©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization and Partial Purification of a Novel Enzymatic Activity
UDP-GlcNAc:Ser-PROTEIN N-ACETYLGLUCOSAMINE-1-PHOSPHOTRANSFERASE FROM THE CELLULAR SLIME MOLD DICTYOSTELIUM DISCOIDEUM(*)

(Received for publication, November 14, 1994; and in revised form, January 3, 1995)

Silvana Merello Armando J. Parodi (§) Roberto Couso (¶)

From the Instituto de Investigaciones Bioquímicas ``Fundación Campomar,'' Antonio Machado 151, 1405 Buenos Aires, Argentina

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

An enzymatic activity that transfers N-acetylglucosamine-1-phosphate residues from UDP-GlcNAc to serine units in proteins (UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase) was detected in membranes of the cellular slime mold Dictyostelium discoideum. The enzyme was partially purified by affinity chromatography in concanavalin A-Sepharose and ion exchange chromatography in a Mono Q column. The enzyme showed an absolute requirement for bivalent cations, Mn being more effective than Mg. It had a broad optimum pH value (6.5-9.0). The K for UDP-GlcNAc was 18 µM. In cell free assays it used apomucin and native or 8 M urea-denatured thyroglobulin but neither bovine serum albumin nor native or denatured uteroferrin as exogenous acceptors. Analysis of proteins isolated from cells grown in the presence of [P]phosphate and from the culture medium showed that the majority of proteins bearing the structure GlcNAc-1-P-Ser were secreted. In equilibrium density centrifugations of microsomes, the enzyme appeared in membranes having lighter densities than the enzyme that phosphorylates high mannose-type oligosaccharides. This showed that the activity that phosphorylates serine residues in proteins (UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase) is different from that phosphorylating protein-linked high mannose-type oligosaccharides (UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase).


INTRODUCTION

The first step in the formation of mannose 6-phosphate markers in mammalian lysosomal enzymes is catalyzed by the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase, an enzyme that efficiently phosphorylates high mannose-type oligosaccharides in lysosomal enzymes and not in other glycoproteins as it recognizes certain determinants only present in the protein moieties of the former glycoproteins. Upon removal of the N-acetylglucosamine by an alpha-N-acetylglucosamine-phosphodiester N-acetylglucosaminidase, the mannose 6-phosphate markers are recognized by specific receptors in the Golgi apparatus and the lysosomal enzymes are subsequently targeted to lysosomes(1) .

The cellular slime mold Dictyostelium discoideum has, as other organisms, an enzyme that transfers N-acetylglucosamine 1-phosphate from UDP-GlcNAc to position 6 of alpha(1,2)-linked mannose units in glycoproteins(2, 3) . This enzyme differs from that described in mammalian cells or in the amoeba Acanthamoeba castellani because it phosphorylates the lysosomal enzyme cathepsin D and other non-lysosomal glycoproteins as ribonuclease B or ovalbumin with the same low efficiency(2) . Surprisingly, another lysosomal enzyme, uteroferrin, is recognized by the D. discoideum enzyme as a very good substrate. It was postulated that this was due to a special conformation of the oligosaccharide in uteroferrin and not to the recognition of a structural feature in the protein backbone by the phosphotransferase because the deglycosylated uteroferrin failed to inhibit phosphorylation of the glycosylated species(2) . In D. discoideum lysosomal enzymes only 2-3% of the mannose 6-phosphate residues are in the form of monoesters(4, 5) . The majority are as methylphosphate mannose diesters. In fact, targeting of lysosomal enzymes in D. discoideum is not mediated by mannose 6-phosphate markers(6) .

In this paper we are reporting the characterization and partial purification of a novel enzyme, the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase from D. discoideum. This enzyme catalyzes the transfer of N-acetylglucosamine-1-phosphate from UDP-GlcNAc to serine residues in proteins and has a subcellular localization different from that mediating phosphorylation of high mannose-type oligosaccharides (UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase). This showed that although both activities use the same donor substrate (UDP-GlcNAc) and both transfer the same residue (GlcNAc-1-P), they correspond to different enzymes.


