From the Department of Biochemistry and Molecular
Biology, University of Melbourne, Parkville, Victoria 3052, Australia
and the § Joint Protein Structure Laboratory, Ludwig
Institute for Cancer Research and the Walter and Eliza Hall Institute
of Medical Research, Parkville, Victoria 3050, Australia
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ABSTRACT |
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Protozoan parasites of the genus
Leishmania secrete a number of glycoproteins and mucin-like
proteoglycans that appear to be important parasite virulence factors.
We have previously proposed that the polypeptide backbones of these
molecules are extensively modified with a complex array of
phosphoglycan chains that are linked to Ser/Thr-rich domains via a
common Man Parasitic protozoa of the genus Leishmania cause a
spectrum of human and animal diseases that are transmitted by a sand
fly vector. During their development in the digestive tract of the sand
fly, these parasites differentiate from non-infective procyclic promastigotes to infective metacyclic promastigotes that target mammalian macrophages when introduced into the host during the insect's blood meal. Following their internalization into the macrophage phagolysosome, metacyclic promastigotes differentiate into a
replicative amastigote stage and eventually rupture the host cell and
perpetuate disease by infecting other host cells. A number of
cell-surface and secreted virulence factors are thought to be crucial
for the survival of these different developmental stages in their
respective host environments. These include an abundant
glycosylphosphatidylinositol-anchored lipophosphoglycan (LPG)1 (1-3) and a number of
secreted glycoproteins and proteophosphoglycans (PPGs) (reviewed in
Refs. 4 and 5). Strikingly, both the cell-surface LPGs and the secreted
molecules are elaborated with structurally related phosphoglycan chains
that are thought to be the major functional determinants of these molecules.
The secreted glycoproteins and proteoglycans of Leishmania
have been shown to form distinct macromolecular complexes in the flagellar pocket and extracellular milieu (4). The most intensively characterized of these molecules are the secreted acid phosphatases (sAPs), which aggregate into large pearl-like filamentous polymers (6,
7). Early studies indicated that sAPs were heavily glycosylated and
phosphorylated enzymes that contained glycan epitopes characteristic of
LPGs (6, 8, 9). We have recently shown that Leishmania mexicana sAP is extensively modified with Man Recent functional studies have shown that these molecules may be
important for parasite infectivity and survival in both the insect and
the mammalian host. L. mexicana aPPG is a potent activator of the complement cascade via the mannan-binding lectin pathway and may
effectively deplete the local supply of complement components needed
for parasite lysis (17). The release of PPGs into the phagolysosome may
also inhibit membrane fusion events and contribute to the formation of
large parasitophorous vacuoles, which are characteristic of New World
species such as L. mexicana (18). On the other hand,
secretion of sAP and promastigote PPG filamentous networks by the
promastigote stages may be responsible for the tendency of cells to
aggregate into large clusters in stationary growth. Cell aggregation
may improve the efficiency of transmission during the sand fly bite or
afford cells within the cluster some protection from complement lysis
during the early stages of infection (4).
As this type of protein glycosylation is unique to
Leishmania, the enzymes involved in initiating and
assembling these glycans may be potential targets for new
anti-leishmanial drugs. At present, several enzyme activities have been
detected in cell-free assays and detergent extracts that are involved
in the synthesis of the phosphoglycan chains of LPG, but that may also
be involved in the synthesis of the shorter phosphoglycan chains of sAP
and PPGs. These include two elongating enzymes, a putative
1-PO4-Ser linkage (Ilg, T., Overath, P.,
Ferguson, M. A. J., Rutherford, T., Campbell, D. G., and
McConville, M. J. (1994) J. Biol. Chem. 269, 24073-24081). In this study, we show that Leishmania
mexicana promastigotes contain a peptide-specific
mannose-1-phosphotransferase (pep-MPT) activity that adds Man
1-P to
serine residues in a range of defined peptides. The presence and
location of the Man
1-PO4-Ser linkage in these peptides
were determined by electrospray ionization mass spectrometry and
chemical and enzymatic treatments. The pep-MPT activity was solubilized
in non-ionic detergents, was dependent on Mn2+, utilized
GDP-Man as the mannose donor, and was expressed in all developmental
stages of the parasite. The pep-MPT activity was maximal against
peptides containing Ser/Thr-rich domains of the endogenous acceptors
and, based on competition assays with oligosaccharide acceptors, was
distinct from other leishmanial MPTs involved in the initiation and
elongation of lipid-linked phosphoglycan chains. In subcellular
fractionation experiments, pep-MPT was resolved from the endoplasmic
reticulum marker BiP, but had an overlapping distribution with the
cis-Golgi marker Rab1. Although Man-PO4
residues in the mature secreted glycoproteins are extensively modified
with mannose oligosaccharides and phosphoglycan chains, similar
modifications were not added to peptide-linked Man-PO4
residues in the in vitro assays. Similarly,
Man-PO4 residues on endogenous polypeptide acceptors were
also poorly extended, although the elongating enzymes were still
active, suggesting that the pep-MPT activity and elongating enzymes may
be present in separate subcellular compartments.
