©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of FP21, a Cytosolic Glycoprotein from Dictyostelium(*)

(Received for publication, May 27, 1994; and in revised form, October 20, 1994)

Emil Kozarov (§) Hanke van der Wel Melvin Field Mikelina Gritzali Ross D. Brown Jr. (1) Christopher M. West (¶)

From the Department of Anatomy and Cell Biology, College of Medicine Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32610-0235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

FP21 is a glycoprotein which, when tracked by radioactivity in its fucosyl moiety, was previously detected in the cytosol of Dictyostelium cells after cell fractionation. This compartmentalization is confirmed by SDS-polyacrylamide gel electrophoresis/Western blotting of cell fractions using three different antibodies. Although a substantial fraction of FP21 is also detected in the particulate fraction using these new antibodies, particulate FP21 is released by disrupting protein-protein interactions, but not membrane disruption. Since purified FP21 is susceptible to aggregation, and purified nuclei do not contain FP21, particulate FP21 is also part of the cytosol. Additional compositional and structural information provides strong evidence that FP21 does not at any time traverse the rough endoplasmic reticulum. First, cDNAs spanning the entire coding region of the FP21 gene predict no hydrophobic motifs expected to promote membrane insertion, but do predict an NH(2)-terminal coiled coil domain which could explain aggregation. Second, monosaccharide composition analysis of the predominant glycoform of FP21 yields 2 mol of galactose, 1 mol of xylose, and 1 mol of fucose/mol of polypeptide; FP21 from a fucosylation-defective mutant contains 1 additional mol of xylose in place of fucose. Thus the N-glycosylation sequon present in FP21 is not utilized by oligosaccharyl transferase, which resides in the rough endoplasmic reticulum. These findings indicate that nascent FP21 remains in the cytosol after synthesis and is therefore glycosylated by unusual cytosolic xylosyl-, galactosyl-, and fucosyltransferases.


INTRODUCTION

An increasing number of proteins which reside in cytosolic and nucleoplasmic compartments of the cell are becoming recognized as glycosylated with a single GlcNAc residue(1, 2) , lending credence to a sizable body of earlier data indirectly suggesting glycosylation of multiple cytosolic and nucleoplasmic proteins(3) . Recently, direct chemical evidence has also been obtained for the existence of oligosaccharides on certain proteins of the cytosol(4, 5) . Since the cytosol is the cellular compartment of protein translation, the simplest mechanism for glycosylating these proteins would involve cytosolic glycosyltransferases, to join several cytosolic glycosyltransferases involved in synthesis of, e.g. dolichol sugars (6) and glycogen(7) . However, since at present all known protein glycosyltransferases which have been cloned are type two membrane proteins with their active sites oriented toward the lumen of either the rER, (^1)the Golgi complex, or a topologically continuous space(8, 9) , an alternative model is that the cytosolic protein translocates into and then back out of a conventional glycosylation compartment.

Consistent with the model that cytosolic glycoproteins are glycosylated in the cytosol, a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase activity has been purified to apparent homogeneity from a cytosolic preparation (10) of the liver. This enzyme catalyzes the glycosidic linkage of GlcNAc to the hydroxyl of serine or threonine of cytosolic and nuclear proteins. The existence of larger tri- and tetrasaccharide modifications suggests that there are additional cytosolic glycosyltransferases capable of synthesizing a variety of interesting oligosaccharide structures, if the compartmentalization of the O-GlcNAc transferase is conserved.

One such cytosolic glycoprotein bearing an oligosaccharide is FP21 from the simple eukaryotic cellular slime mold Dictyostelium discoideum. FP21 possesses a tetra- or pentasaccharide whose polypeptide linkage is susceptible to mild alkaline degradation(5) . Characteristics of the FP21 polypeptide reported here strongly indicate that it is glycosylated in the cytosolic compartment and, using a sensitive micromethod, we find that it contains Gal, Xyl, and Fuc and lacks GlcNAc and GalNAc. This suggests that the Dictyostelium cytosol contains novel galactosyl and xylosyltransferases in addition to the FP21-fucosyltransferase already detected there (5) and that these transferases comprise a glycosylation pathway independent of the GlcNAc modification pathway cited above.


EXPERIMENTAL PROCEDURES

Cells

Strain Ax3 of D. discoideum was grown axenically on HL-5 and was used as the normal strain(11) . Strain HL250, derived from strain Ax3 by mutagenesis, is unable to synthesize GDP-Fuc from GDP-Man and hence FP21 from this strain contains no Fuc (5, 11) . Ax3 was alternatively grown on Klebsiella aerogenes where indicated. Development of axenically grown cells was induced as described(11) .

Preparation of Anti-FP21 Antibodies and Antibody Affinity Columns

FP21 from strain HL250 was purified as described(5) . Gel slices containing approximately 2 µg of protein/injection/animal were macerated using a pestle in a microcentrifuge tube, emulsified with Freund's complete (primary immunization) or incomplete (secondary immunization) adjuvant, and injected subcutaneously into 4-month-old BALB/c mice. The tertiary injection consisted of phenyl-Sepharose purified FP21 (5) in phosphate-buffered saline introduced intraperitoneally 3 days prior to harvest of the spleen. Hybridoma colonies were prepared after fusion of SP2/0 plasmacytoma and spleen cells according to standard protocols(12) , and positive wells were identified by an ELISA screen using partially purified FP21 preparations adsorbed onto the wells of Immulon 2 plates (Dynatech Laboratories, Chantilly, VA) and alkaline phosphatase-conjugated goat anti-mouse secondary antibody. A single spleen yielded 21 positive colonies. Colony 3F9 was subcloned and isotyped as IgG(1).

mAb 3F9 IgG was purified from ascites by a caprylic acid/(NH(4))(2)S0(4) differential precipitation method(17) , and a 15 mg/ml solution was coupled to CNBr-activated Sepharose (Pharmacia Biotech Inc.) according to the manufacturer's instructions. Purified rabbit IgG (Sigma) was coupled in a similar manner.

Additional mAbs were developed after immunization of mice with FP21 purified from strain Ax3 according to the protocol described below. Two of these antibodies, 3F5 and 3D11, show similar binding to wild-type and mutant FP21 in Western blots of cell extracts, compared with mAb 3F9.