EXPERIMENTAL PROCEDURES

Materials

Escherichia coli alkaline phosphatase, Streptomyces griseus protease type XIV (Pronase), MES, (^1)phenylmethylsulfonyl fluoride, trans-epoxysuccinyl-L-leucylamido (4-guanidino) butane (E-64), alpha-methylmannoside, UDP-GlcNAc, GlcNAc, dithiothreitol, ATP, wheat germ agglutinin-Sepharose, UDP-hexanolamine-Sepharose, bovine submaxillary mucin, bovine serum albumin, Man-6-P, GlcNAc-1-P, UDP-Gal, UDP-Glc, Ser-P, and Thr-P were from Sigma. Griffonia simplicifolia I-agarose was purchased from E. Y. Laboratories (San Mateo, CA). Proteose-peptone and yeast extract were from Difco (Detroit, MI). UDP-[^3H]GlcNAc (5.25 Ci/mmol), [P]P(i) (9,000 Ci/mmol), and [-P]ATP (3,000 Ci/mmol) were purchased from DuPont NEN (Wilmington, DE). [beta-P]UDP-GlcNAc was prepared as described(7) . Concanavalin A-Sepharose and the Mono Q HR 5/5 column were from Pharmacia (Uppsala, Sweden).

Cells

D. discoideum strain Ax3 (wild type) cells were grown at 22 °C in a rotatory shaker in a medium containing per liter of solution: 10 g Proteose-Peptone, 10 g of glucose, 1.3 g of MES, and 5 g of yeast extract. pH was adjusted to 6.6.

Crude Cell Lysate and Soluble Enzymatic Preparations

Cells were harvested when the culture reached a density of 10^7 cells/ml, resuspended in 50 mM Tris-HCl, pH 8.0, and centrifuged. Approximately 5 times 10^8 cells were resuspended in 3 ml of the same buffer but containing 1 mM phenylmethylsulfonyl fluoride and 1 µM E-64 and the suspension was first submitted to freezing and thawing and then homogenized with a glass-Teflon homogenizer (20 strokes). The suspension was centrifuged at 12,000 times g for 10 min and the supernatant was used as a source of enzyme (crude cell lysate). For obtaining the soluble preparation the suspension was centrifuged at 100,000 times g for 60 min. The pellet was resuspended in the same solution as above but with the addition of 1% Triton X-100. The tubes were then centrifuged at 100,000 times g for 60 min. The enzyme-containing supernatants had 6-10 mg of protein per ml.

Enzymatic Assays

The incubation mixtures contained, in a total volume of 50 µl, 10 mM MgCl(2), 10 mM MnCl(2), 50 mM Tris-HCl buffer, pH 7.5, 0.25 mM dithiothreitol, 50 mM GlcNAc, 2 mg/ml bovine serum albumin, enzyme protein, 2 nmol of UDP-GlcNAc, 100 nmol of ATP, pH 7.0, and 5 times 10^5 cpm of [beta-P]UDP-GlcNAc or 1.5 times 10^5 cpm of UDP-[^3H]GlcNAc. Apomucin (0.2 mg) or other proteins or alpha-methylmannoside (0.2 M) were added when indicated. Reactions were performed at 22 °C for 60 min except where otherwise indicated. Reactions where transfer to Ser residues was assayed were stopped by the addition of 1 ml of 0.75% phosphotungstic acid in 0.37 N HCl. After 5 min at 0 °C, tubes were centrifuged at 5,000 rpm for 5 min. The precipitates were resuspended in 1 ml of phosphotungstic acid, sonicated, and centrifuged. This procedure was repeated twice more. Precipitates were resuspended in 450 µl of 0.1 M Tris-HCl, pH 8.0, to which 50 µl of the same buffer containing 30 mg/ml Pronase and 10 mg/ml calcium acetate was added. Tubes were incubated for 30 min at 56 °C and then heated at 100 °C for 10 min. Tubes were then centrifuged at 12,000 times g for 10 min. To the supernatants 0.5 ml of 10 mM sodium phosphate buffer, pH 7.0, 0.15 M NaCl was added and solutions were applied to 0.6-ml concanavalin A-Sepharose columns. They were washed with three 1-ml fractions of phosphate-buffered saline. Radioactivity in these fractions corresponded to the activity of the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase. Label retained by the column and measured after mechanical extrusion of the Sepharose-coupled lectin corresponded to the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase. When incorporation into alpha-methylmannoside was measured, incubations were stopped by the addition of 1 ml of sodium EDTA, pH 7.8. Solutions were applied to 1-ml QAE-Sephadex columns equilibrated and washed with 2 mM Tris. Elution of the reaction product was performed with 30 mM NaCl.

