Proteinase-1 was described previously as an abundant lysosomal
cysteine protease from Dictyostelium discoideum amoeba growing
on bacteria(1) . More recently, we found that proteinase-1 is
the predominant member of a family of developmentally regulated
cysteine proteinases whose expression is dramatically increased at the
very end of vegetative growth on bacteria and then plummets as
development begins(2) . These enzymes are weakly expressed in
axenically growing cells(3) . Proteinase-1 originally attracted
attention because it has multiple GlcNAc-
-1-P residues bound to
serine(4, 5) . No other carbohydrate modifications
were reported for this protein, which makes it unusual for two reasons.
First, animal lysosomal cysteine proteases typically contain one or
more N-linked oligosaccharides(6) , and second, all
other known Dictyostelium lysosomal enzymes have
phosphorylated and sulfated N-linked chains(7) . The
presence of mannose-6-phosphate as a phosphomethyl diester
(Man-6-P-OCH
) (
)(8, 9) mediates
the binding of these enzymes to the 275-kDa mammalian phosphomannosyl
receptor(10, 11) . However, no analogous receptor is
known in Dictyostelium. Sulfate occurs as a cluster of
mannose-6-SO
residues that define an antigenic determinant
called common antigen 1, which is recognized by a panel of monoclonal
antibodies(12) . Previous studies showed that antibodies raised
against proteinase-1 also recognize a determinant shared with
-N-acetylglucosaminidase (GlcNAc), a typical lysosomal
enzyme known to contain phosphorylated and sulfated N-linked
chains(13) . This cross-reactivity suggested that either
GlcNAc-
1-P is also present on
-N-acetylglucosaminidase or perhaps proteinase-1 contains
previously unrecognized N-linked chains typical of other Dictyostelium lysosomal enzymes. To resolve this issue and
begin to explore the function of phosphoglycosylation, we purified
proteinase-1 from vegetative cells and characterized its carbohydrate
modifications. We confirmed the presence of GlcNAc-
-1-P and found
that proteinase-1 also contained sulfated N-linked chains but
not mannose-6-P. We developed a monoclonal antibody against
GlcNAc-
-1-P and also showed that an entire family of vegetative
cysteine proteinases shares this modification, but all lack
mannose-6-P. Conversely, the typical lysosomal enzymes with mannose-6-P
do not have GlcNAc-
-1-P, except for a small portion of N-acetyl-
-D-glucosaminidase.
EXPERIMENTAL PROCEDURES
Materials
PNGase-F was purified as
described(14) . Alkaline phosphatase-conjugated goat
anti-rabbit and anti-mouse IgG were from Promega (Madison, WI).
Nitrocellulose membranes and protein A-agarose were obtained from
Schleicher & Schuell. Nitro blue tetrazolium,
5-bromo-4-choloro-3-indolyl phosphate, wheat germ agglutinin-agarose,
concanavalin A-Sepharose, bovine serum albumin, L- and D-fucose, UDP-GlcNAc, GlcNAc-
-1-P, UDP-Glc, UDP-GalNAc, N-t-(N-tert-butoxy-carbonyl)-Val-Leu-Lys-(7-amido-4-methylcoumarin),
and goat anti-mouse IgG (Fc-specific) were purchased from Sigma.
Dulbecco's modified Eagle's medium, 8-azoguanine,
hypoxanthine/aminopterin/thymidine supplement, and nonessential amino
acids were from Life Technologies, Inc. Fetal bovine serum was from
HyClone Laboratories, Inc. (Logan, UT). Pansorbin was purchased from
Calbiochem Novabiochem Corp. (La Jolla, CA). All other chemicals were
of reagent grade.
Growth of Cells
All experiments used D.
discoideum strain AX4. In most experiments, cells were grown on
agar plates in association with Klebsiella aerogenes and
harvested prior to the onset of development(15) . Cells were
also grown axenically in synthetic HL5 medium and harvested in late log
phase (15) . Cells at various times of vegetative growth and
development were prepared as described(2) .
Lectin Chromatography
Concanavalin A-Sepharose
binding of proteinase-1 was done on 2
0.5-cm columns
equilibrated in TBS at pH 7.2. Sample was loaded in 0.5 ml of TBS and
allowed to bind for 15 min. Four 1-ml fractions were collected followed
by elution with 4
1-ml fractions of 100 mM
-methyl mannoside in TBS. Each fraction was assayed for
proteinase-1 activity as described(1) . Wheat germ agglutinin
affinity chromatography was performed on a 1
0.5-cm column by
adding the sample in 0.5 ml of TBS and allowing it to bind for 15 min.
