was varied in the presence of 200 µM SP85-peptide-2 during a 1-h reaction period. E, double-reciprocal plot of the data shown in D, which yielded an apparent Km for UDP-GlcNAc of 38 µM. A Km of 39 µM was estimated using SP85-peptide-1 (data not shown).

 
Using SP85 peptide-2, activity was proportional to concentration at lower concentrations, but, at higher concentrations, activity decreased (Fig. 5B). A similar effect was observed for SP85 peptide-1 (data not shown), which contains one fewer tetrapeptide repeat (Table I). This suggests peptide aggregation or substrate inhibition. In contrast, activity exhibited the expected hyperbolic dependence on the concentration of another peptide, SP29 peptide-1 (Table I) from a different protein for which O-glycosylation is also dependent upon pp {alpha}-GlcNAc-T2 in vivo (Fig. 3, C and D). SP29 peptides had been previously shown to exhibit acceptor activity in vivo when expressed appended to a carrier protein (38). The pp {alpha}-Glc-NAc-T2 activity was specific for the peptides derived from Dictyostelium proteins, as a peptide substrate for an {alpha}-GlcNAc-Tase from extracts of T. cruzi (6), T. cruzi mucin peptide-1 (Table I), was not an acceptor in this assay (Fig. 5B). A Michaelis-Menten analysis of the activity with respect to SP29-peptide-1 yielded an apparent Km of 84 µM (Fig. 5C), which is expected to reflect contributions from the modification of each of the Thr residues modified in the peptide (see below).

Enzyme activity also exhibited a hyperbolic dependence on UDP-GlcNAc concentration (Fig. 5D). A double-reciprocal plot of the data yielded an apparent Km of 38 µM (Fig. 5E). This is ~2 orders of magnitude higher than that of pp {alpha}-GlcNAc-T1 (23), the cytoplasmic GlcNAc-Tase that modifies Skp1, but consistent with values reported for Golgi glycosyltransferases (9). The Vmax of the enzyme calculated from these data was ~20 µmol/mg of protein/min, which dramatically exceeds the value for pp {alpha}-GlcNAc-T1; however, this enzyme has not been assayed with a native acceptor (23). Therefore, cis4c(gntB) encodes a pp GlcNAc-Tase that is able to modify, in vitro, peptides from the same proteins the O-glycosylation of which depends on this gene in vivo.

Characterization of the Reaction Product—As described above, the enzyme is predicted to catalyze formation of one or more GlcNAc{alpha}1-Thr linkages in the acceptor peptide. To count the number of sugars added, 50 nmol of SP85-peptide-2 was modified to completion as determined by a time-course analysis of incorporation from UDP-[3H]GlcNAc. Based on the specific activity of the UDP-[3H]GlcNAc and the concentration of the peptide as determined spectroscopically, the modified SP85 glycopeptide-2 contained 3.2 mol of GlcNAc/mol of peptide, suggesting that each of the four available Thr residues could be modified.

To confirm modification of the peptide by multiple GlcNAc residues, the reaction mixture was fractionated on a reverse phase HPLC column in the presence of 0.1% trifluoroacetic acid (Fig. 6A). The glycopeptide eluted heterogeneously at 18% acetonitrile, slightly ahead of the elution position of the unreacted peptide (22% acetonitrile). The elution profile of the unreacted peptide was also somewhat heterodisperse, which might be the result of partial isomerization of prolyl bonds. The exact mass of the peptide from selected fractions was examined by MALDI-TOF-MS. As expected, the unreacted peptide (fraction 23) yielded a singly charged ion (m/z 1804), which corresponds exactly to the predicted MNa+ of the unmodified peptide (Fig. 6B). The earliest eluting reacted peptide fraction (fraction 20) contained a mixture of ions corresponding to modification by 3 or 4 GlcNAc residues, whereas later eluting fractions were enriched in ions corresponding to modification by 3 GlcNAc residues (fraction 21) or 2 GlcNAc residues (fraction 22). Therefore, SP85-peptide-2 was modified by 2, 3, or 4 GlcNAc residues, with the more highly substituted peptides eluting earlier as expected based on the hydrophilic character of the sugar modification.