An antiserum was prepared against a synthetic peptide consisting of CQGDDKKDEKRLDDIP, which corresponds to residues 68-82 of FP21 (see Fig. 5) plus an NH(2)-terminal Cys for coupling. The peptide was coupled to maleimide-activated keyhole limpet hemocyanin and ovalbumin using the Imject(TM) Activated Immunogen Conjugation Kit (Pierce). Rabbits were immunized with the keyhole limpet hemocyanin conjugate, and sera were titered using an ELISA assay based on the ovalbumin conjugate. Anti-FP21-(68-82) from rabbit L97 gave a 50% reaction in the ELISA assay at a dilution of 1:7500. The antiserum was affinity-purified on a column prepared by conjugating approximately 5 mg of the original peptide to 2 ml of 2-nitro-5-thiobenzoate-thiol agarose (Pierce), according to the manufacturer's protocol. After acid elution and neutralization, anti-FP21-(68-82) showed a specificity for FP21, based on Western blots blocked in milk, which was similar to the monoclonal antibodies.


Figure 5: FP21 cDNA sequence and translation of the single, long open reading frame. The sequence of the overall cDNA group of overlapping clones assembled from primer-dependent amplification products from the ZAP cDNA library, and partially confirmed by corresponding amplification products from genomic DNA, is shown, together with translation of the single long open reading frame. The underlined amino acid sequence from residues 4-38 was previously determined by Edman degradation of the intact purified protein(5) . The underlined amino acid sequence from residues 31-43 was obtained from the M(r) 11,000 CNBr fragment described in Table 1. A synthetic peptide encompassing the underlined sequence, including residues 68-82, was found to induce an antiserum which, after affinity purification against the peptide, was monospecific for FP21 (data not shown). The underlined nucleotide sequences represent two consensus polyadenylation signals.





Cell Fractionation and Extractions

Vegetative axenically grown cells were filter-lysed, and S100 (cytosol) and P100 (particulate) fractions were prepared as described(5) . All manipulations were at 0-4 °C. The P100 pellet was extracted by resuspension using probe sonication in the solution described, except for the control pellet, which was gently resuspended in the original lysis buffer (0.25 M sucrose, 50 mM Tris-HCl, pH 7.4). The suspensions were then recentrifuged at 100,000 times g times 60 min and the supernatants concentrated on a Centricon-10 ultrafiltration device (Amicon, Beverly, MA).

A Triton X-100-resistant cytoskeletal fraction, and associated soluble fraction, were prepared as described previously(13) . An actomyosin fraction was prepared(14) , as modified according to (15) , except that cells were lysed by forced passage through a 5-µm pore diameter nuclepore filter.

Nuclear fractions were prepared as described(16) , using both the digitonin and Nonidet P-40 methods. Nuclei were detected by staining with 30 µg/ml propidium iodide, diluted from a 100 times stock solution in water, and visualized epifluorescently through a rhodamine filter channel. To improve the purity of nuclei, nuclear preparations were layered on a 1.0-2.0 M linear gradient of sucrose in nuclear lysis buffer and centrifuged in a swinging bucket rotor at 70,000 times g for 20 min. Nuclei were most enriched in turbid bands near the bottom of the gradient, well separated from other organelles, but still partially contaminated by intact cells.

Analysis of Association of FP21 with Cytoskeletal or Nuclear Fractions

Cytoskeletal and nuclear fractions were, together with the whole cell starting fraction and associated soluble supernatant fraction generated, resolved by SDS-PAGE at 100 µg of protein/lane, Western blotted, and immunoprobed with mAb 3F9. A parallel gel was stained with Coomassie Blue to confirm sample loadings and effective fractionation of cellular proteins, including actin and myosin heavy chain.

Modified Purification of FP21

The S100 fraction from axenically grown vegetative cells was made 50 mM in NH(4)Ac from a 2 M stock solution, applied to a bed of DEAE-Fast Sepharose, and eluted with a 0.1-1.5 M gradient of NH(4)Ac, as described previously(5) , or with a gradient of 0-0.25 M NaCl in lysis buffer (later trials). Analysis of fractions using a mAb 3F9 ELISA/dot blot assay revealed that FP21 from both strains Ax3 and HL250 eluted in two peaks from 0.2-0.4 M NH(4)Ac or 0.15-0.22 M NaCl, with <10% of total FP21 eluting in the nonbound fraction. The second peak (II) contained 3-4-fold more FP21 than peak I. Unless indicated otherwise, the two FP21 peaks were pooled and applied to immobilized rabbit IgG and mAb 3F9 columns, which were connected in series and preequilibrated in 10 mM sodium phosphate, 140 mM NaCl, pH 7.4. After loading of the sample followed by washing with the same buffer until all nonbound protein was washed out, the mAb 3F9 column was disconnected and eluted with 0.1 M glycine HCl into tubes containing 0.1 fraction volumes of 1 M Tris-HCl, pH 8.2. All manipulations were performed at 0-5 °C. The method worked similarly starting with strains Ax3 or HL250. In some cases, FP21 was further purified by SDS-PAGE. Slices from gels which had been soaked in 4 M NaAc for 20 min, or fixed and stained in standard methanol/acetic acid/Coomassie Blue R-250 solutions, were excised, briefly equilibrated in H(2)0, suspended in 200 mM NH(4)HC0(3), 0.025% SDS, 0.5 mM dithiothreitol, pH 7.4, macerated with a pestle in a matching microcentrifuge tube, and incubated overnight at 37 °C. Protein was recovered by centrifugation for 5 min through a 0.2-µm nylon-66 basket in a 1.5-ml microcentrifuge tube (Rainin, Woburn, MA), and precipitated with 10 volumes of ice-cold acetone. Recovery was routinely >75%.

FP21 Cleavage and Sequencing

Cyanogen bromide cleavage of FP21 in gel slices was as described(18) , except that the incubation temperature was 37 °C rather than 25 °C. The reaction mixture was subjected to SDS-PAGE using a Tris-Tricine buffer system (19) and Western blotted onto PVDF paper (Immobilon(TM)-P; Millipore Corp., Bedford, MA)(20) , and the largest (11 kDa) band was subjected to Edman degradation as described previously(5) .