4
1-ml fractions were collected followed by elution with 4
1 ml of 100 mM
-methyl Gal and finally by elution
with 3
1 ml of 100 mM GlcNAc. Lysosomal enzymes were
prepared from axenic cells after their secretion into non-nutrient
phosphate buffer for 3 h(16) .
Carbohydrate Analyses
The carbohydrate
composition was determined on a 50-100-µg sample that was
hydrolyzed in 2 N trifluoroacetic acid for 4 h at 100 °C
and then injected into a CarboPac PA-1 column and detected with a
Dionex DX-500 system (Sunnyvale, CA) equipped with a pulsed
amperometric detector (UCSD Glycobiology Core). Identification and
quantitation was based on co-elution and detector response using known
monosaccharide standards (17) . Sugars obtained by
-elimination were directly injected on a CarboPac PA-1 column
eluted for 5 min with 100 mM sodium hydroxide followed by a
60-min linear gradient up to 400 mM sodium acetate. Standard
Fuc, GlcNAc, and GlcNAc-
-1-P were used for calibration. Sugar
alcohols were analyzed on a CarboPac MA-1 column following
-elimination or acid hydrolysis (as above) of the glycoprotein.
Elution was done with a linear gradient from 20 to 500 mM sodium hydroxide over 53 min. In these conditions, FucOH,
GalNAcOH, GlcNAcOH, and their de-N-acetylated derivatives are
separated. Monosaccharides were analyzed after acid hydrolysis (same as
above) on a CarboPac PA-1 column eluted isocratically with 16 mM sodium hydroxide. The elution position and detector response were
determined with authentic standards. Composition analysis was also
performed on a Finnigan GLC mass spectrometer using trimethylsilyl
derivatives of the re-N-acetylated methyl glycosides and
arabitol as internal standard(18) . Identity of the individual
sugars was confirmed by retention time and by comparison of the mass
spectra with authentic standards.
-Elimination and Analysis of Proteinase-1
500 µg of purified proteinase-1 was dialyzed against water,
lyophilized, and dissolved in 0.05 ml of 0.05 N NaOH at 22
°C for 2 h, neutralized with 0.05 N HCl, and passed over a
0.7
20-cm column of Biogel P-2 equilibrated in water. Fractions
were taken and aliquots were monitored at 241 nm (measures the double
bond formation after
-elimination from Ser or Thr) or assayed for
reducing sugar by the method of Park and Johnson (19) following
acid hydrolysis. Carbohydrate positive fractions were pooled and
subjected to carbohydrate analysis using a CarboPac PA-1 column as
described above. Alternatively, the pooled sample was used for LSIMS as
described below. To determine the presence of fucitol, a similar sample
of 200 µg was analyzed following reductive
-elimination with
0.2 N NaOH at 22 °C in the presence of NaBH
for 24 h. The released material was isolated following
neutralization and passage over a mixed bed ion exchange resin (MB-3).
Fucitol was analyzed using the pulsed amperometric detection system.
LSIMS
Liquid secondary ion mass spectrometry
analysis was performed with a VG 70-SE (VG Instruments) magnetic sector
mass spectrometer in the negative ion mode. The instrument was equipped
with a cesium ion gun operated at 23 kV and with an emission current of
4-5 mA. Spectra were recorded in the mass range 100-1000 at
constant magnetic field. The sample (10 µg) dissolved in 5 µl
of 40% methanol was applied to the stainless steel target holding 2
µl of glycerol/thioglycerol matrix, and 1 µl of 1 M HCl was added. The scans were repeated after the addition of
sodium chloride to confirm the identity of the molecular ion. An
authentic standard was analyzed using the same conditions.
Immunological Procedures
Polyclonal antibodies
against proteinase-1 were prepared as described(1) . IgG
fraction was prepared by passing the serum over a protein A-agarose
column (Schleicher & Schuell) according to the manufacturer's
directions and eluted at pH 2.5. All active antibodies bound to the
column. Affinity purification of antibodies against GlcNAc-
-1-P
was done on a UDP-GlcNAc column as described previously(20) .