INTRODUCTION
Top
Abstract
Introduction
References
1-PO4
residues that are linked to serine residues in the polypeptide backbone
(10). This unusual type of linkage, in which a monosaccharide is linked to protein via a phosphodiester bridge, has been termed
phosphoglycosylation (11) and may be widespread in several lower
eukaryotes (12). In L. mexicana, the Man
1-PO4
residues can be further elaborated with
1-2-linked mannose
oligosaccharides or short chains of phosphorylated di- or
trisaccharides (10), which are also found in the long phosphoglycan
chains (as capping structures or internal repeat units, respectively)
of L. mexicana LPG (see Fig. 1). Interestingly, the nature
of these modifications may be influenced by the size of the
Ser/Thr-rich repeat domains in the polypeptide backbone. For example,
sAP-1 contains a relatively short Ser/Thr-rich domain and is modified
primarily with mannose oligomers, whereas sAP-2, which contains a
longer stretch of Ser/Thr-rich repeat sequences, is extensively
modified with short phosphoglycan chains (7, 10). In addition to sAP,
two heavily phosphoglycosylated PPGs have been recently characterized
from leishmanial promastigotes and amastigotes (13-16). Promastigote
PPGs are produced by all species of Leishmania and form a
network of unbranched filaments in the center of aggregated
promastigotes (14, 16). Amastigote PPGs (aPPGs) are produced by most
species and accumulate to very high levels in the lumen of the
parasitophorous vacuole and in the extracellular space of lesions (13,
15). The polypeptide backbones of PPGs differ from sAPs in being rich
in proline and alanine as well as serine and threonine and are more
extensively modified with phosphoglycan chains, which may account for
as much as 90% of the molecule (13-15). Whereas the phosphoglycan
chains of Leishmania major promastigote PPG are similar to
those of the corresponding LPG, L. mexicana aPPGs are
elaborated with novel stage-specific phosphoglycans that are distinct
from those found on both promastigote sAPs and LPGs (15). However, all
these glycans are thought to be linked exclusively to the polypeptide backbone via a Man
1-PO4-Ser core sequence (14, 15).
-mannose-1-phosphotransferase (MPT) and a
1-4-galactosyltransferase, that transfer Man
1-PO4 and Gal from GDP-Man and UDP-Gal, respectively, to form the repeating Gal
1-4Man
1-PO4 backbone of both the LPG and PPG
glycans (Fig. 1) (19, 20). In addition, a
1-3-galactosyltransferase activity that adds the L. major-specific side chains to these repeat units has also been
characterized (21). Interestingly, a separate MPT activity is thought
to be involved in adding the first Man-PO4 residue to the
LPG anchor precursor and thus to be required to initiate LPG
phosphoglycan biosynthesis (22). As one of the enzymes involved in
synthesizing the LPG anchor is localized to the Golgi apparatus (23),
it is likely that both the initiating MPT and phosphoglycan
chain-elongating and -branching enzymes are also localized in either a
Golgi or post-Golgi compartment. This is consistent with the finding
that a GDP-Man transporter required by these MPTs is also localized to
the Golgi apparatus (24) and that phosphoglycan chain elongation is
inhibited by the Golgi-perturbing ionophore, monensin (25).
View larger version (13K):
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Fig. 1.
Structure of the phosphoglycan chains of
L. mexicana promastigote LPG and sAP. The
glycosylphosphatidylinositol (GPI) anchor of LPG is modified
with a single phosphoglycan chain comprising a long domain of
phosphodi- and phosphotrisaccharide repeat units and a mannose
oligosaccharide cap at the nonreducing terminus (2, 3). In contrast, a
Ser/Thr-rich domain in the secreted sAP glycoproteins is modified
either with mannose oligosaccharides or short phosphoglycan chains that
are capped with mannose oligosaccharides. Both classes of glycans are
thought to be attached to serine residues via a Man 1-PO4
linkage (10). The more complex branched phosphoglycans of the L. mexicana aPPGs are not shown (15).