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  		FIG. 6.
HPLC and MALDI-TOF-MS analysis of the reaction product. SP85 peptide-2 was maximally modified in a time-course study. The degree of substitution was estimated to be 3.2 mol of GlcNAc/mol of peptide based on a parallel reaction using UDP-[3H]GlcNAc. A, the reaction mixture was separated by HPLC on a C18 column eluted with an ascending gradient of acetonitrile in the presence of 0.1% trifluoroacetic acid, and fractions were collected as indicated based on A280. An unreacted aliquot of peptide was chromatographed in parallel. B, fractions indicated in A were analyzed by MALDI-TOF-MS. The unreacted peptide from fraction 23 contained predominantly a singly charged sodiated ion corresponding to the unmodified peptide, whereas fractions from the reacted peptide were enriched in singly charged sodiated ions corresponding to peptides containing 2, 3, or 4 GlcNAc residues.

 
To determine the anomericity of the GlcNAc linkage formed and the amino acids modified, the modified peptide was subjected to Edman degradation analysis. This method of N-terminal sequencing has been previously employed to analyze GlcNAc-Thr linkages in a Milligen sequenator (15), and to analyze GalNAc-Thr linkages in an ABI 494 Procise sequenator (39, 40). Here the peptides were examined in a Procise sequenator.

Data from the unreacted peptide confirmed the predicted sequence of the synthetic peptide (data not shown). For the reacted peptide, cycles corresponding to the positions of Thr yielded low levels of Thr, and high levels of peaks corresponding to the elution positions of G and S, as shown for cycle 5 (Fig. 7A). These new peaks (G:S) had a molar ratio of 1.36. The new peak at the position of Gln was attributed to contamination from position 6 of the peptide (data not shown). Cycle 5 in the unreacted peptide yielded a peak only at the position of Thr (Fig. 7D). Analysis of a peptide from a T. cruzi mucin, containing authentic GlcNAc{alpha}1-Thr (6), yielded peaks at the positions of G and S, and these also were present at ratio of 1.36 (Fig. 7B). A standard of GlcNAc{beta}1-Thr yielded primarily Thr, presumably a breakdown product as {beta}-linked GlcNAc is labile to Edman chemistry (41). These results indicate that the Thr from SP85-peptide-2 was modified by {alpha}-linked GlcNAc and not {beta}-linked GlcNAc. Quantitation of the level of GlcNAc{alpha}1-Thr and Thr throughout the peptide showed that 80-95% of the internal Thr residues and ~30% of the N-terminal Thr are modified (Table II). This is consistent with the radioactivity incorporation and mass spectrometry data showing a preponderance of tri-substituted peptides. Therefore, highly purified recombinant pp {alpha}-GlcNAc-T2 is able to catalyze the modification of all four Thr residues in the peptide with {alpha}-linked GlcNAc, and does not modify the Tyr residues.



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  		FIG. 7.
Characterization of the reaction product by Edman degradation. Reacted and unreacted peptides from the HPLC separation shown in Fig. 6A were applied to an ABI 494 (Procise) sequenator and processed for 20 cycles. A, cycle 5 from reacted SP85-peptide-2 (fraction 21 from Fig. 6A), at the position of Thr-2, yielded peaks eluting at the positions of S, Q, T, and G. The ratio of areas of peaks G:S was 1.36. B, cycle 8 from a T. cruzi mucin peptide, containing authentic GlcNAc{alpha}1-Thr, yielded peaks eluting at the positions of S, T, and G. The ratio of areas of peaks G:S was 1.36. C, cycle 6 from a synthetic Myc peptide, containing GlcNAc{beta}1-Thr58, yielded a main peak eluting at the position of Thr and a minor peak eluting slightly earlier. D, cycle 5 from unreacted SP85-peptide-2 (fraction 23 from Fig. 6A), at the position of Thr-2, yielded only Thr as expected.