Monosaccharide and Amino Acid Analysis

Gel-purified, acetone-precipitated FP21 (2-5 µg) was dissolved in 0.05 M NH(4)Ac, pH 7.4, in 20% (v/v) methanol, spotted onto PVDF membrane, which was dried and washed in H(2)0. Comparison of acid hydrolyzed (see below) Millipore Immobilon(TM)-P, Immobilon(TM)-P, Bio-Rad Trans-Blot, Applied Biosystems ProSpin(TM), and Applied Biosystems ProBlott(TM), showed that Immobilon(TM)-P contained the lowest level of Glc-, Xyl-, Fuc-, and Gal-like substances after chromatography (see below). Alternatively, protein was precipitated from 90% acetone, 0.1 N HCl by incubation for 15-30 min at -20 °C and recentrifugation, to reduce SDS. Crystalline bovine serum albumin (Schwarz-Mann) and fetuin (Life Technologies, Inc.) were resolved by SDS-PAGE, electroblotted onto Immobilon(TM)-P or Immobilon(TM)-P (later trials), and localized by staining with Coomassie Blue. For monosaccharide analysis, the dried membranes were transferred to a 1-ml Reactivial(TM) (Pierce) followed by 10 µl of MeOH to wet the membrane, 400 µl of H(2)0, and 75 µl of trifluoroacetic acid (to a final concentration of 2 M), capped, heated at 100 °C for 4 h(21) , and dried in a vacuum centrifuge. The sample was taken up in a small volume of H(2)0, and 50-500-pmol aliquots were subjected to monosaccharide composition analysis on a Dionex chromatography system equipped with a pulsed amperometric detector as described previously (11, 21) . For analysis of most neutral and amino sugars, a CarboPac PA-1 column was eluted isocratically in 14-18 mM NaOH. Man and Xyl, which coelute under these conditions, were resolved by eluting with 4 mM NaOH, or by a program consisting of 2.5 min 2 mM NaOH, followed by elution with 100% H(2)0, as suggested by the manufacturer. For detection of uronic acids, a 20-min ascending gradient of 100 mM to 200 mM NaAc in 200 mM NaOH was employed, as modified from (22) . To determine total protein, parallel membrane-bound samples handled in identical fashion were acid hydrolyzed and subjected to amino acid composition analysis (20) using ninhydrin with norleucine as an internal standard. Total protein was calculated based on the fractional molar content of proline or valine.

Mass Spectrometry

Gel-purified samples were repetitively spotted and dried onto a film of nitrocellulose deposited on a metal sample holder, in order to build up sample mass, followed by 1 µl of sinapinic acid or alpha-cyano(4-0H)cinnamic acid as the matrix, as described(23) . Mass was estimated in a Finnegan Lasermat time-of-flight instrument, and the peak value is reported. Cytochrome c (M(r) 12,400), lysozyme (M(r) 14,136), and myoglobin (M(r) 16,860) served as internal or external standards.

SDS-PAGE and Western Blotting

SDS-PAGE on 7-20% linear gradient slab gels, using a Tris-glycine buffer system, and Western blotting were performed as described(5) , using nitrocellulose or PVDF membranes with 0.2 or 0.45 µm pore sizes as indicated.

Protein Determination

Protein was estimated using a micro-adaptation of the bicinchoninic acid-based assay (24) distributed by Pierce.

Phosphorylation and Sulfation

To detect phosphorylation of FP21, vegetative amoebae were metabolically labeled with PO(4), as described(25) , in HL-5 medium modified to contain 0.4 mM phosphate and 5 mM MES-NaOH, and a soluble extract was prepared from 1.7 times 10^9 cells by resuspension of washed cells in 20 ml of lysis buffer and centrifugation at 38,000 times g for 15 min by centrifugation as described(25) . To detect sulfation, cells were grown for three generations in HL-5 containing 25 µCi/ml Na(2)SO(4), and extracts for purification of FP21 were prepared as above using the same buffers supplemented with 20 mM Na(2)SO(4). The supernatants from 10^9 cells were applied to a 1-ml mAb 3F9 column as described above. Acid-eluted FP21 containing fractions and the original supernatant were electrophoresed on both standard one-dimensional and two-dimensional O'Farrell gels, electroblotted to nitrocellulose, immunoprobed with mAb 3F9, and subjected to autoradiography(11) . One-dimensional gels were also sliced into 1-mm segments and counted in a liquid scintillation counter, after swelling in scintillation mixture (for S) (9) or by Cerenkov counting (P). No evidence for the presence of PO(4) or SO(4) was obtained, despite efficient labeling of numerous protein species of much lower apparent abundance as estimated by Coomassie Blue staining.

Preparation and Sequencing of FP21 cDNA

Degenerate and normal oligonucleotides (listed in Table 4) were synthesized at an Interdisciplinary Center for Biotechnology Research core laboratory at the University of Florida and used to prime the amplification of cDNA from three different nucleic acid templates. The cDNA library template was prepared from mRNA of 4-h developing cells and cloned in a ZAP vector (generously provided by R. A. Firtel, University or California, San Diego, La Jolla, CA). After phage amplification, DNA was isolated using LambdaSorb(TM) phage absorbent according to the manufacturer's protocol (Promega Corp., Madison, WI). The genomic DNA template was isolated from nuclei of axenically grown vegetative strain Ax3 cells and purified on a CsCl gradient, as described(26) . cDNA was also synthesized from total RNA (see next paragraph) using Moloney murine leukemia virus reverse transcriptase (Promega Corp.) and either fp16 (see Table 4) or oligo(dT) as the primer(27, 28) . Identical results were obtained when the total RNA template was predigested with 0.05-0.1 of RNase-free DNase/µg of RNA (RQ-1; Promega Corp.), performed as recommended by the manufacturer to remove DNA from in vitro transcription preparations. This treatment destroyed the residual PCR template activity of the non-reverse-transcribed RNA preparation (data not shown).



cDNA was amplified by a PCR protocol (cycle profile: 1 min, 94°; 1 min, 49°; 5 min, 72°) using Taq polymerase (Promega) in 3 mM MgCl(2). PCR-derived DNA fragments were gel purified and subcloned into either pBluescript (Stratagene, La Jolla, CA) or pGEM-T (Promega). Dideoxy sequencing was performed at an Interdisciplinary Center for Biotechnology Research core laboratory at the University of Florida on an ABI 373a DNA sequencer system. All regions reported were sequenced two to six times in each direction from two or more different PCR amplifications.