In this study, we have identified a peptide-specific MPT (pep-MPT)
activity from L. mexicana promastigotes that is most likely involved in initiating protein phosphoglycosylation. Mass spectrometry of the glycopeptides containing the Man-PO4 modification
provides the first direct characterization of this novel linkage.
We show that pep-MPT is most active against peptides containing the
Ser/Thr-rich sequences of endogenous polypeptide acceptors and that it
is not inhibited by glycan acceptors of the LPG
phosphoglycan-initiating and -elongating MPTs. Furthermore, we provide
evidence that this enzyme occurs in a distinct subcompartment of the
Golgi apparatus from enzymes involved in phosphoglycan chain elongation.
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EXPERIMENTAL PROCEDURES |
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Materials--
Alkaline phosphatase, GDP-Man, UDP-Gal, and
stachyose were from Sigma; jack bean -mannosidase was from
Boehringer Mannheim. The synthetic oligosaccharide acceptor L2
(Gal
1-4Man
1-PO4-(CH2)8CH=CH2) was generously provided by Professor Michael Ferguson (University of
Dundee). GDP-[3H]Man was prepared using previously
described methods (26, 27). Briefly,
[2-3H]Man-6-PO4 was synthesized enzymatically
from [2-3H]Man (NEN Life Science Products) using yeast
hexose kinase and then converted to the sugar nucleotide using a
mixture of yeast proteins supplemented with snake venom pyrophosphatase
and glucose-1,6-diphosphate. The yeast proteins, corresponding to a
50-70% ammonium sulfate cut, were prepared from protease A-deficient
yeast strain SC295. GDP-[3H]Man was purified on a column
(1 ml) of concanavalin A-Sepharose (Amersham Pharmacia Biotech) that
was washed with 15 mM ammonium acetate, 1 mM
MgCl2, and 1 mM CaCl2 and then
eluted with the same buffer containing 50 mM
-methylmannoside. Fractions containing radioactivity were desalted
on a column of Sephadex G-10 eluted in water. Polyclonal antibodies
against L. major Rab1 protein (28) and Trypanosoma
brucei BiP (29) were generously provided by Dr. Emanuela Handman
(Walter and Eliza Hall Institute of Medical Research, Melbourne,
Australia) and Dr. James Bangs (Department of Biochemistry, University
of Wisconsin, Madison, WI), respectively.
Synthesis of Peptide Substrates-- Peptides were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry on a Applied Biosystems Model 431A peptide synthesizer. Peptides were cleaved from the resin using 95% trifluoroacetic acid (3 h, 25 °C), recovered by extraction with diethyl ether, and purified on a C18 reversed-phase column (4.6, inner diameter, × 100 mm; Brownlee) eluted with acetonitrile (0-60%) in 0.1 M ammonium acetate over 20 min at a flow rate of 1 ml/min. Reductive methylation of sapp-1 was carried out in 0.2 M borate buffer, pH 9.0 (140 µl), containing 16 mM formaldehyde and 0.1 mM NaB3H4 (5 mCi) at 0 °C for 10 min (30). The mixture was acidified with 1 M acetic acid to remove excess NaB3H4, and the radiolabeled peptides were recovered after application to a Sep-Pak C18 cartridge (Waters) following elution with 40% acetonitrile.
Preparation of Parasite Membranes--
Promastigotes of L. mexicana (strain MNYC/BZ/62/M379) were cultivated at 27 °C in
RPMI 1640 medium supplemented with 10% fetal bovine serum and
harvested at mid-log growth unless otherwise stated. Amastigotes were
isolated from tail lesions in BALB/c mice as described by Bates
et al. (31). Cells were harvested by centrifugation
(750 × g, 10 min, 27 °C); washed once with
phosphate-buffered saline; repelleted; and suspended in 50 mM HEPES-NaOH, pH 7.4, containing 50 mM KCl, 10 mM MgCl2, 10 mM MnCl2,
5 mM Na2EGTA, 2 mM dithiothreitol,
1 mM ATP, 0.1 mM phenylmethylsulfonyl fluoride, 2 µM leupeptin, and 0.2 mM
N-p-tosyl-L-lysine
chloromethyl ketone (buffer A). Cell disruption was carried out by
probe sonication (MSE Soniprep 150 Ultrasonic Disintegrator) at 0 °C
using 8 × 10-s bursts at an amplitude of 24 µ with 20-s
intervals between each burst. Greater than 90% cell lysis was
confirmed by microscopy. Intact cells and large cell fragments were
removed by centrifugation (750 × g, 5 min, 4 °C),
and microsomal membranes in the supernatant were recovered by
ultracentrifugation at 100,000 × g (30 min, 4 °C).