Affinity Chromatographies

Columns of 1 ml of wheat germ agglutinin-Sepharose, UDP-hexanolamine-Sepharose, G. simplicifolia-I-agarose were equilibrated with 50 mM Tris-HCl buffer, pH 7.5, 1% Triton X-100, and concanavalin A-Sepharose with 50 mM Tris-HCl buffer, pH 8.0, 1% Triton X-100, 1 mM MgCl(2), 1 mM MnCl(2), 1 mM CaCl(2), and 100 mM KCl. Solubilized enzyme (0.5 ml) was applied three times to the columns. Columns were washed with 5 ml of the respective equilibrating solutions. Fractions of 0.5 ml were collected. The enzyme was not retained by the first three lectins. In the case of concanavalin A-Sepharose, elution was performed with equilibrating solution containing 0.5 M alpha-methylmannoside. The column plus the solution were first kept at 0 °C for 2 h and then for 1 h at room temperature before further addition of the eluting solution.

Ion Exchange Column

The Mono Q column was equilibrated with 50 mM Tris-HCl buffer, pH 7.5, 1% Triton X-100. Solubilized membranes (0.5 ml) were applied and the column washed with 3.5 ml of the same solution (1 ml/min). Fractions of 1 ml were collected. Elution was performed with a 15-ml linear gradient of 0-0.5 M KCl at the same rate. When preparations eluted from a concanavalin A-Sepharose were used, they were dialyzed against the equilibrating solution prior to application to the column.

Subcellular Fractionation

Cells were harvested and washed once with 50 mM Tris-HCl buffer, pH 7.5, 25 mM KCl, 5 mM MgCl(2) (TKM) plus 0.25 M sucrose and resuspended at a concentration of 2-4 times 10^8 cells/ml in the same solution. Cells were broken with a Dounce homogenizer (20 strokes) and the suspension centrifuged for 1000 times g for 5 min. Two homogenization cycles resulted in the rupture of 70-80% of cells. The postnuclear supernatant (3 ml) was layered on 7 ml of TKM plus 0.5 M sucrose, 2.5 ml of TKM plus 2.5 M sucrose. Tubes were centrifuged at 100,000 times g for 30 min. The supernatant was removed and membranes in the 0.5-2.5 M sucrose interphase applied to a discontinuous gradient containing 2 ml of 1.8, 1.5, 1.1, and 0.8 M sucrose in TKM. Tubes were centrifuged for 150 min at 100,000 times g. Membranes in the interphases were removed, diluted with an equal volume of TKM, and centrifuged at 100,000 times g for 30 min. Pellets were resuspended in 50 mM Tris-HCl, pH 7.5, 1% Triton X-100 at protein concentrations of 1-6 mg/ml.