 


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  		TABLE II
Yields of Thr and GlcNAc{alpha}1-Thr from Edman degradation analysis of the reacted peptide

From the separation shown in Fig. 6A, approximately 500 pmol (based on A280) of reacted (fraction 21) and unreacted (fraction 23) SP85-peptide-2 were subjected to Edman degradation as described in Fig. 7. Values for GlcNAc-Thr were derived from the integrated value of the peak eluting at the position of G in Fig. 7B. The total value listed includes contributions from the preceding and subsequent cycles to account for the effects of incomplete degradation resulting from the intervening Pro residues.

 
cis4c(gntB) Corresponds to the modB Locus—As shown in Fig. 3, mutations of the cis4c(gntB) and modB loci have, within the resolution of the method, identical selective, negative effects on the glycosylation of SP85 and SP29. To determine whether the cis4c(gntB) locus is altered in modB mutants, the coding region of the cis4c(gntB) locus was amplified by PCR from two modB mutant strains and sequenced. A single difference from that of the normal strain (Ax3), a nonsense mutation immediately upstream of the DXD-like DXH motif located in the putative catalytic domain, was found in DNA from the modB mutant strains DL118(H) and HU2421, which have a common genetic origin (Table III). This would be expected to lead to nonsense-mediated decay of the cis4c(gntB) message or to the expression of a truncated, inactive protein. The cis4c(gntB) coding region from strain HW11 contained three missense substitutions. One, M166T, occurs in a highly conserved hydrophobic cluster also just upstream of the DXH motif. The others, A87V and F243S, occur in less well conserved regions of the protein. These substitutions are consistent with partial function of the modB allele in this strain, which exhibits a ladder of lower Mr underglycosylated glycoforms of SP29 (29).


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  		TABLE III
The cis4c(gntB) locus is altered in two modB-mutant strains

Strain HW11 also contains the nucleotide substitutions C392T and T860C, resulting in the amino acid changes A87V and F243S.

 
To determine whether the underglycosylation of SP29 and SP85 in the modB and cis4c(gntB) mutant strains (Fig. 3) is the result of reduced pp {alpha}-GlcNAc activity, detergent-permeabilized membrane preparations were tested for activity using SP29-peptide-1 (Fig. 8). Previous studies had shown that crude membranes have robust activity toward endogenous acceptors (42), which were predicted to be increased in level in the modB mutant and might confound the analysis. Therefore, the particulate material was removed after the incubation period by centrifugation, and the supernatant was used to determine incorporation of [3H]GlcNAc into the peptide. Extracts from either growing or slug stage normal strain cells exhibited maximal activity. Similar results were obtained for a second strain that had inserted the bsr resistance marker elsewhere of the cis4c(gntB) coding region (data not shown). In contrast, only background incorporation (<5%) was observed using extracts from strain DL118(H) or the cis4c(gntB) mutant strain, confirming that the pp {alpha}-GlcNAc-Tase that modifies the SP29 peptide was deficient.



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  		FIG. 8.
pp {alpha}-GlcNAc-T2 activity in normal and mutant cell extracts. Growing or slug stage cells from the strains indicated were lysed in the presence of Sucrose Buffer and particulate fractions were prepared by ultracentrifugation as described under "Experimental Procedures." These were resuspended in Sucrose Buffer containing 25 µM UDP-[3H]GlcNAc, 50 µM SP29-peptide-1, and 0.1% (v/v) Tween 80, and incubated for 1 h. Parallel reactions contained no added peptide. The reaction mixture was centrifuged, and incorporation of radioactivity into material in the supernatant fraction was determined using the C18 Sep-Pak assay. Activity in Ax3 extracts was dependent on added peptide (data not shown).