Production of Monoclonal Antibodies
Four-week-old
BALB/c mice were injected intraperitoneally with 100 µg of purified
proteinase-1 in complete Freund's adjuvant. Three consequent
boosters were given at 3-week intervals with 35-50 µg of
proteinase-1 in incomplete Freund's adjuvant. Mice were given 50
µg of proteinase-1 in phosphate-buffered saline 3 days prior to the
fusion. Spleen cells were fused with P3
63-Ag8.653 myeloma
cells at 2:1 ratio in the presence of 42% polyethylene glycol in
serum-free medium. Growth and maintenance of hybridomas, cloning by
limiting dilution, and ascites production was performed as
described(21) . IgG was purified from ascites fluid using the
caprylic acid method(22) . Mouse isotyping kit (Bio-Rad
Laboratories) was used to determine the IgG subclass.
ELISA Assays
Titer of the antiserum, screening of
hybridoma supernatants, and inhibition of antibody binding to
proteinase-1 were measured by an ELISA assay. Proteinase-1 was dried at
50 °C for 1 h at the bottom of a plastic 96-well plate. Blocking
was done with 3% bovine serum albumin in TBS for 1 h. An appropriate
dilution of the antibody was added and incubated for 1 h in the
presence or the absence of the indicated amount of inhibitor. The plate
was washed and incubated with a 1:1000 dilution of alkaline
phosphatase-conjugated secondary antibody for an additional hour. After
washing with TBS containing 0.05% Tween-80 the plates were incubated
with 1 mM 4-methylumbelliferyl phosphate for 30-60 min
and diluted to 3 ml, and the fluorescence was read on a Turner
fluorometer (23) . When 3 mMp-nitrophenyl
phosphate was used as the substrate, the resultant color was read on an
ELISA plate reader.
Immunoprecipitations
Vegetative Dictyostelium cell extracts were made by lysing cells (grown on bacteria and
harvested on clearing) with buffer A (0.1 M Tris/HCl, pH 7.5,
containing 150 mM NaCl, 0.1% Nonidet P-40, and 1 mM dithiothreitol) and then precleared with Pansorbin for 2 h at 4
°C. 10 µg of precleared proteins were mixed with different
aliquots of purified mAb AD7.5 and incubated overnight with rocking at
4 °C. 2-fold excess of goat anti-mouse IgG over the mAb was then
added and further incubated at 4 °C for 2 h. Pansorbin was added in
excess to pellet all the antibodies and allowed to incubate for 2 h at
4 °C. The pellet was collected by centrifugation at 10,000
g for 5 min and washed 3-4 times with buffer A. It was
then used either for proteinase assay using N-t-(N-tert-butoxy-carbonyl)-Val-Leu-Lys-(7-amido-4-methylcoumarin) (2) or for the lysosomal enzymes(24) . Specificity was
determined by incubations with 1 mM UDP-GlcNAc.
Immunoprecipitation with CI-M6PR and anti-CI-M6PR was done with
Pansorbin precleared extract prepared as above. Varying amount of
purified CI-M6PR was added to 0.5 µg of precleared extract and
incubated for 5-6 h at 4 °C followed by the addition of
Pansorbin coated with anti-M6PR and further incubated overnight at 4
°C. Specific precipitation was determined by competition with 5
mM Man-6-P.
Western Blots
Mini 12.5% SDS-gels were blotted
onto nitrocellulose paper and blocked with 5% powdered milk TBS
containing 0.05% Tween-80(25) . After washing in TBS containing
0.05% Tween-80, the blots were incubated for 1 h with appropriate
dilutions of antibodies. Blots were washed and incubated for 1 h in
alkaline phosphatase-conjugated goat anti-rabbit IgG or goat anti-mouse
IgG (dilution 1:5000) prior to developing with
5-bromo-4-choloro-3-indolyl phosphate and nitro blue
tetrazolium(25) . Man-6-P containing oligosaccharides on
blotted proteins were detected by incubation with 1 µg/ml bovine
CI-MPR in TBS containing 0.05% Tween-80, followed by rabbit antiserum
against CI-MPR (1:1000 dilution) and developed with alkaline
phosphatase-conjugated goat anti-rabbit IgG. In control experiments,
normally positive bands were not seen if CI-MPR was omitted from the
incubation sequence or if CI-MPR is co-incubated in the presence of 5
mM Man-6-P.