In Vivo Labeling of Cells

To 25 ml of a culture of D. discoideum cells in the exponential phase, 1 mCi of [P]P(i) was added. Cells were centrifuged at 1000 times g for 5 min after 12 h with the label. The supernatant was precipitated with 80% ammonium sulfate and the suspension centrifuged at 12,000 times g for 20 min. The precipitate was resuspended in a minimal volume of 50 mM Tris-HCl, pH 8.0, and exhaustively dialyzed against the same solution (secreted proteins). The cell pellet was resuspended in 170 mM potassium phosphate buffer, pH 7.0, and centrifuged at 1000 times g for 5 min. The pellet was resuspended in 50 mM Tris-HCl buffer, pH 8.0, and sonicated. The suspension was centrifuged at 12,000 times g for 10 min. The supernatant was centrifuged at 100,000 times g for 30 min. Proteins in this supernatant were precipitated with 80% ammonium sulfate. The precipitate of a 12,000 times g centrifugation was resuspended in a minimal volume of 50 mM Tris-HCl buffer, pH 8.0, and exhaustively dialyzed against the same solution (intracellular soluble proteins). The membrane pellet of sonicated material obtained by 100,000 times g centrifugation for 30 min was resuspended in 1 ml of water. Two ml of methanol and 3 ml of chloroform were successively added. The protein interphase was subjected to chloroform/methanol/water (3:2:1) partition twice more. The protein interphase was extracted three times with chloroform/methanol/water (1:1:0.3), washed twice with methanol, and once with water (membrane proteins). The three fractions, soluble, intracellular, and membrane proteins were further analyzed as described under ``Enzymatic Assays'': they were degraded with Pronase and applied to a QAE-Sephadex column. Material that eluted with 30 mM NaCl was applied to a concanavalin A-Sepharose column. Substances not retained by the lectin were desalted on a 60 times 1-cm column of Bio-Gel P-2 equilibrated with 7% 2-propanol.

Proteins Glycosylated in Cell-free Assays

The standard incubation mixture containing a crude cell lysate as enzyme source was divided in three aliquots of 15 µl each after 60 min at 22 °C. One of them was diluted with 190 µl of 0.25 M sucrose, 10 mM EDTA, the second one was treated the same as the first one but it was sonicated, and the third one was diluted with 190 µl of 0.1 M Na(2)CO(3). The three aliquots were then centrifuged at 100,000 times g for 60 min in an Airfuge and incorporation into Ser residues in proteins in the supernatants and pellets was measured as described above.

Methods

Strong and mild acid hydrolysis were performed in 1 N HCl for 4 h at 100 °C or in 0.01 M HCl for 15 min at the same temperature. Solutions were dried and the resulting material resuspended in water and dried several times to eliminate the acid. Basic hydrolysis was performed in 0.4 M NaOH for 20 h at room temperature. The solution was neutralized with HCl and desalted with a 60 times 1-cm Bio-Gel P-2 column equilibrated with 7% 2-propanol. Apomucin was prepared from mucin as described(8) . The samples were treated with alkaline phosphatase as follows: material in 0.1 M Tris-HCl, pH 8.0, was incubated with 2 µl of commercial alkaline phosphatase for 60 min at 30 °C. Reactions were stopped by addition of 2 volumes of ethanol and the supernatants were obtained after a 5-min centrifugation at 5,000 rpm and desalted through a Bio-Gel P-2 column as described above. Chromatographies and electrophoresis were performed on Whatman 1 papers with the following solvents: 1) ethyl acetate/pyridine/acetic acid/water (5:5:1:3), and 2) 95% ethanol, 1 M ammonium acetate, pH 7.4 (5:2). Denaturation of proteins was performed in 8 M urea as described previously(9) .


RESULTS

Characterization of the Reaction Products

A crude cell lysate from D. discoideum was incubated with [beta-P]UDP-GlcNAc or UDP-[^3H]GlcNAc as described under ``Experimental Procedures.'' The reactions were stopped by the addition of phosphotungstic acid and the precipitates were washed and degraded with an unspecific protease (Pronase). Resulting glycopeptides were applied to concanavalin A-Sepharose columns. Unbound radioactivity was characterized as GlcNAc-1-P-Ser as follows.

Results obtained with the P-labeled glycopeptides were: (a) strong acid hydrolysis (1 N HCl, 4 h, 100 °C) of glycopeptides yielded a substance that migrated as Ser-P on paper chromatography. It behaved differently from mannose 6-P and Thr-P (Fig. 1A); (b) weak basic hydrolysis (beta-elimination) of glycopeptides (0.4 N NaOH for 20 h at room temperature) yielded a substance migrating as GlcNAc-1-P on paper chromatography (Fig. 1B); (c) all the label migrated as P(i) if glycopeptides were first treated with alkali (thus producing GlcNAc-1-P, Fig. 1B) and then with alkaline phosphatase (Fig. 1C), this result indicating that the alkaline treatment had produced a substance with an exposed phosphate unit; (d) the same result shown in Fig. 1C was obtained if the product of the alkaline hydrolysis (GlcNAc-1-P) was treated with mild acid (0.01 N HCl, 15 min, 100 °C, not shown), thus indicating that the alkaline treatment had produced a substance with an exposed phosphate linked through an acid-labile bond; (e) glycopeptides first treated with alkaline phosphatase and then with alkali produced a substance migrating as GlcNAc-1-P, thus indicating that the phosphate units were covered in the original sample (Fig. 1D).