 
To establish that the pp {alpha}GlcNAc-Tase deficiency was the result directly of the mutation in the cis4c(gntB) locus, the ability of cis4c(gntB) to rescue the glycosylation deficiency was examined. For this test, the cis4c(gntB) expression strain (HW210) used to produce soluble recombinant pp {alpha}-GlcNAc-T2 (Fig. 4) was examined for recovery of expression of the GA-XX glycoantigen detected by mAb 54.2 that is dependent on {alpha}-GlcNAc-T2 and absent in the modB mutants (Fig. 3). The discoidin 1{gamma} promoter used to control expression of the recombinant pp {alpha}-GlcNAc-T2 is maximally active as proliferating cells exhaust nutrients in the growth medium (stationary cells) and in early development (preaggregation). A comparison of late growth phase cells from strains DL118(H), HW210 (DL118(H) containing the modified cis4c/gntB expression plasmid), and strain Ax3 (normal strain) by Western blotting showed that HW210 expressed normal levels of almost all of the mAb 54.2-reactive bands detected in Ax3, whereas DL118 exhibited no reactivity (Fig. 9, lanes a-c). Although pp {alpha}-GlcNAc-T2 was expressed as a soluble protein lacking its N-terminal signal anchor in this strain, its high level of continuous overexpression is likely to have maintained enzymatically functional levels of enzyme in the Golgi apparatus. The expressed pp {alpha}-GlcNAc-T2 partially mimicked the endogenous enzyme as cells entered the aggregations stage (lanes d-f). However, the pattern of mAb 54.2-reactive bands later in development diverged greatly between strains HW210 and Ax3 (lanes g-i), and SP85 and SP29 were not glycosylated (data not shown), as expected because the discoidin 1{gamma} promoter is inactive later in development. Because modB mutant strains have inactivating mutations in the cis4c(gntB) locus, cannot modify SP29 and SP85 peptides in vitro, fail to O-glycosylate SP29 and SP85 in vivo, but can be partially complemented with respect to glycoantigen formation by expression of a truncated pp {alpha}-GlcNAc-T2 protein, it is concluded that the modB-gene encodes pp {alpha}-GlcNAc-T2, and that mutation in the cis4c(gntB) locus is the primary cause of defective O-glycosylation in modB mutant cells.

modB and cis4c(gntB) Mutant Spores Have Similar Spore Phenotypes—Spores produced by modB mutants exhibit differences compared with normal spores, which, if consequential to the modB mutations, are also predicted to occur in the cis4c(gntB) mutant. One difference is that modB mutant spores are less elongated than those of the normal strain Ax3, which have an average axial ratio of 2.0 (32). A comparison of strain DL118(H) and cis4c(gntB) mutant spores reveals lower average axial ratios of 1.3-1.6. A second difference is that modB mutant strains have permeable spore coats as measured by accessibility of the Gal/GalNAc-polysaccharide, localized internally near the plasma membrane, to labeling with the fluorescent lectin FITC-RCA-I (120) (22). Parallel labeling of spores revealed that the normal strain Ax3 was labeled weakly in comparison to both mutant strains (Fig. 10). In contrast, when spores were pre-extracted with 8 M urea, 1% 2-mercaptoethanol to strip external layer barrier proteins from the coat (5), spores were similarly labeled (Fig. 10) indicating that reduced labeling was caused by inhibition of accessibility rather than reduced level of the Gal/GalNAc-PS target. The similar phenotypes of the two mutant strains confirms that the coat barrier defect in modB mutants is the direct result of improper O-glycosylation. Because SP85 contributes to outer layer barrier function (43), this might be the result of inhibition of O-glycosylation of SP85.