Enzyme Digestions
PNGase-F digestions were
performed on heat and SDS denatured samples of proteinase-1 using 1
milliunit of enzyme in a total volume of 50 µl at pH 7.0 as
described previously (26) . After digestion, the protein was
analyzed by Western blotting.
RESULTS
Carbohydrate Composition and Preliminary Carbohydrate
Chain Characterization
Previous studies showed that proteinase-1
contains only GlcNAc-
-1-P linked to serine, and it accounted for
nearly 20% of the mass of the protein(4, 5) . No other
sugars were reported. We purified proteinase-1 using an established
method and found that it bound to a wheat germ agglutinin-agarose
column and was eluted by 100 mM GlcNAc. Because Dictyostelium does not synthesize sialic acids(35) ,
this supported the presence of clustered GlcNAc residues in the protein
that is consistent with the presence of GlcNAc-
-1-P. We
re-examined the carbohydrate composition of purified proteinase-1 by
gas chromatography/mass spectrometry analysis of the trimethylsilylated
methyl glycosides and by direct high performance anion exchange
chromatography-pulsed amperometric detection analysis as described
under ``Experimental Procedures.'' The results obtained by
two methods agreed well, and the average compositions are shown in Table 1. In contrast to previous studies(4, 5) ,
these analyses showed the presence of fucose and mannose and a small
amount of xylose. Variable amount of glucose is most likely a
contaminant. Significantly, these analyses showed no evidence for
mannose-6-phosphate, which is normally found on N-linked
chains of Dictyostelium lysosomal enzymes. To determine
whether the various sugars occurred on N- and O-linked chains, the carbohydrate composition was determined
before and after digestion with PNGase F to release N-linked
oligosaccharides. The released chains were isolated by gel permeation
chromatography, and their composition was determined. Table 2shows that the majority of the mannose and xylose was
sensitive to PNGase F, whereas less than 10% of the fucose and GlcNAc
were released.
Identification of GlcNAc-
-1-P in
Proteinase-1
To determine the type of linkage involved in GlcNAc
and fucose, purified proteinase-1 was treated with 0.05N NaOH at 22
°C for varying times. This treatment caused a dramatic increase in
absorbance at 241 nm, which reached a maximum by 1 h (not shown). This
change is due to the formation of an unsaturated bond in the linkage
amino acid during
-elimination of the carbohydrate
moiety(27) . The sample was neutralized and fractionated on a
Biogel P-4 column. The void region containing the protein was pooled
separately from the released material. The latter was analyzed by LSIMS
(mass accuracy of ± 0.5 mass unit) as described under
``Experimental Procedures.'' As shown in Fig. 1, a
major molecular ion was detected at m/z 300. The
pattern observed corresponded exactly to that obtained for the
GlcNAc-
-1-P standard. The predicted theoretical mass of GlcNAc-1-P
was 299.17 and the observed [M+H] value was 300, which
is within the error of the instrument. No peaks were detected beyond m/z 500, indicating the absence of larger structures.
Complete acid hydrolysis of the sample followed by high pressure liquid
chromatography analysis showed only GlcNH
in this fraction.
Because no fucose was released,
-elimination was repeated using
harsher conditions of 0.2 N NaOH for 24 h at 22 °C in the
presence of NaBH
. This is sufficient to release O-linked fucose from mammalian glycoproteins as
fucitol(28) , but HPAEC-PAD in a CarboPac PA-1 column showed
the presence of only GlcNAc-
-1-P and no evidence of fucitol. Taken
together, these results show that all of the base released GlcNAc
occurs as GlcNAc-
-1-P. However, the linkage of fucose to the
protein or to other sugars remains unresolved. Fucose is not likely to
be retained in a PNGaseF-resistant N-linked oligosaccharide,
because nearly all mannose was released by digestion with this enzyme.
Figure 1:
LSIMS of the material released by
-elimination from proteinase-1. 200 µg of proteinase-1 was
prepared as described under ``Experimental Procedures,'' and
the released material was subjected to LSIMS. Top panel,
standard GlcNAc-
-1-P. Lower panel, material released from
proteinase-1 after
-elimination in 0.02 N NaOH.