Figure 1: Characterization of reaction products. Crude D. discoideum membranes were incubated with [beta-P]UDP-GlcNAc (A-D) or with UDP-[^3H]GlcNAc (E and F), proteins degraded with a protease and glycopeptides not retained by concanavalin A-Sepharose were subjected to strong acid (A) or weak basic hydrolysis (B and E). The products in B and E were treated with alkaline phosphatase (C and F, respectively) or glycopeptides were first treated with the phosphatase and then with alkali (D). In G and H a partially purified enzymatic preparation was employed, apomucin was the exogenous acceptor and [beta-P]UDP-GlcNAc the sugar donor. Glycopeptides were submitted to strong acid (G) or weak basic (H) hydrolysis. Solvent 1 was used for paper chromatography in A and G, and solvent 2 in all the other chromatographies. Abscissa scale on top right corresponds to E and F. For further details see ``Experimental Procedures.'' Standards: 1, Ser-P; 2, P(i); 3, Man 6-P; 4, GlcNAc-1-P; 5, UDP-GlcNAc; 6, GlcNAc; and 7, Thr-P.



When the sugar nucleotide used was UDP-[^3H]GlcNAc the following results were obtained: (a) glycopeptides submitted to basic hydrolysis yielded substances migrating as GlcNAc-1-P on paper chromatography (Fig. 1E); (b) when glycopeptides were first treated with alkali and then with alkaline phosphatase the resulting products migrated as GlcNAc on paper chromatography (Fig. 1F), thus confirming transfer of GlcNAc-1-P by the enzymatic activity. In order to ensure that a N-acetylglucosamine-1-phosphotransferase and not a N-acetylglucosaminyltransferase was being measured, [beta-P]UDP-GlcNAc was used in all experiments described below.

The stability of the phosphate-amino acid bond in the strong acid medium discards the presence of acid labile phosphoamino acids as Lys-P, His-P, or Arg-P. Moreover, the lability of the phosphate-amino acid bond under weak alkaline conditions precludes the presence of Tyr-P(10) . The migration of the product of strong acid hydrolysis on paper chromatography as Ser-P and not as Thr-P confirms the identity of the amino acid involved.

Partial Purification of the Enzymatic Activity and Dependence on Exogenous Acceptors

All the enzymatic activity was found in a particulate fraction that sedimented at 100,000 times g in 60 min. The enzyme was solubilized with a non-ionic detergent and purified by affinity chromatography through concanavalin A-Sepharose or by ionic exchange through a Mono Q column in a fast protein liquid chromatography system (Fig. 2, A and B). The extreme lability of the enzyme first purified by affinity chromatography and then by the Mono Q column precluded further purification steps. All classical procedures to stabilize the enzyme (addition of bovine serum albumin, sucrose, glycerol, etc.) were unsuccessfully attempted so the enzyme preparation used in experiments described below was that only purified by affinity chromatography through a concanavalin A-Sepharose column. The enzyme was not retained by wheat germ agglutinin-Sepharose, UDP-hexanolamine-Sepharose, and G. simplicifolia I-agarose columns.


Figure 2: Partial purification of the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase and dependence on exogenous acceptors. A soluble preparation was purified by affinity chromatography through concanavalin A-Sepharose (A) or by ion exchange chromatography through a Mono Q column (B). Activity in the presence (circle) or absence (bullet) of apomucin is indicated. C, the activity of the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase purified by affinity chromatography was measured in the presence of denatured (bullet) or native (circle) thyroglobulin, apomucin (up triangle), or native uteroferrin (box). D, the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase present in the preparation purified by affinity chromatography was measured in the presence of native (box) or denatured () uteroferrin.