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  		FIG. 10.
Permeability of normal and mutant spore coats. Untreated spores or spores pre-extracted with urea and 2-mercaptoethanol from the different strains (indicated at left) were incubated in the presence of FITC-RCA-I (120) and washed, as described under "Experimental Procedures." Representative fields of view were photographed using phase contrast optics to image spores, or epifluorescence optics through a fluorescein filter to detect lectin binding to the coat of each spore. Increased fluorescence labeling implies greater accessibility of the lectin probe to the Gal/GalNAc-rich polysaccharide associated with the inner layer of the coat (22).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
cis4c(gntB) Encodes pp {alpha}-GlcNAc-T2—The Dictyostelium cis4c gene product has been identified as a pp {alpha}-GlcNAc-Tase based on the ability of a highly purified, soluble recombinant form of the protein (Fig. 4), secreted from Dictyostelium, to transfer [3H]GlcNAc from UDP-[3H]GlcNAc to synthetic acceptor peptides (Fig. 5). GlcNAc is attached in {alpha}-linkage to the hydroxyl side chain of Thr residues based on mass spectrometry of the modified peptide, and co-chromatography of the modified amino acid with authentic standards after release using Edman chemistry (Figs. 6, 7). The synthetic peptides model mucin-type repeat sequences in the cell surface proteins SP29 and SP85, and the Thr residue of each tetrapeptide repeat in SP85 is modified (Table II). Extracts from a mutant strain in which the cis4c gene is interrupted by insertion of an antibiotic resistance marker lack the pp {alpha}-GlcNAc-Tase activity (Fig. 8), and SP29 and SP85 are not correctly O-glycosylated in the cells (Fig. 3), showing that the cis4c {alpha}-GlcNAc-Tase activity defined in vitro is required for O-glycosylation in vivo. Except for the cytoplasmic enzyme pp {alpha}-GlcNAc-T1 encoded by gntA, there are no other sequences closely related to that of the cis4c(gntB) gene product, now referred to as pp {alpha}-GlcNAc-T2, in the nearly completed Dictyostelium genomic sequencing project. This is supported by a Southern blot analysis (27). Therefore, it is likely that pp {alpha}-GlcNAc-T2 directly mediates the {alpha}-GlcNAc-modification of each of the Thr residues in SP85 and probably also SP29 in slug cells. An ability to modify the Ser residues found in the SP85- and SP29-repeats, known to be modified in SP29 in vivo (14, 15), remains to be examined.

pp {alpha}-GlcNAc-T2 Is a Type 2 Membrane Protein Probably Localized to the Golgi—pp {alpha}-GlcNAc-T2 appears to be a type 2 membrane protein associated with the Golgi apparatus based on indirect evidence. The sixth amino acid of the coding sequence is basic and followed by a stretch of 21 uncharged, predominantly hydrophobic residues predicted to target co-translational association with the rER. The absence of a small side-chain residue at the C terminus of the hydrophobic region suggests that it is not cleaved and therefore constitutes a signal anchor for a type 2 membrane protein with a short, N-terminal cytoplasmic domain of 4-5 residues, and the bulk of the protein, including the putative catalytic domain, projecting into the lumen. Several observations support this model. 1) pp {alpha}-GlcNAc-T2 activity is associated with the particulate fraction in cell extracts (Fig. 8). 2) Expression of the catalytic domain of pp {alpha}-GlcNAc-T2 with a cleavable signal peptide known to direct secretion (30) rescues pp {alpha}-GlcNAc-T2 deficiency (Fig. 9). 3) Treatment of cells with EDTA inhibits pp {alpha}-GlcNAc-T2-dependent O-glycosylation (29), consistent with the presence of pp {alpha}-GlcNAc-T2 in an endosome-accessible compartment and its dependence, shown here, on MnCl2 (Fig. 5A). 4) Mucin-type O-glycosylation of SP85 and other cell surface glycoproteins in Dictyostelium, including contact sites A/gp80 and SP29, is posttranslational based on developmental and pulse-chase studies (18, 5), and the morphological equivalent of Golgi stacks are seen in developing prespore cells at a time when O-glycosylation of spore coat proteins is maximal (44). Together these observations strongly support the model that pp {alpha}-GlcNAc-T2 is a type 2 membrane protein, in contrast to pp {alpha}-GlcNAc-T1 but like other known Golgi glycosyltransferases (35), and that it resides in a post-rER secretory compartment, probably the Golgi, as seen for the animal pp {alpha}-GalNAc-Tases (45, 9).