Northern and Southern Blotting

Total RNA was isolated using guanidinium thiocyanate as described previously(29) . For Northern blotting, 5-15 µg was electrophoresed on 1% agarose-formaldehyde gels(30) , blotted onto GeneScreen Plus, and probed with random-primed labeled FP21 cDNA (0.5 kilobase) (30) amplified using primers fp12 and fp16 (see Table 4). Oligonucleotide probes were end-labeled using [P]ATP and T4 polynucleotide kinase (New England Biolabs, Beverly, MA)(30) . Gels were calibrated using the 0.24-9.5-kilobase ladder from Life Technologies, Inc. For Southern blots, restriction digests and PCR products were electrophoresed on GTG agarose (FMC, Rockland, ME), blotted, and probed as above.


RESULTS

Anti-FP21 Antibody Confirms a Cytosolic Localization for FP21

An S100 fraction from a fucosylation-defective mutant (strain HL250) was trace-labeled with GDP-[^3H]Fuc, and labeled FP21 was purified (5) and used to immunize a mouse. This mouse yielded 21 reactive hybridoma colonies, identified by positive reaction in an ELISA screen using partially purified FP21 and SDS-PAGE/Western blotting of purified FP21. mAb 3F9 was chosen for further analysis. mAb 3F9 reacted with a predominant band of M(r) 21 times 10^3 in strain Ax3 (normal) vegetative (axenically grown) cells, and a band at a slightly lower M(r) (20.5 times 10^3) in the fucosylation mutant strain HL250 (Fig. 1, lanes c and d), as expected for FP21 based on previous metabolic labeling studies(5) . A similar intensity of labeling was observed in both strains. There was no evidence for FP21 precursors of higher M(r), although a weaker reactive signal was often detected at the position of the dimer M(r), 42 times 10^3. New mAbs were subsequently induced against wild-type FP21, and a rabbit antiserum was induced against a synthetic peptide from the FP21 sequence (see below and ``Experimental Procedures''). Each of these antibodies recognized the same pair of FP21 bands in wild-type and mutant cells (data not shown).


Figure 1: FP21 is found in S100 and P100 fractions at different stages of the life cycle. Lanes a-f, growth phase cells grown either on K. aerogenes (lanes a and b) or axenically (lanes c and d), or developed to the preculmination (18 h) stage (lanes e and f), were resolved by SDS-PAGE and analyzed by Western blotting using mAb 3F9. The use of hemoglobin as the blocking agent led to a higher level of nonspecific binding than that observed using 5% non-fat dry milk (see Fig. 2and Fig. 3). Lanes g-j, S100 (lanes g and h) and P100 (lanes i and j) fractions were prepared from axenically grown vegetative cells and analyzed in like fashion. The normal strain Ax3 (lanes a, c, e, g, and i) and fucosylation-defective strain HL250 (lanes b, d, f, h, and j) samples are arranged in pairwise fashion in alternate lanes. Each lane contains 100 µg protein. The region of the gel from approximately M(r) 10,000-35,000 is shown. The position of FP21 is indicated by a double bar, since its apparent M(r) differs slightly between the two strains.




Figure 2: Changes in ionic strength or pH release FP21 from the P100 (particulate) fraction. The P100 fraction of axenically grown vegetative Ax3 cells was resuspended either in original lysis buffer, 0.25 M sucrose, 50 mM Tris-HCl, pH 7.4 (lanes a and b), or in 0.1 M Na(2)C0(3), pH 11.5 (lane c), 1 M NaCl, 10 mM Na(2)EDTA, 50 mM Tris-HCl, pH 7.4 (lane d), 10 mM Na(2)EDTA-Na0H, pH 7.4 (lane e), or 0.5% Triton X-100, 50 mM Tris-HCl, pH 7.4 (lane f). Samples in lanes b-f were probe-sonicated to facilitate resuspension. To determine whether FP21 was released from the particulate fraction by these treatments, the samples were recentrifuged. The resulting P100 (upper panel: new pellet) and S100 (lower panel: new supt) fractions were examined by SDS-PAGE and Western blotting with mAb 3F9.




Figure 3: Bulk soluble FP21 is not accessible to in vitro fucosylation. An S100 fraction from axenically grown HL250 vegetative cells was labeled with GDP-[^3H]Fuc and precipitated with progressively higher concentrations of (NH(4))(2)S0(4). Successive cuts, listed as percent saturation, were collected by centrifugation, dissolved, and dialyzed. The distribution of total S100 protein among cuts is given in the bar graph (light shading). Equal amounts of total protein from each cut were resolved by SDS-PAGE and analyzed either by Western blotting with mAb 3F9 for FP21 (shown in lower panel) or by counting gel slices containing FP21 (bar graph, dark shading)(5) . Whereas FP21 is fairly equally distributed over all cuts on a per protein basis, only a small fraction of total FP21 (<5%) resides in the 70-80% (NH(4))(2)S0(4) fraction, because <5% of total protein precipitates in this fraction. In contrast, >80% of FP21 which can be fucosylated in vitro precipitates in this fraction, suggesting the bulk of FP21 is protected from in vitro fucosylation.



In order to demonstrate that mAb 3F9 reacts with FP21, a mAb 3F9 affinity column was prepared and used to purify [^3H]fucosyl-FP21, prepared by metabolic labeling of strain Ax3 vegetative cells with [^3H]fucose, or by labeling FP21 in a strain HL250 S100 preparation with GDP-[^3H]fucose(5) . In each case, radioactive FP21 was recovered from the S100 fraction at >70% yield, whereas <5% of total protein was bound to the column. Thus mAb 3F9 binds to FP21 and is highly specific for FP21 with respect to total cell protein.

mAb 3F9 was used to assay FP21 at different stages of development. FP21 was present after 18 h of development (the preculmination stage), and axenically grown vegetative cells at a similar level, and was also present in bacterially grown vegetative cells (Fig. 1, lanes a, c, and e). A similar result was found in mutant strain HL250 (Fig. 1, lanes b, d, and f), except that a strongly reactive protein of a smaller size was also detected reproducibly with mAb 3F9 only in bacterially grown vegetative HL250 cells (Fig. 1, lane b). The identity of this band is unknown.