Preparation and Characterization of Rabbit Antibodies
against Proteinase-1
We prepared an antiserum against
proteinase-1 and purified the IgG fraction by repeated passage over a
column of immobilized UDP-GlcNAc to select for antibodies that
recognize GlcNAc-
-1-P(1, 19) . The affinity
purified antibody bound to proteinase-1 and was 50% inhibited by 0.08
mM UDP-GlcNAc, 0.1 mM GlcNAc-
-1-P, 0.7 mM UDP-GalNAc, and >1 mM UDP-Glc. No inhibition was seen
with GalNAc, Glc, Man, Gal-1-P, Man-1-P, Glc-1-P, UDP-Gal, or serine
phosphate at 0.5 mM. On Western blots with proteinase-1, these
antibodies recognize a major band at 38 kDa and two faint bands at 36
and 55 kDa. Binding to the three bands was competed by 12.5 mM UDP-GlcNAc (Fig. 2A).
Figure 2:
Competition between the GlcNAc-
-1-P
antibody and different sugars for the carbohydrate epitope. A,
50 ng of proteinase-1 was separated by SDS-polyacrylamide gel
electrophoresis, blotted, and analyzed with UDP-GlcNAc purified
antibodies in the presence and the absence of 12.5 mM UDP-GlcNAc. B, 0.5 µg of proteinase-1 run as
described above and probed with mAb 83.5 (recognizes a fucose
containing epitope on Dictyostelium non-N-linked
glycans) in the presence and the absence of L- and D-fucose.
Further Analysis of N-linked Chains
Proteinase-1
bound to concanavalin-Sepharose and was eluted with
-methyl
mannoside (not shown). As mentioned above, digestion of proteinase-1
with PNGase-F releases a portion of the carbohydrate and reduces the
apparent size of the major band by approximately 1.5 kDa (Fig. 3), but the binding of GlcNAc-
-1-P specific rabbit
antibody is not affected. This is consistent with the results obtained
by composition analysis of the PNGase-F released material and suggests
the presence of probably a single N-linked chain. However, as
expected, proteinase-1 still retains full reactivity with the affinity
purified GlcNAc-
-1-P-specific antibody. In contrast, PNGase-F
digestion greatly reduces the binding of monoclonal antibody mLE2
directed against common antigen 1, a group of mannose-6-sulfate
residues on N-linked oligosaccharides of Dictyostelium lysosomal enzymes(12) . These sugar chains typically also
contain Man-6-P-OCH
residues that mediate their binding to
the mammalian CI-MPR(8, 9, 10, 11) .
As shown in Fig. 3, immunoblots with the CI-MPR showed no
binding of purified Proteinase-1 but reacted strongly with a similar
amount of a mixture of Dictyostelium lysosomal enzymes with
phosphorylated oligosaccharides. The binding to CI-MPR is lost if the
proteins are first treated with PNGase-F (Fig. 3) or if binding
is carried out in the presence of 5 mM Man-6-P (not shown).
These results further confirm the direct carbohydrate compositional
analysis, showing that proteinase-1 does not contain Man-6-P, which
makes proteinase-1 quite unusual compared with other lysosomal enzymes
in Dictyostelium.
Figure 3:
Probing the carbohydrate structures of
proteinase-1 with various antibodies. 1 µg of pure proteinase-1 (lanes 1-8) was run on 10% SDS-polyacrylamide gel
electrophoresis, blotted onto nitrocellulose, and probed with either,
polyclonal antibodies against proteinase-1 purified over UDP-GlcNAc
column (lanes 1 and 2), mAb against the CA-1, a
cluster of mannose-6-sulfate (lanes 3 and 4), mAb
83.5 against fucose (lanes 5 and 6), and CI-M6PR (lanes 7 and 8). Lanes 9 and 10 contained 2 µg of Dictyostelium secreted lysosomal
enzymes and were probed with CI-M6PR. C, control lanes; NG, PNGase-F digests.
Presence of Fucose on Non-N-linked
Structures
Monoclonal antibody 83.5 recognizes
-fucose
residues in an unknown linkage to several proteins in Dictyostelium(29) . As shown in Fig. 3,
proteinase-1 also contains the epitope recognized by mAb 83.5 in a
PNGase-F-resistant linkage that is competed with L-fucose but
not D-fucose (Fig. 2B). This result confirms
that fucose occurs on proteinase-1 and is not a contaminant in the
preparation. Digestions with almond
-L-fucosidase, which
cleaves
(1-3,4) linked residues(30) , or chicken
liver
-L-fucosidase, which cleaves
(1-2,4,6)
linkages (31) , could not remove these fucose residues. We have
been unable to identify how fucose residues are linked to the protein.