The pelleted crude microsomal membranes were resuspended in buffer A or
in buffer A containing 1% Triton X-100 and either used immediately or
stored frozen at
70 °C. Detergent-extracted material (10 min, 0 °C) was centrifuged at 14,000 × g (5 min,
4 °C) to remove insoluble material. Triton X-100 proved to be the
most effective of several detergents tested for recovery of pep-MPT
activity; these included Nonidet P-40, CHAPS, Zwittergent 3-12,
nonanoyl-N-methylglucamide (MEGA-9), and n-octyl
-D-glucopyranoside. Protein was precipitated in
chloroform/methanol/water (32) prior to quantitation using the BCA
assay (Pierce).
Mannose-1-phosphotransferase Assays-- pep-MPT activity was measured using two assays. In the first method, microsomal membranes (2 × 107 cell eq, 20-60 µg of protein) were resuspended in buffer A (50 µl) containing GDP-[3H]Man (160,000 cpm, 0.1-0.5 mM) and 0.4 mM synthetic peptide and incubated at 32 °C for 30 min. Triton X-100-solubilized membranes were assayed under identical conditions, except that the reaction mixture contained 0.1% Triton X-100 and assays were performed at 16 °C. The reaction mixture was diluted with 0.1 M ammonium acetate (950 µl), and the enzyme reaction was terminated by boiling (3 min). Precipitated proteins was removed by centrifugation (14,000 × g, 5 min), and the supernatant was loaded onto Sep-Pak C18 cartridges pre-equilibrated in 0.1 M ammonium acetate. After washing the cartridge with 0.1 M ammonium acetate (10 ml) to remove unincorporated GDP-[3H]Man, [3H]Man-labeled peptide was eluted with 40% acetonitrile (3 ml), and eluted radioactivity was measured by scintillation counting.
In the second method, Triton X-100-solubilized membranes (2 × 107 cell eq, 20-60 µg of protein) were diluted in buffer A (15 µl) containing 0.1% Triton X-100, GDP-Man (0.5 mM), and 3H-labeled peptide (500,000 cpm, 0.16 mM). Assay mixtures were incubated at 16 °C for 15 min. In the substrate competition experiments described in the legend to Fig. 6, the reaction mixtures contained unlabeled peptide or glycan acceptors at 0-5 mM final concentration. Reactions were terminated by boiling (3 min), and the precipitated protein was removed by centrifugation at 14,000 × g for 5 min. Underivatized and mannosylated 3H-labeled peptides in the reaction mixture were resolved from each other by analytical thin-layer electrophoresis of 4 µl of the reaction mixture. Alternatively, 3H-labeled peptides were recovered on Sep-Pak C18 cartridges as described above for subsequent chemical and enzymatic analysis.
HPLC and HPTLC Analyses--
Underivatized and
Man-PO4-modified peptides were separated on a
C18 reversed-phase HPLC (RP-HPLC) column (4.6, inner
diameter, × 100 mm; Brownlee) eluted with 0-35% acetonitrile in 0.1 M ammonium acetate at 1 ml/min over 30 min. Peptides were
detected by UV absorbance at 280 nm (for sapp 1, 2, 4-6) or 216 nm
(for Ser11 and sapp-3). Radioactivity in 0.2-ml fractions was
determined by scintillation counting. Man-PO4-modified
peptide and lipo-oligosaccharide L2 acceptors were resolved on
aluminum-backed Silica Gel 60 HPTLC sheets (Merck) developed in
chloroform, methanol, and 0.25% KCl (10:10:3, v/v) over 10 cm. HPTLC
sheets were sprayed with EN3HANCE (NEN Life Science
Products) and exposed to Biomax MR film (Eastman Kodak Co.) at
70 °C.
Thin-layer Electrophoresis-- Man-PO4-modified 3H-labeled peptides were resolved from unmodified 3H-labeled peptides by thin-layer electrophoresis on plastic-backed cellulose thin-layer sheets (20 × 20 cm; Macherey Nagel). Aliquots of the reaction mixture (containing 100,000 cpm) were spotted onto the cellulose sheets, which were then saturated with pyridine/acetic acid/water (1:10:89, v/v) and subjected to electrophoresis (500 V, 30 mA, 75 min) under a layer of petroleum spirit and pyridine/acetic acid/water (1:10:89, v/v) in each electrophoresis chamber. Radioactivity on the dried cellulose sheets was detected using an automatic TLC scanner (Berthold) and/or by fluorography as described above. Incorporation of radioactivity into phosphoglycosylated peptides was quantitated by scraping the cellulose containing the bands using the fluorograph as template, extracting the cellulose with 30% acetonitrile (2 × 250 µl), and liquid scintillation counting of the extracts.