pp {alpha}-GlcNAc-T2 Is Homologous to the Animal pp {alpha}-GalNAc-Tases—The predicted catalytic domain of ~341 amino acids begins ~35 residues downstream of the putative signal anchor sequence (Fig. 2), based on an alignment of the pp {alpha}-GlcNAc-T2 sequence with that of pp {alpha}-GlcNAc-T1 (the cytoplasmic Skp1-Hyp pp {alpha}-GlcNAc-Tase) and the animal Golgi pp {alpha}-GalNAc-Tases (Fig. 2 of Ref. 25; Fig. 3 of Ref. 26). Although the first 285 amino acids of this putative domain exhibit very low sequence identity with the pp {alpha}-GalNAc-Tases (~14% compared with murine pp {alpha}-GalNAc-T1), they have 41% similarity, allowing for conservative substitutions, and key motifs are shared. Although the sequence similarities cannot be detected using standard BLAST, a PSI-BLAST approach in the NCBI non-redundant data base, based on support from pp {alpha}-GalNAc-T1 and other related microbial pp {alpha}-GlcNAc-T2-like sequences, identified pp {alpha}-GalNAc-Tase sequences with significant similarity scores (data not shown). The N-terminal 125 amino acids of this region comprise the so-called NRD2 (nucleotide recognition domain-2) subdomain (46), which is found in many UDP-sugar-dependent glycosyltransferases including those from families GT2, GT23, GT27, and GT60,2 and contributes determinants for binding a divalent cation and the donor substrate. The DXD-like DXH sequence (see Figs. 1 and 2), a key motif of animal pp {alpha}-GalNAc-Tases (family GT27) likely to help coordinate the Mn2+ ion, is conserved in this region. The C-terminal subdomain includes a region that is similar to the so-called Gal/GalNAc-subdomain found in the animal pp {alpha}-GalNAc-Tases and {beta}4-GalTases (7). The canonical subsequence DX5-WGGENXE of this subdomain is represented as D278X6FEGEEXL in pp {alpha}-GlcNAc-T2 (Fig. 1) and DX6FFGEEXS in pp {alpha}-GlcNAc-T1. The variation may reflect use of UDP-GlcNAc in place of the UDP-GalNAc donor substrate, based on comparison with related sequences in other microbial eukaryote genomes (see below). In contrast, the sequences of Golgi pp Fuc-Tase and Man-Tase, and cytoplasmic O-{beta}-GlcNAc-Tase (for which there is a possible Dictyostelium ortholog based on genomic sequence data),3 which are inverting rather than retaining enzymes, are so different that no meaningful alignment has been found. Like the core catalytic domain of the pp {alpha}-GalNAc-Tases, pp {alpha}-GlcNAc-T2 possesses six conserved Cys residues (relative to related microbial sequences predicted to be Golgi-expressed) that might participate in disulfide bonding, although their positions are not conserved. pp {alpha}-GlcNAc-T2 and the pp {alpha}-GalNAc-Tases also show similar retention of configuration of the anomeric linkage of GlcNAc, and similar dependence on MnCl2. pp {alpha}-GlcNAc-T2 appears to lack the ricin-like C-terminal domain found in most pp {alpha}-GalNAc-Tases and thought to be involved in acceptor substrate recognition; however, this domain is also missing in the putative Gly-8 pp {alpha}-GalNAc-Tase from C. elegans (9). Therefore, it is likely that the modification of mucin-type repeats in the Golgi of Dictyostelium and animals is mechanistically conserved except for the substitution of GlcNAc for GalNAc.