As shown in Fig. 2, A and B, the enzyme was heavily dependent on an exogenous acceptor for activity after either the concanavalin A or Mono Q purification procedures. Apomucin, native or 8 M urea-denatured thyroglobulin were good acceptors, whereas uteroferrin had no acceptor capacity (Fig. 2C). Bovine serum albumin and denatured uteroferrin gave identical results as uteroferrin (not shown). The product obtained with apomucin as acceptor was characterized. Apomucin is a mucin from which all monosaccharides have been removed by chemical procedures and is, therefore, very rich in free Ser and Thr residues. Paper chromatography of the product obtained upon strong acid hydrolysis of glycopeptides derived from proteolytic degradation of phosphorylated apomucin showed that it migrated as Ser-P, differently from Thr-P (Fig. 1G). A weak alkaline hydrolysis of the same glycopeptides produced substances migrating as GlcNAc-1-P (Fig. 1H), thus confirming the identity of the glycopeptide as GlcNAc-1-P-Ser.

As previously reported(2) , native but not denatured uteroferrin was a good acceptor for the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase (Fig. 2D). Native thyroglobulin behaved the same as denatured uteroferrin (not shown).

Some Properties of the Partially Purified Enzyme

The enzyme required the presence of bivalent cations for activity, Mn being more effective than Mg (Fig. 3A). Unlabeled UDP-GlcNAc inhibited the incorporation of label much more efficiently than UDP-Gal or UDP-Glc (Fig. 3B). The optimum pH value was rather broad, between 6.5 and 9.0 (Fig. 3C). The apparent K(m) for UDP-GlcNAc was about 18 µM (Fig. 3D) and the optimum temperature was 20-22 °C (not shown).


Figure 3: Properties of UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase. A, metal requirement. The indicated cations or EDTA, at 10 mM concentration, were added to incubation mixtures. B, the indicated unlabeled sugar nucleotides (100 nmol) were added to incubation mixtures. C, optimum pH. The following buffers at 50 mM concentrations were used: MES (bullet), HEPES (circle), and Tris-HCl (up triangle). D, K of the enzyme for UDP-GlcNAc. Lineweaver-Burk representation of kinetic data. For further details see ``Experimental Procedures.''



Proteins Glycosylated in Vivo and in a Crude Cell Lysate

D. discoideum cells were grown in the presence of sodium [P]phosphate, secreted proteins were collected, and cells were lysed and cellular proteins were divided into intracellular soluble and particulate fractions. All three fractions were proteolytically degraded and the resulting material applied to a QAE-Sephadex column. In order to select phosphodiesters as GlcNAc-1-P-Ser, only material eluted with 30 mM NaCl was collected and further applied to a concanavalin A-Sepharose column to eliminate labeled high mannose-type oligosaccharides. Unbound material was submitted to strong acid (Fig. 4, A-C) or weak basic hydrolysis (Fig. 4, E-G) followed by paper chromatography. Material migrating as Ser-P was present in all samples originated in acid hydrolysis, but higher amounts were found among secreted proteins (Fig. 4A). Material migrating as Ser-P in Fig. 4A was degraded to P(i) when treated with alkaline phosphatase (Fig. 4D).


Figure 4: In vivo phosphorylation of proteins. D. discoideum cells were grown in the presence of [P]P(i) and secreted (A and E), intracellular soluble (B and F), and membrane proteins (C and G) were degraded with a protease, applied to QAE-Sephadex columns, and material eluting with 30 mM NaCl was applied to concanavalin A-Sepharose columns. Unbound material was submitted to strong acid (A-C) or weak basic (E-G) hydrolysis. Material migrating as Ser-P in A was treated with alkaline phosphatase (D). The substance migrating as GlcNAc-1-P in E was submitted to mild acid hydrolysis (H). Paper chromatographies were in solvent 1 (A-D) and 2 (E-H). For further details see ``Experimental Procedures.'' Standards: 1, Ser-P; 2, P(i); 3, Man-6-P; 4, GlcNAc-1-P.



