(Received for publication, November 14, 1994; and in revised form, January 3, 1995)
From the
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).
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
-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 (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.
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
(
-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
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
[-
P]UDP-GlcNAc (A-D) or with
UDP-[
H]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
[
-
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
; 3, Man 6-P; 4, GlcNAc-1-P; 5,
UDP-GlcNAc; 6, GlcNAc; and 7,
Thr-P.
When the sugar nucleotide used was
UDP-[H]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,
[
-
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.
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 () or absence (
) 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 (
) or
native (
) thyroglobulin, apomucin (
), or native uteroferrin
(
). D, the UDP-GlcNAc:glycoprotein N-acetylglucosamine-1-phosphotransferase present in the
preparation purified by affinity chromatography was measured in the
presence of native (
) 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).
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 (), HEPES (
),
and Tris-HCl (
). D, K
of
the enzyme for UDP-GlcNAc. Lineweaver-Burk representation of kinetic
data. For further details see ``Experimental
Procedures.''
Figure 4:
In vivo phosphorylation of
proteins. D. discoideum cells were grown in the presence of
[P]P
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
; 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 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, 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
[-
P]UDP-GlcNAc and the reaction mixture
centrifuged at 100,000
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
CO
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.
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.
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
-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.