The Dictyostelium enzyme appears to modify multiple sites in multiple proteins based on the biochemical and genetic analyses (Figs. 3 and 9; Table II). With evidence that Dictyostelium cells can faithfully modify expressed human MUC1 in a pp {alpha}-GlcNAc-T2-dependent manner similar to that of mammalian cells (15), the pp {alpha}-GlcNAc-T2 catalytic domain, lacking the companion ricin-like C-terminal domain seen in most pp {alpha}-GalNAc-Tases, may represent the minimal functional unit required for both primary and secondary modification of the same acceptor substrate. The animal pp {alpha}-GalNAc-Tases represent a multigene family of 8-24 members in C. elegans, Drosophila melanogaster, and mammals (9), in contrast to the apparent single member in Dictyostelium, suggesting that diversification of pp {alpha}-GalNAc-Tase paralogs in animals reflects specialization of function rather than an intrinsic requirement of processing multiple substrates.

A Similar Enzyme in Other Unicellular Eukaryotes?—A pp {alpha}-GlcNAc-T2-like enzyme activity has been reported in Golgi preparations from T. cruzi (6, 12) and a pp {alpha}-GlcNAc-T2-like sequence is present in the T. cruzi genome3 (26). This sequence is more similar to Dictyostelium pp {alpha}-GlcNAc-T2 than to pp {alpha}-GlcNAc-T1 or the pp {alpha}-GalNAc-Tases. The results from the present study implicate this sequence as a candidate for the pp {alpha}-GlcNAc-Tase activity in T. cruzi extracts. Related sequences are present in the genomes of other trypanosomatids including T. brucei and Leishmania major (Fig. 3 of Ref. 26), a diatom, and a soybean stem and root rot,3 suggesting that a similar O-glycosylation step exists in the secretory organelles of these organisms.

This glycosylation step is traceable back to Gram-positive and Gram-negative bacteria based on the presence of related sequences predicted to encode soluble cytoplasmic enzymes in their genomes (25, 26), suggesting an evolutionary origin for the initial step of mucin-type O-glycosylation that is cytoplasmic, and much more ancient than previously realized.

pp {alpha}-GlcNAc-T2 Modifies Many Glycoproteins throughout the Life Cycle—Mutations affecting O-glycosylation in Dictyostelium have been identified by screening chemically induced mutant cell libraries for strains that fail to label with anticarbohydrate antibodies (47, 37). Together with biochemical and immunochemical studies, this approach has defined four pathways in the secretory compartment based on the initiating (reducing) sugar linkage to a hydroxyamino acid: GlcNAc{alpha}1-, Fuc1-, GlcNAc{alpha}1-PO4-, and Fuc{beta}1-PO4- (reviewed in Ref. 5). In the present work, the modB locus is concluded to encode pp {alpha}-GlcNAc-T2, which forms the GlcNAc{alpha}1-Thr type of linkage, based on 1) the detection of nonsense and missense mutations in the cis4c(gntB) locus of two independent modB mutants (Table III), 2) identical serologically and biochemically defined glycosylation phenotypes of modB and cis4c(gntB) mutants (Figs. 3, 8), 3) the ability of a modified cis4c(gntB) gene to partially complement the modB glycosylation defect (Fig. 9), and 4) the similar spore coat phenotypes of modB and cis4c(gntB) mutant spores (Fig. 10). Therefore, it can be concluded that glycoproteins recognized by mAbs 54.2, 5.2, MUD50, 178, E28D8, MUD102, and 16.1, which recognize GlcNAc-associated glycans dependent on the modB locus (14, 16-18, 22, 29, 30, 36, 37), are glycosylated by pp {alpha}-GlcNAc-T2. These include the mucin-type domains in SP29/PsA (14) and SP85/PsB (30), the previously characterized type 2 structures on cell contact site A glycoprotein gp80 (18), and the many additional mAb 54.2-reactive proteins detected in Fig. 9. pp {alpha}-GlcNAc-T2 Contributes to Many Functions—Mucin-type O-glycosylation supports many functions in animals depending on the type of protein modified (9, 48). The present findings indicate that this is true for free-living lower eukaryotes as well. Although previous genetic studies in Dictyostelium have correlated significant mutant phenotypes with glycosylation defects, they are unable, until the mutant gene is characterized, to differentiate whether mutant phenotypes result from glycosylation defects or vice versa. Now that the modB locus is shown to encode pp {alpha}-GlcNAc-T2, which is likely to be the major if not only pp {alpha}-GlcNAc-Tase in the Dictyostelium Golgi, the interesting phenotypes assigned to modB mutations (see Introduction) can be attributed to a proximate defect in the initiation of mucin-type O-glycosylation, i.e. attachment of {alpha}-linked GlcNAc to the polypeptide. This conclusion has been verified here (Fig. 10) for the modB mutant spore coat permeability phenotype (22). Although Dictyostelium and T. cruzi use {alpha}-GlcNAc rather than {alpha}-GalNAc as found in animals and certain apicomplexans (49), both sugars appear to stabilize polypeptide conformation in a similar way (10), and therefore might support similar functions for mucin-type domains such as protection from proteolysis, supramolecular assembly, and presentation of recognition motifs. In SP29, the {alpha}-linked GlcNAcs are clustered and further modified by phosphodiester- and phosphomonoester-linked sugars (reviewed in Ref. 5), resulting in an anionic character, which, although based on different chemistry, also mimics a common feature of mammalian mucin-type glycans.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF509501 [GenBank] (gntB locus)