To determine the compartmentalization of FP21, cells were filter-lysed in sucrose/Tris buffer and separated by centrifugation into S100 (cytosolic) and P100 (total particulate) fractions. mAb 3F9 detected FP21 in both the S100 (Fig. 1, lanes g and h), where it was originally found(5) , and at a similar but slightly lower level, on a per total protein basis, in the P100 (Fig. 1, lanes i and j). Although previously [^3H]fucosyl-FP21 was not detected in the P100 fraction (5) , as if P100-associated FP21 is not fucosylated, the M(r) difference between wild-type and mutant isoforms of FP21 was conserved in the P100 fraction. This discrepancy could be explained if the fucose turned over more rapidly, in the cell, on FP21 in the cytosol relative to the insoluble form. To evaluate the nature of the association of FP21 with the particulate fraction, aliquots of the P100 were resuspended in various solubilizing agents and recentrifuged, and the new pellets and supernatants were compared by SDS-PAGE/Western blotting. Lane a (Fig. 2) shows that almost half of the P100-bound FP21 was eluted by simply washing in the same sucrose/Tris buffer. The remaining P100-bound FP21 was not eluted by sonication (lane b), indicating that particulate FP21 is not a soluble protein trapped within a vesicle. In contrast, high pH (lane c), high salt/EDTA (lane d), and low salt/EDTA (lane e) each nearly completely released FP21 into the supernatant. Finally, non-ionic detergent stabilized FP21's association with the pellet (lane f). Similar results were observed for HL250-derived FP21 (not shown). If FP21 is associated with membranes, it must be external and peripheral.

To address the possibility that soluble FP21 might be particulate-associated in the intact cell in a manner which is sensitive to the conditions of cell lysis, cells were lysed in buffers which have been optimized for the preservation of cytoskeletal components. FP21 was not detected in a Triton X-100 cytoskeletal fraction which enriches for actomyosin or in a purified actomyosin fraction (data not shown), prepared as described under ``Experimental Procedures.'' Finally, FP21 was not present in either of two nuclear fractions prepared as described under ``Experimental Procedures'' (data not shown). If FP21 is concentrated in the nucleus of intact cells, it must be weakly bound, allowing it to diffuse out during the isolation procedure. Because purified FP21 tends to self-aggregate (see below), it is likely that particulate FP21 consists simply of aggregates of FP21 which are sensitive to high pH, high salt/EDTA, or low salt/EDTA.

Purification and Aggregation of FP21

The original purification of FP21 (5) had been optimized for the recovery of in vitro fucosylated [^3H]fucosyl-FP21 from HL250 S100. Reexamination of the purification scheme with the benefit of mAb 3F9 to screen FP21 showed that [^3H]fucosyl-FP21 does not copurify with bulk FP21. Whereas >80% of [^3H]fucosyl-FP21 precipitated above 70% (NH(4))(2)SO(4), <5% of total FP21 precipitated above this cutoff (see Fig. 3and legend). It is unlikely that the change in glycosylation (see below) would dramatically alter the precipitability of FP21 because a similar distribution of bulk FP21 among different (NH(4))(2)S0(4) cuts was observed in extracts of strain HL250 (data not shown). This observation indicates that the majority of S100 FP21 is inaccessible to fucosylation by the cytosolic FP21-fucosyltransferase, perhaps due to the existence of FP21 in a molecular complex, such as that form present in the P100 fraction.

Since (NH(4))(2)SO(4) precipitation did not enrich for bulk FP21, a new purification protocol was developed which bypassed precipitation, retained DEAE ion exchange chromatography, and employed a mAb 3F9 affinity column (see ``Experimental Procedures''). Fig. 4compares the Coomassie Blue-stained SDS-PAGE profiles of the wild-type prerun (pr), flow-through (ft), and acid-eluted fractions (1, 2, 3, 4, 5) from the affinity column (after warming and centrifugation, see next paragraph), in addition to acid eluted fractions (3, 4) from a column containing mutant FP21. FP21 is the predominant protein in the eluted fractions for both wild-type and mutant FP21.


Figure 4: Purification and aggregation of FP21. FP21 was purified separately from the S100s of axenically grown Ax3 and HL250 vegetative cells by DEAE-Sephadex chromatography and adsorbed onto mAb 3F9 affinity columns. Each affinity column was eluted with 0.1 M glycine HCl, pH 2.5, and fractions were neutralized and precentrifuged at 4 °C. After warming the supernatants to 22 °C, aggregated protein was collected by centrifugation at 13,000 times g for 10 min and compared with remaining soluble material by SDS-PAGE and staining with Coomassie Blue. Shown in the first two lanes are the prerun (pr), flow-through (ft), followed by successive acid-eluted fractions (1-5) from the strain Ax3 sample, resolved into pelleted (left member of each pair) and soluble (right member of each pair) subfractions. An M(r) ladder is shown in the middle, consisting of, from top to bottom, M(r) values 66,200, 45,000, 36,000, 29,000, 24,000, 20,100, and 14,300, as marked in the right-hand margin. Shown to the right of the M(r) ladder is a mixture of fraction 4 from Ax3 and fraction 3 from HL250 (M for mix), followed by the acid-eluted fractions (3 and 4) from a parallel affinity column containing a sample from strain HL250. Greatest sedimentability of FP21 is observed for the fractions with the highest protein concentration, and differences between the two strains in the contaminating minor proteins which cosediment with FP21, suggest that their association is nonspecific.



Fig. 4also illustrates the tendency of FP21 to aggregate. Protein recovered in the neutralized, acid-eluted fractions from the mAb 3F9 column was observed to undergo rapid clouding upon warming to 23 °C, which was reversible upon returning to 0 °C. This behavior was visible through at least five cycles of warming/cooling. To determine which proteins were aggregating, after two cycles, rewarmed fractions were centrifuged, and the pellet and the clear supernatant for each fraction were electrophoresed in pairs (Fig. 4). FP21 was partially sedimentable, and when its concentration exceeded approximately 50 µg/ml, the majority of FP21 was pelleted. A similar concentration dependence was observed for FP21 from both strains Ax3 and HL250. Although other proteins were also sedimented, their involvement appears to be nonspecific, because each is present in a lower quantity, and proteins of different apparent M(r) were sedimented in the Ax3 and HL250 preparations. This behavior suggests that FP21 self-aggregates, although it cannot be ruled out that other minor proteins contribute to this process by a substoichiometric mechanism.