Similarly, substances migrating as GlcNAc-1-P on paper chromatography were produced upon basic hydrolysis of glycopeptides originated from all fractions but higher amounts were found in those derived from secreted proteins (Fig. 4E). Material migrating as GlcNAc-1-P in Fig. 4E was degraded to P(i) when treated with 0.01 N HCl for 15 min at 100 °C (Fig. 4H).

In all panels shown in Fig. 1and Fig. 4the indicated standards were run in both lanes adjacent to those having the labeled samples. This precaution was taken to properly identify the products obtained, independently from the distances migrated by the labeled substances. For instance, the labeled substance in Fig. 4H was identified as P(i), although it migrated far ahead of the same standard in other chromatographies (Fig. 4, E-G) It may be concluded that higher amounts of phosphorylated proteins were found among secreted proteins and that membrane-bound and soluble intracellular proteins were phosphorylated in vivo to lesser extents.

In order to confirm that both soluble and membrane bound proteins were phosphorylated, a crude cell lysate was incubated with [beta-P]UDP-GlcNAc and the reaction mixture centrifuged at 100,000 times g for 60 min. As shown in Table 1, incorporation into Ser residues was found both in the supernatant and the precipitate. A slight increase in the amount of labeled soluble proteins was found when the incubation mixture was sonicated or treated with 0.1 M Na(2)CO(3) before centrifugation. Both procedures are known to liberate proteins in sealed vesicles and those loosely attached to membranes(11) . Results shown in Table 1confirm, therefore, that both soluble and membrane bound proteins were phosphorylated.



Subcellular Distribution of UDP-GlcNAc:Ser-Protein N-Acetylglucosamine-1-phosphotransferase

A crude microsomal membrane fraction was submitted to equilibrium density centrifugation in tubes containing layers of 0.8, 1.1, 1.3, 1.5, and 1.8 M sucrose. As depicted in Fig. 5A the enzyme mainly sedimented in the 0.8-1.1 and 1.1-1.3 M sucrose interphases. The distribution was different from that of the activity that phosphorylates high mannose-type oligosaccharides (UDPGlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase) that mainly sedimented at heavier densities (1.3-1.5 and 1.5-1.8 M sucrose interphases) (Fig. 5B). The ratio of both total activities in the different fractions clearly showed a different subcellular distribution (Fig. 5C). This is the only evidence that indicated that both phosphotransferases were different enzymes as they had similar optimum pH curves, metal requirements, and they behaved similarly in all affinity chromatographies mentioned above as well as in the Mono Q ion exchange chromatography. Moreover, thermal inactivation curves at 28, 32, and 37 °C at pH 7.5 and 9.0 gave similar results for both enzymes.


Figure 5: Subcellular distribution of N-acetylglucosamine-1-phosphotransferases. D. discoideum membranes were submitted to equilibrium density centrifugation in discontinuous sucrose gradients as indicated under ``Experimental Procedures.'' The percentages of total activities of the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase (A) and the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase (B) found in the interphases of different sucrose molarities are represented. In C the ratio of the activities of the first over the last enzyme are represented.




DISCUSSION

The enzyme that transfers of N-acetylglucosamine-1-phosphate to serine residues in proteins (UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase) appeared to be bound to light membranes that have been found to derive from a Golgi compartment(12) . This location differs from that of the phosphotransferase that phosphorylates mannose units (UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase). Although targeting of lysosomal enzymes in D. discoideum is not mediated by Man-6-P markers, the latter enzyme was found bound to heavier membranes, probably those belonging to the endoplasmic reticulum-Golgi intermediate compartment and/or the cis-Golgi cisternae, the same as in mammalian cells(13) .

Results obtained both in cell free incubations and in vivo showed that soluble and membrane bound proteins were phosphorylated. Secreted proteins appeared to be the species mainly modified by the addition of GlcNAc-1-P units to Ser residues in vivo. D. discoideum cells efficiently secrete lysosomal enzymes during vegetative growth in axenic media(14) . In fact, the presence of GlcNAc-1-P-Ser in a lysosomal enzyme (cysteine proteinase I) of this cellular slime mold was described several years ago(15) . This indicates that the presence of the phosphate-linked GlcNAc residues in the enzyme is permanent and not transient as in mammalian lysosomal enzymes.