Monoclonal Antibody Recognizing
GlcNAc-
-1-P
Monoclonal antibodies against GlcNAc-
-1-P
were made in mice immunized with proteinase-1. 26 positive hybridomas
bound to purified proteinase-1 by ELISA screening, and three of these
were specifically inhibited with UDP-GlcNAc or GlcNAc-
-1-P. One of
these, AD7.5, was used for further studies, and as shown in Fig. 4, its binding to proteinase-1 is inhibited best by
UDP-GlcNAc at amounts comparable with that required for inhibition of
the affinity-purified GlcNAc-
-1-P-specific rabbit antibody. AD7.5
was also inhibited with UDP-Glc and GlcNAc-
-1-P and partially with
UDP-GalNAc but was unaffected by 5 mM Glc-6-P, GlcNAc, GalNAc,
and serine phosphate. The three GlcNAc-
-1-P-specific antibodies
recognized the protein on Western blots, suggesting that most of the
other antibodies recognize peptide portions of native proteinase-1. mAb
AD7.5 recognized the same three bands on Western blots as seen by the
polyclonal antibody, and this reactivity could be competed with 10
mM UDP-GlcNAc (not shown).
Figure 4:
Inhibition of mAb AD7.5 with different
sugars. ELISA was performed with 50 ng of immobilized proteinase-1 and
analyzed with mAb AD7.5 in the presence of various competitors at the
indicated concentrations. The results are expressed as percentages of
control without competing sugar.
mAb AD7.5 recognizes other
proteins during various times of growth and development, but
densitometer scans showed that more than 90% of the GlcNAc-
-1-P
reactivity is found at the end of vegetative growth and proteinase-1 is
the major carrier of GlcNAc-
-1-P (Fig. 5). However, other
cysteine proteinases in Dictyostelium are also known to have
this modification(2, 3) .
Figure 5:
Presence of GlcNAc-
-1-P epitope
during vegetative growth and development of Dictyostelium. 10
µg of total cell protein at various stages of vegetative growth and
development was separated by 12.5% SDS-polyacrylamide gel
electrophoresis and after blotting onto nitrocellulose was probed with
1:2000 dilution of mAb AD7.5 tissue culture supernatant, followed by
1:5000 dilution of alkaline phosphatase-conjugated secondary antibody.
The blots were developed with 5-bromo-4-choloro-3-indolyl
phosphate/nitro blue tetrazolium. Axenic indicates cells
growing in axenic medium, whereas points from 29 to 48 represent cells growing on bacteria. Lanes labeled +4 to +24 correspond to cells undergoing development on
2% phosphate-buffered agar plates. The numbers represent the
time in hours.
Reactivity of mAb AD7.5 with Other Lysosomal
Enzymes
Because proteinase-1 did not contain Man-6-P, we wanted
to determine whether other lysosomal enzymes with phosphorylated
oligosaccharides also had GlcNAc-
-1-P. To do this, monoclonal
antibodies against
-mannosidase(32) ,
-glucosidase(33) , and acid phosphatase (34) were
used to precipitate the activities from extracts of cells growing in
liquid medium. The precipitates were then solubilized and analyzed in
immunoblots using CI-M6PR and anti-M6PR or polyclonal GlcNAc-
-1-P
antibody. Each enzyme bound CI-M6PR and its antibody, but none reacted
with the GlcNAc-
-1-P antibody (not shown). To expand this finding,
cell extracts made from bacterially grown Dictyostelium were
mixed with either increasing amounts of CI-M6PR and anti-CI-M6PR or mAb
AD7.5. As shown in Fig. 6A, mAb AD7.5 completely
precipitated all of the cysteine proteinase activity, which includes
proteinase-1 and at least two other cysteine proteinases(3) .
However, it did not precipitate any acid phosphatase,
-glucosidase, or
-glucosidase activities. Technical
difficulties prevented a similar analysis of
-mannosidase in these
samples. Instead, a partially purified preparation of
-mannosidase
was analyzed. Increasing amounts of mAb AD7.5 that completely
precipitated an equal amount of purified proteinase-1 did not
precipitate any
-mannosidase activity (Fig. 6B).