Chemical Characterization of FP21 from Normal and Fucosylation-defective Cells

FP21 purified by the new rapid protocol and SDS-PAGE was not, in contrast to earlier results using FP21 purified by a longer scheme(5) , susceptible to Edman degradation. To confirm the identity of the two proteins, the new preparation was subjected to CNBr cleavage and the products resolved on another gel. The largest band, with an apparent M(r) of about 11,000 (Table 1), was found to have an NH(2)-terminal sequence (residues 31-43; see Fig. 5) overlapping with the NH(2)-terminal sequence obtained previously for FP21(5) . The FP21 cDNA sequence reported (Fig. 5) predicts the existence of a CNBr-generated fragment of M(r) 10,428 (residues 31-122) with the NH(2)-terminal sequence determined for the M(r) 11,000 protein fragment. Thus the newly purified protein is the originally purified FP21.

The M(r) of gel-purified FP21 was determined independently by MALDI-TOF-MS. As summarized in Table 1, the M(r) of normal FP21 averages 20,500 and mutant FP21 averages 19,800, in close agreement with the M(r) differences estimated by SDS-PAGE. The M(r) values of both proteins appear heterodisperse, possibly due to the presence of SDS. Alternatively, heterodispersity may reflect variability in the extent of multiple posttranslational modifications.

To estimate the isoelectric point, a whole cell soluble (S100) fraction, and a mAb 3F9 column-purified FP21 fraction, were subjected to two-dimensional O'Farrell gel electrophoresis and either stained with Coomassie Blue, or silver, or Western-blotted with mAb 3F9 as described under ``Experimental Procedures.'' In both fractions, FP21 electrophoresed as a monodisperse spot with no evidence for multiple isoelectric isoforms, with an apparent pI of 5.5.

To confirm and extend earlier data on the glycosylation of FP21, identical aliquots of the above gel-purified preparation were spotted onto PVDF membranes in parallel, hydrolyzed, and subjected to monosaccharide and amino acid composition analysis. Separate elution programs were required to optimize the separation of amino and neutral sugars, Xyl and Man, and uronic acids. As summarized in Table 2, each mole of normal FP21 contains approximately 2 mol of Gal, 1 mol of Xyl and 1 mol of Fuc; substoichiometric amounts of GlcNH(2) and GalNH(2) were detectable at <0.2 mol; Glc could not be determined owing to high background in the standard protein controls and the PVDF blanks. No other peaks greater than 0.1 mol/mol polypeptide were present in the chromatogram from 4-24-min retention time. Since FP21 resolves into two peaks after DEAE-Sepharose chromatography, the sugar composition of these two peaks was compared. The majority of FP21 (75-80%) is present in peak II, and material from this peak had approximately the same molar ratios of Gal, Xyl, and Fuc, 2:1:1, as the pooled material. A lower level of Glc (1 mol/mol Fuc) is detected, presumably because these samples were acetone-precipitated rather than blotted onto PVDF paper, which contains variable levels of Glc after acid hydrolysis. In contrast, peak I contained Gal, Xyl, and Fuc in a 1:2:1 ratio, and Glc:Fuc was approximately 3:1. It is not known whether Glc is covalently attached to FP21.



The amino acid composition (Table 3) of FP21 was consistent with the prediction from the FP21 cDNA sequence (see below), given that Met, Ser, and Thr are typically underestimated by the technique employed, and that Ala and Gly are typically overestimated, with high levels of Gly likely deriving from the SDS-PAGE purification step, which included glycine in the buffers. After the predicted composition was used to estimate the levels of amino acids not quantified chemically, FP21 was found to consist of about 3% carbohydrate, if glucose is absent.



The molar Fuc content of pooled peaks I and II of mutant FP21 was, as expected(5) , less than 0.05 (Table 2). The relative molar content of Xyl was increased to approximately 2. The amino acid composition of mutant FP21 was indiscernible from that of the normal form (Table 3).

No phosphate or sulfate could be detected under conditions of metabolic labeling that efficiently labeled other proteins (see ``Experimental Procedures''), including minor contaminating proteins which coeluted with FP21 from the mAb 3F9 column (data not shown).

FP21 cDNA Encodes a Protein with Cytosolic Features

A PCR strategy was used to prepare FP21 cDNA from Dictyostelium genomic DNA. Two, opposite sense 12-fold degenerate oligonucleotides (fp7 and fp8, see Table 4) were synthesized based on the sequence of the amino-terminal 35 amino acids. These oligonucleotides were capable, as expected, of priming the amplification of a unique 86-mer which, upon subcloning into pBluescript, yielded a nucleotide sequence predicting the known intervening amino acid sequence. These same degenerate oligonucleotides were then each used in combination with arm-specific primers (based on T3 and T7 promoter sequences, see Table 4) to amplify additional FP21-specific upstream and downstream cDNA from the cDNA library. Desired amplification products were identified by probing Southern blots of PCR products using the alternative end-labeled degenerate oligonucleotide as a probe. Amplification reactions containing the fp8 primer yielded 200- and 800-bp products in both directions, and the fp7 primer yielded a reaction product of 700 bp in both directions. These were subcloned, sequenced, and found together to contain only a single long open reading frame predicted to encode the FP21 polypeptide (Fig. 5). The 800-bp clone contained the sequence of the 200-bp clone in addition to 600 bp of upstream (relative to the open reading frame) sequence which did not resemble the A/T content of Dictyostelium DNA. A synthetic oligonucleotide from the distal (5`) end of 800-bp sequence (fp17; Table 4), together with the degenerate oligonucleotide primer (fp8) used initially to amplify the 800-bp product or fp11, did not prime the PCR amplification of any DNA fragments from genomic DNA. Since these combinations of primers did amplify the expected fragment length of DNA from the cDNA library (data not shown), the unconfirmed, 5` end of the 800-bp fragment is not included in the sequence group of overlapping clones shown in Fig. 5.