An unanswered question is whether the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase phosphorylates all free Ser residues in proteins or if alternatively, it recognizes special features in the acceptor protein. On one hand the fact that proteins having free Ser units as uteroferrin (a very good substrate for D. discoideum UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase)(2) , denatured uteroferrin, or bovine serum albumin were completely ineffective as acceptors would indicate that the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase does indeed recognize special features in the acceptor protein.

On the other hand, results both reported previously and presented here would suggest a lack of specificity of the enzyme: it has been communicated that D. discoideum cysteine proteinase I contains 0.7 µmol of phosphate per mg of protein and that all phosphate is as GlcNAc-1-P-Ser(16) . Moreover, from the known amino acid sequence of cysteine proteinase I, the enzyme has a molecular mass of 36,000 daltons(17) . It can be calculated, therefore, that the cysteine proteinase has 25 GlcNAc-1-P-Ser residues per molecule. As the enzyme has exactly 25 Ser units it may be concluded that all Ser residues are phosphorylated. This suggests a lack of specificity of the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase with respect to the acceptor protein backbone as the Ser units are evenly distributed in the protein moiety and no special features can be recognized in sequences vicinal to them. This agrees with the fact that proteins totally foreign to D. discoideum as apomucin or thyroglobulin were phosphorylated. The fact that denatured thyroglobulin was phosphorylated even better than the native species was probably due to a difference in the Ser units exposed and discards any special conformational requirement of the protein moiety for phosphorylation as previously shown for the mammalian UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase(18) . This lack of specificity strongly suggests that all proteins traversing the subcellular compartments containing the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase, as probably all lysosomal enzymes do, may contain GlcNAc-1-P-Ser units. It is suggestive that a peptide isolated by papain digestion of cysteine proteinase I completely inhibited immunoprecipitation of beta-N-acetylglucosaminidase from D. discoideum by an antiserum prepared against the former enzyme(19) . The peptide had a high concentration of GlcNAc-1-P-Ser units. The presence of these units would represent another modification of D. discoideum lysosomal enzymes in addition to the already known methylphosphate mannose (4, 5) and sulfated mannose in high mannose-type oligosaccharides(20) .

The specificity of the UDP-GlcNAc:Ser-protein N-acetylglucosamine-1-phosphotransferase with respect to the acceptor protein merits, therefore, further studies as contradictory evidence does not allow us to clearly decide whether or not the enzyme recognizes special features in the acceptor protein.

There are also indications suggesting that a nonlysosomal enzyme, the contact A glycoprotein from the plasma membrane of D. discoideum, might contain GlcNAc-1-P-Ser residues. It has been reported that this glycoprotein contains Ser-P but not Thr-P residues (21) . The procedure employed for the isolation of phosphoamino acids (hydrolysis of the glycoprotein in 6 M HCl at 110 °C for 1 h) should have liberated the GlcNAc residues if they had been present in GlcNAc-1-P-Ser groups. Moreover, the glycoprotein had sulfated high mannose-type oligosaccharides and was recognized by wheat germ agglutinin, a lectin that binds N-acetylglucosamine residues. Addition of tunicamycin to the growth medium abolished the appearance of the sulfated oligosaccharides but not the capacity to bind the lectin(22) . It may be speculated that the N-acetylglucosamine residues were linked to Ser through phosphodiester bridges. The function of GlcNAc-1-P-Ser residues is unknown but they may confer proteins bearing them increased resistance to proteolysis.


FOOTNOTES

*
This work was supported by the Swedish Agency for Research Cooperation with Developing Countries (SAREC), the National Research Council (Argentina), and the University of Buenos Aires. 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.

§
Career Investigator of the National Research Council (Argentina). To whom reprint requests and correspondence should be addressed: Instituto de Investigaciones Bioquímicas, Fundación Campomar, Antonio Machado 151, 1405 Buenos Aires, Argentina. Tel.: 54-1-88-4015; Fax: 54-1-865-2246.

Deceased on August 15, 1992.

(^1)
The abbreviation used is: MES, 2-(N-morpholino)ethanesulfonic acid.


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