Conversely, CI-M6PR and anti-CI-M6PR precipitated 30-70% of the
typical lysosomal enzymes containing Man-6-P, including
-mannosidase,
-glucosidase, and
-glucosidase, but none
of the cysteine proteinase activity (Fig. 6C). These
results show that a representative selection of lysosomal enzymes have
either GlcNAc-
-1-P or Man-6-P but not both modifications on the
same protein.
Figure 6:
Immunoprecipitation of lysosomal enzymes
from bacterial cell lysates with mAb AD7.5 and CI-M6PR. Lysates were
made from cells growing on bacteria and harvested when the bacterial
lawn was cleared. Cell lysate (A and C) or purified
-mannosidase (B) was immunoprecipitated with varying
amounts of the mAb AD7.5 (A and B) or CI-M6PR and
anti-CI-M6PR (C), and the pellets and supernatants were
analyzed for lysosomal enzymes as described under ``Experimental
Procedures.'' The activities are plotted as percentages of the
total.
An exception to this pattern was NAG, which had been
previously shown to react with the affinity purified
GlcNAc-
-1-P-specific rabbit antiserum(13) . In crude
extract, we found that about 50% of NAG activity was precipitated with
AD7.5. However, 95% of NAG activity was lost during the preclearing
step, prior to the addition of antibody. This large loss of activity
could mean that the sample we analyzed was not a representative. To
determine whether NAG also contains GlcNAc-
-1-P, we used a
preparation of lysosomal enzymes that was highly enriched in NAG and
-glucosidase activities and also contained
-glucosidase and
proteinase-1 activity. Fig. 7shows that mAb AD7.5 completely
precipitates cysteine proteinase activity, whereas it precipitates only
15% of NAG, none of the
-glucosidase, and less than 3% of
-glucosidase activities. All of the precipitations were inhibited
with 1 mM UDP-GlcNAc. The reverse pattern is seen with CI-M6PR
and anti-CI-M6PR. It precipitates 40-90% of the glycosidases, but
none of the cysteine proteinase activity.
Figure 7:
Immunoprecipitation of partially purified
NAG with mAb AD7.5 and CI-M6PR. Partially purified NAG was
immunoprecipitated either with GlcNAc-1-P mAb (A) or CI-M6PR
and anti-CI-M6PR (B) as described under ``Experimental
Procedures.'' After extensively washing the pellets, they were
analyzed for NAG,
- and
-glucosidases, and cysteine
proteinase activity.
NAG activity is coded by
two different genes(35) . The major form in axenically growing
cells accounts for 90% of the activity and has a half-life of
15-17 min at 55 °C(35) , whereas the minor form is
thermostable at 55 °C(35) . Because only a minor portion of
NAG was immunoprecipitated, we wanted to determine whether the major
isoform that is known to have Man-6-P also contained the
GlcNAc-
-1-P epitope. More than 85% of the partially purified
GlcNAc was thermolabile at 55 °C (t
=
12 min) and is therefore the major isoenzyme (data not shown). The
thermostable activity (<15%) is probably the minor isozyme. After
inactivating the major enzyme at 55 °C, mAb AD7.5 still only
immunoprecipitated 10% of the remaining NAG activity. If
GlcNAc-
-1-P occurred only on the minor isozyme, the proportion of
the thermostable precipitable activity should have increased. Because
it did not, this suggests that a small fraction of the major isozyme
contains GlcNAc-
-1-P. The 10% of thermostable activity that is
precipitated could represent the residual activity of the major form.
DISCUSSION
Proteinase-1 is rich in GlcNAc-
-1-P as shown by
composition analysis and LSIMS of this sugar released by
-elimination ( Table 1and Fig. 1). This agreed with
previous studies by others(4, 5) . In contrast to
earlier reports, we also found evidence for the presence of N-linked oligosaccharides based on compositional analysis,
lectin binding, and PNGaseF sensitivity, which released the great
majority of the mannose and some of GlcNAc ( Table 1and Table 2). These results clearly show that GlcNAc-
-1-P is not
a part of N-linked chains. Fucose residues were also detected
in hydrolysates of proteinase-1 (Table 1), and a fucose-specific
monoclonal antibody confirmed that it was bound to the protein (Fig. 2B and Fig. 3). However, these residues
were not found on N-linked chains (Table 2), nor did
they appear to be bound to GlcNAc-
-1-P residues. They were not
released by any
-fucosidase digestion or as fucitol following base
treatment. (Fig. 1). Thus, although fucose is clearly present,
it may be linked to the protein via another base-resistant linkage.