The start of the long open reading frame is preceded by a 93-bp A/T-rich sequence and possesses a typical translation start context (31) . The long open reading frame contains an A/T content and codon usage typical of Dictyostelium coding regions(32) . The open reading frame ends with an ochre (TAA) codon, also typical for Dictyostelium, and is followed by about 15 bp of nucleotides having a coding region base composition, followed by 90 A/T-rich bp. This is followed by a 60-bp open reading frame with an A/T content typical of coding regions. This short, isolated open reading frame, flanked on both sides by A/T-rich sequences, is followed shortly by two AATAAA polyadenylation consensus signals within a 55 bp A/T rich 3` segment. The overall sequence contains 801 bp, in agreement with the length of the single hybridizing band observed by Northern blotting of total RNA (800 bp; see Fig. 6) taken from cells under two different nutritive conditions and two stages of development.


Figure 6: Northern blot of FP21 expression. Northern blot analysis of Ax3 RNA. Lanes marked 0 and 13 h contain total RNA from axenically grown vegetative cells (26 µg) and 13-h filter-developed cells (15 µg), respectively; the size of Dictyostelium rRNAs in kilobases are indicated on the left. The lane marked Lad (ladder) contains 5 µg of RNA standards with sizes in kilobases labeled as indicated. Left-hand panel, low-stringency wash; right-hand panel, subsequent high-stringency wash of the same blot. The probe was a random prime-labeled 0.5-kilobase fragment amplified with primers 12 and 16 from cDNA.



The long open reading frame is concluded to encode FP21 for the following reasons. 1) The message length predicted by the cDNA group of overlapping clones is similar to the length of the mRNA determined by Northern blotting (800 bp; see Fig. 6). The FP21 message is present during development and during growth on either bacteria or axenic medium (Fig. 6), consistent with the protein accumulation data (Fig. 1). 2) The cDNA sequence correctly predicts the 40-amino acid sequence determined by Edman degradation of the isolated protein. 3) The chemically determined amino acid composition of purified FP21 is accurately predicted by the long open reading frame (Table 2). 4) The long open reading frame predicts a polypeptide with a length of 162 amino acids and an M(r) of 18,689, which accounts for about 91% of the M(r) estimated by MALDI-TOF mass spectrometry (Table 1). 5) The nucleotide sequence predicts the NH(2)-terminal amino acid sequence and the size of FP21's largest CNBr fragment (Table 1). 6) As a further test of the validity of the predicted FP21 amino acid sequence, a rabbit antiserum was generated against a synthetic peptide, spanning predicted residues 68-82 (see Fig. 5), which was covalently coupled to keyhole limpet hemocyanin. Anti-FP21-(68-82) recognized both wild-type and mutant glycoforms of purified FP21 after SDS-PAGE and Western blotting at dilutions of greater than 1:5000. After affinity purification of the antiserum against the peptide coupled to 2-nitro-5-thiobenzoate-thiol-agarose beads, the antibody was specific for FP21 by Western blotting (data not shown).

The short, isolated open reading frame, 3` to the long open reading frame and 5` to the polyadenylation signal, is an unusual feature of 3`-untranslated RNA. Several observations indicate that this feature was not an amplification or recombination artifact. First, oligonucleotides which hybridize with the short open reading frame (fp9, fp14, and fp16; Table 4) each, in pairwise combination with oligonucleotides hybridizing with the long open reading frame (fp7, fp10, fp12, fp13, and fp15), primed unique PCR products of identical and expected lengths whether the template was 1) DNA from the ZAP cDNA library, 2) unamplified cDNA reverse-transcribed from total mRNA, or 3) nuclear genomic DNA (data not shown). This result argues that the second open reading frame exists in the genome and that the sequence intervening between the two open reading frames is not an unspliced intron. Second, the length of the FP21 mRNA (800 bp) is not consistent with removal of any conceptual intron in this region which can be constructed from conventional Dictyostelium splice sites. Third, the amino acid composition of purified FP21 is inconsistent with the composition for a protein predicted to result if any conceptual intron is spliced out and the resultant protein were to reflect the small open reading frame.

The most notable feature of the FP21 sequence is that it is devoid of a signal peptide (33) for translocation across the rER or any other sequences which are known to directly or indirectly promote membrane translocation or anchorage. Based on comparison of predicted and determined (5) NH(2) termini, only up to two NH(2)-terminal amino acids are removed from cellular FP21 after translation. These observations are consistent with the model in which FP21 is both synthesized and glycosylated in the cytosolic compartment, by glycosyltransferases which also reside in the cytosolic compartment(5) .

No significant homology was detected with other proteins in release 83.0 of the GenBank data base. The amino acid sequence from 120-128 (Fig. 5) matches the ATP/GTP-binding site motif A (P-loop) consensus sequence ((A/G)times4GK(S/T))(34) . A sequence (amino acids 32-49, see Fig. 5) conforms to the prediction of the PEST hypothesis for targeting rapid protein turnover(35) . Significant alpha-helical folding is predicted for the first 40 amino acids according to both Chou-Fasman and Garnier-Robson criteria, and this region contains four heptad repeats which are predicted to fold into a coiled coil domain (score = 1.33; (36) ) capable of interacting with another polypeptide. This domain may explain the aggregation characteristics of FP21 observed biochemically.


DISCUSSION

FP21, when tracked by radioactivity in its fucosyl moiety, was found previously to be highly enriched in the cytosolic fraction of lysed cells(5) . In this report, FP21's compartmentalization has been confirmed using antibodies, and new data about its carbohydrate composition and amino acid sequence provide additional strong evidence that this protein does not at any time enter the rER and is thus also glycosylated in the cytosolic compartment. The evidence can be summarized as follows.