* This work was supported in part by National Institutes of Health Grant R01-GM37539 and National Science Foundation Grants MCB-9730036 and MCB-0240634. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 940 Stanton L. Young Blvd., BMSB 853, Oklahoma City, OK 73104. Tel.: 405-271-2227 (ext. 1247); Fax: 405-271-3139; E-mail: christopher-west{at}ouhsc.edu.

1 The abbreviations used are: pp {alpha}-GalNAc-Tase, polypeptide {alpha}-N-acetylgalactosaminyltransferase; FITC-RCA-I(120), fluorescein isothiocyanate-conjugated R. communis agglutinin-I; mAb, monoclonal antibody; MALDI-TOF, matrix-assisted, laser desorption, time-of-flight; MS, mass spectrometry; pp {alpha}-GlcNAc-Tase, polypeptide {alpha}-N-acetylglucosaminyltransferase; nt, nucleotide(s); MES, 4-morpholineethanesulfonic acid; HPLC, high performance liquid chromatography; TBS, Tris-buffered saline; EST, expressed sequence tag; ORF, open reading frame; rER, rough endoplasmic reticulum. Back

2 P. M. Coutinho and B. Henrissat, Carbohydrate-active Enzymes server (afmb.cnrs-mrs.fr/CAZY/GT.html). Back

3 H. van der Wel and C. M. West, unpublished data. Back


   ACKNOWLEDGMENTS
 
We are grateful to Steve and Hannah Alexander for providing the cis4c mutant strain, Jose Previato for the GlcNAc-rich peptide from T. cruzi, Natasha Zachara and Gerald Hart for the glycosylated Myc peptide, Bill Loomis for strain DL118, and Keith Williams for strain HU2421 and mAb MUD1. We thank Lee Kaplan for her assistance with the Western blot analyses, and members of the UF ICBR Protein Chemistry Core Lab for synthesis of the peptides (Alfred Chung), Edman degradation analyses (Scott McClung), and assistance in MALDI-TOF-MS (Scott McMillen). For EST sequence data, we acknowledge the University of Tsukuba (Japan) cDNA Sequencing Initiative. For gDNA sequence data, we acknowledge the Institute of Biochemistry I (Cologne, Germany) and the Genome Sequencing Centre Jena (genome.imb-jena.de/dictyostelium/; supported by Deutsche Forschungsgemeinschaft Grants 113/10-1 and 10-2), the Baylor College of Medicine (supported by the NICHD, National Institutes of Health), and the National Biomedical Computation Resource at the San Diego Supercomputer Center (DKTY.Sdsc.edu/, supported by National Institutes of Health Grant P41-RR80605).



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2003 by the American Society for Biochemistry and Molecular Biology.
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