The N-linked chains of proteinase-1 contain a modification
that is recognized by a series of monoclonal antibodies that bind to a
cluster of mannose-6-SO
residues found in Dictyostelium(12) . This modification is found almost
invariably along with Man-6-P-OCH
in the previously
analyzed Dictyostelium lysosomal enzymes (8, 9) and is analogous to that seen in mammalian
cells. It accounts for the binding of Dictyostelium lysosomal
enzymes to the mammalian CI-MPR (10, 11) . However,
direct analysis of acid hydrolysates showed no mannose-6-P residues (Table 1), and immunoblots using the CI-MPR showed no reactivity
against proteinase-1 (Fig. 3). This was highly unusual for a Dictyostelium lysosomal enzyme. Presence of xylose is known to
occur in Dictyostelium proteins (36) and is usually in
a
1-2 linkage to the mannose of an N-linked chain.
Although we have not analyzed this in detail, it is tempting to
speculate that xylose could be in a similar linkage.
The
GlcNAc-
-1-P-specific monoclonal antibody showed that an entire
family of at least 3-4 co-regulated proteins all contain this
modification (3) (Fig. 5). The major bands recognized by
this antibody show cysteine proteinase activity (2, 3) and are the major carriers of this modification
in Dictyostelium. Although most cysteine proteinases are well
conserved in eukaryotes, at least two members of this cysteine
proteinase family in Dictyostelium have an unusual serine-rich
domain that probably accommodates the GlcNAc-
-1-P
residues(3, 37) . However, none of these proteinases
contain Man-6-P residues ( Fig. 6and Fig. 7). This
contrasts with
-mannosidase and
and
-glucosidases that
have been shown to contain Man-6-P (7, 8, 9) but do not have GlcNAc-
-1-P (Fig. 6). An apparent exception to this pattern is seen with
NAG, because it is known to contain Man-6-P but was also precipitated
with the GlcNAc-
-1-P antibody (Fig. 7). Earlier studies
with polyclonal serum against proteinase-1 also showed cross-reactivity
with NAG(13) . One possible explanation is that the reactivity
is due to a contamination with a minor NAG isozyme. However,
differential thermolability of the two forms showed that the minor
enzyme could not account for the GlcNAc-
-1-P reactivity and
implied that only a small portion of the major form is modified by
GlcNAc-
-1-P.
We have not examined all lysosomal enzymes for the
presence of GlcNAc-
-1-P and Man-6-P, but our initial results
suggest that they occur on different types of lysosomal enzymes. There
is no information on how the two types of enzymes are distinguished
from each other, but two other Dictyostelium cysteine
proteinases recently cloned by our lab (3) and proteinase-1 (2) all contain serine-rich regions that could be sites of
phosphoglycosylation.
-Mannosidase also has a serine-rich region,
but it clearly does not contain GlcNAc-
-1-P (38) . A
peptide containing a series of Ser-Gly repeats serves as a good
acceptor (K
= 200 µM) for
GlcNAc-
-1-P transferase(37) , showing that the intact
protein is not required for recognition. This transferase is clearly
distinct from GlcNAc-
-1-P transferase catalyzing the first step in
biosynthesis of Man-6-P on N-linked
oligosaccharides(39) .
The mechanism of lysosomal enzyme
targeting in Dictyostelium has not been resolved, especially
in regard to the role of Man-6-P-OCH
. Clearly, if it can be
used for targeting, the cysteine proteinases do not use it. Even though
these enzymes comprise 1-2% of the protein of cells growing on
bacteria, they have received little attention compared with the
glycosidases. This is, in part, because the lysosomal enzyme studies
have all used cells growing in a convenient axenic medium where the
Man-6-P containing enzymes are highly expressed(40) , whereas
the cysteine proteinase activity is lower in axenic
medium(2, 3, 41) . The large increase in
cysteine proteinase activity at the very end of vegetative growth (2) as cells are about to begin development would not be
expected if these enzymes are being used only for digesting bacteria.
The rapid decrease in activity at the onset of development may be
linked to some specialized function at the growth-development
transition to their unusual carbohydrate modification or to the protein
domain that carries these modifications. Preliminary studies in our
laboratory suggest that the two carbohydrate modifications have
distinct roles during phagocytosis of bacteria.