First, when FP21 is tracked using specific antibodies, the majority of FP21 is found in the cytosolic fraction. This method of detection reveals, in addition, a substantial fraction of FP21 in the particulate fraction of the cell (Fig. 1). However, purified nuclei and purified actomyosin cytoskeletal preparations, isolated from cells extracted with different lysis mixtures, do not contain FP21, and membrane-disruptive agents do not release FP21 from the particulate fraction (Fig. 2). Rather, FP21 is released by high or low salt treatments (Fig. 2), which tend to dissociate protein-protein interactions. Consistent with this observation, soluble FP21 is capable of undergoing aggregation as determined by sedimentation studies (Fig. 4), SDS-PAGE, gel filtration high performance liquid chromatography (5) , and inaccessibility to in vitro fucosylation (Fig. 3). Possibly, FP21 is associated with a cytoskeletal fraction other than the actin-based system. Thus particulate FP21 appears to be in equilibrium with soluble FP21 in the cytosolic compartment.

Second, the deduced amino acid sequence of FP21 (Fig. 5) predicts no signal peptide which might promote translocation of FP21 into the rER or other hydrophobic domains which might directly or indirectly promote membrane association. No more than 2 NH(2)-terminal amino acids are normally cleaved after synthesis. The amino-terminal region of the polypeptide is predicted to fold into a coiled coil domain, which could support protein-protein interactions leading to aggregation.

Third, monosaccharide composition analysis after acid hydrolysis shows that FP21 does not contain GlcNAc or Man above background levels and thus does not contain any N-linked glycans. Since FP21 contains an N-glycosylation sequon at amino acid residues 45-47 (Fig. 5) which might be utilized if FP21 was exposed to the lumen of the rough endoplasmic reticulum, the absence of N-glycosylation provides a functional verification of the structural evidence that this protein does not visit the lumen of the rER. FP21 glycosylation utilizes a pathway distinct from that using the previously characterized cytosolic O-GlcNAc transferase (10) , based on the absence of GlcNH(2) after acid hydrolysis. The FP21 glycan is also significantly different from an O-linked oligosaccharide on the cytosolic enzyme phosphoglucomutase(4) , which is present in Dictyostelium(^2)and in other organisms. M(r) determinations (Table 1) indicate that additional unknown posttranslational modifications occur on FP21. Since the M(r) difference between wild-type and mutant FP21 is not explained by the differences in sugar or amino acid composition, it follows that the unknown additional posttranslational modifications are distinct in the wild-type and mutant strains.

The monosaccharide analyses were performed on 5-10 µg of FP21 adsorbed to PVDF membrane, an amount comparable with that used for protein microsequencing, even though FP21 contains only about 3% carbohydrate. Of the commercial membranes tested, Immobilon(TM)-P contained the lowest background levels of Glc and Xyl, but even this membrane contains sufficient endogenous Glc to account for the level of Glc detected in FP21. Protein was determined for parallel replicated adsorbed samples by amino acid analysis. Protein determination measurements, and comparison of acetone-precipitated and PVDF membrane-adsorbed samples, allowed us to conclude that the methodology described by Weitzhandler et al.(21) can determine accurately for glycoproteins the absolute level as well as the relative levels of neutral sugars, as previously reported using gas chromatographic methods(38) . However, modified elution programs are required to distinguish Xyl and Man, which often coelute.

FP21 resolves into two fractions, peaks I (20-25% of total) and II (75-80%), during DEAE anion exchange chromatography. These peaks contain distinct glycoforms as determined by monosaccharide composition analysis. Peak II contains approximately 2 mol of Gal, 1 mol of Xyl, 1 mol of Fuc, and 0-1 mol of Glc/mol of polypeptide chain, whereas peak I contains 1 mol of Gal, 2 mol of Xyl, 1 mol of Fuc, and 0-3 mol of Glc. Previous data indicated the presence of an O-linked, fucosylated tetra- or pentasaccharide on total pooled FP21(5) , and thus all sugars may occur as a single glycan. If so, the composition results suggest the existence of alternative glycosylation pathways. The negative charge previously reported (5) on this oligosaccharide is not due to phosphate or sulfate substituents, since FP21 could not be metabolically labeled with precursors of these substituents. It is unlikely to be due to a uronic acid, since no sugars with retention times similar to glucuronic or galacturonic acid were detected. It is also unlikely to be due to sialic acid, since there are no confirmed reports of this sugar in Dictyostelium. In the absence of fucosylation as a result of mutation, a single extra Xyl is applied to FP21. This is reminiscent of glycosylation pathways in human cells, where specific sialylation and fucosylation steps are mutually exclusive modifications of a core oligosaccharide structure(37) .

A transferase activity capable of fucosylating FP21 was previously detected in the cytosol(5) . The antibody tracking and sequence data reported here for FP21 substantiate the cytosolic localization of this enzyme, and the sugar analysis data strongly suggest the existence, in addition, of at least one novel galactosyltransferase and one novel xylosyltransferase, each capable of modifying proteinaceous acceptor substrates in the cytosol of Dictyostelium. Further evidence for these enzyme activities is currently being sought.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant R01-GM37539, National Institutes of Health Training Grant 2-T35-HL07489, and grants to the Interdisciplinary Center for Biotechnology Research at the University of Florida. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U18063[GenBank].

§
Permanent address: Dept. of Molecular Genetics, Institute of Genetics, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria.

To whom correspondence should be addressed: Dept. of Anatomy & Cell Biology, University of Florida College of Medicine, 1600 SW Archer Rd., Gainesville, FL 32610-0235. Tel.: 904-392-3329; Fax: 904-392-3305; westcm{at}anatomy.med.ufl.edu.

(^1)
The abbreviations used are: rER, rough endoplasmic reticulum; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; MALDI-TOF-MS, matrix-assisted laser desorption time-of-flight mass spectrometry; PCR, polymerase chain reaction; all sugars are of the D configuration, with the exception of fucose, which is of the L configuration.

(^2)
B. Gonzalez and C. M. West, unpublished results.


ACKNOWLEDGEMENTS

We are grateful to R. A. Firtel (University of California, San Diego) for providing the cDNA library, Adriana Manzi (University of California, San Diego) for her valuable advice on monosaccharide separations, Gary Lafleur for preparing genomic DNA, Gerry Shaw for interpreting sequence motifs, Scherwin Henry for preparing the hybridomas, Nancy Denslow for advice on gel extraction and Western blotting, Marc Forgione for excellent technical support, Benne Parten for excellent protein sequencing and composition analysis, and Ed Siden for advice on RNA preparation.


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