Enzyme and Chemical Analyses of Phosphoglycosylated
Peptides--
Phosphoglycosylated peptides were digested with jack
bean -mannosidase (30 µl, 50 units/ml) in 0.1 M sodium
acetate buffer, pH 5.0 (18 h, 37 °C), with or without subsequent
digestion in alkaline phosphatase (2000 units/ml) in ammonium
bicarbonate, pH 8.5 (7 h, 37 °C). For recovery of
3H-labeled glycans, the enzyme digests were desalted on a
column (400 µl) of AG 50-X12 (H+) over AG 3-X8
(OH
). Alternatively, 3H-labeled peptides were
recovered on a Sep-Pak C18 cartridge (as described above),
which was eluted with 40% acetonitrile. Specific cleavage of the
Man
1-PO4 bond was achieved with mild acid hydrolysis in
40 mM trifluoroacetic acid (12 min, 100 °C) (3). The
acid was removed by drying under high vacuum in a Speed-Vac
concentrator. 3H-Labeled peptides were analyzed by
thin-layer electrophoresis as described above, whereas
3H-labeled glycans were analyzed by HPTLC using
1-propanol/acetone/water (9:6:5, v/v) as the solvent system (33).
Electrospray Ionization Mass Spectrometry--
Unmodified and
phosphoglycosylated peptides were analyzed with a triple quadrupole
mass spectrometer (Finnigan MAT Model TSQ-700) equipped with a Finnigan
MAT electrospray ionization (ESI) source and rapid capillary RP-HPLC.
The column used in this study (0.2, inner diameter, × 150 mm; Vydac
C18) was fabricated and operated as described elsewhere
(34). The column was developed with a linear 30-min gradient at 1.6 µl/min from 0 to 100% solvent B, where solvent A was 0.1%
trifluoroacetic acid and solvent B was 60% acetonitrile containing
0.1% trifluoroacetic acid (35). The ESI needle was operated at 4.5
kV. The sheath liquid was methoxyethanol delivered at 3 µl/min.
Nitrogen sheath and auxiliary gasses were supplied at 30 p.s.i.
and 15 units (arbitrary units), respectively. The heated capillary was
set at 150 °C. Mass spectra were collected every 3 s in
centroid mode. Peptide masses were calculated using Finnigan MAT
BIOMASSTM software. For tandem mass spectrometric peptide
sequence analysis, quadrupole Q1 was operated with a resolution of
2-2.5 Da and Q3 with a resolution of 1-1.5 Da (35). The collision
cell offset voltage was calculated by multiplying the mass of the ion
selected for collision-induced dissociation by 0.04. The daughter ion
offset voltage was set at twice the collision cell offset value. The parent ion offset voltage was set at one-third the value of the daughter ion offset voltage. Argon was used as the collision gas at a
pressure of 2-2.5 millitorr.
Characterization of Endogenous Polypeptide and LPG
Acceptors--
Sonicated microsomal membranes were incubated with
GDP-[3H]Man in the presence of exogenous peptide
acceptors and UDP-Gal as described above. Membranes were recovered by
centrifugation (14,000 × g, 5 min), washed with water
to remove residual peptide, and recentrifuged, and the supernatants
were combined for recovery of the peptide on a Sep-Pak column as
described previously. The membrane pellet was subsequently extracted
twice in 1% Triton X-100 (200 µl) for 10 min at 30 °C to remove
LPG and soluble phosphoglycan and proteoglycans. Insoluble material was
recovered by centrifugation; the supernatants were combined and
subjected to two-phase partitioning with 1-butanol; and the
detergent-free aqueous phase was dried in a Speed-Vac concentrator.
This material was resuspended in 0.1 M NH4OAc
containing 5% 1-propanol and loaded onto a minicolumn of
octyl-Sepharose equilibrated in the same buffer. The column was washed
with 0.1 M NH4OAc containing 5% 1-propanol and
then with 40% 1-propanol to elute bound LPG (36). The Sep-Pak-eluted peptide, octyl-Sepharose-purified LPG, and Triton X-100-insoluble material were hydrolyzed in 40 mM trifluoroacetic acid (12 min, 100 °C), and the acid was removed under reduced pressure in a Speed-Vac concentrator (36). Acid-released glycans were
dephosphorylated with alkaline phosphatase and desalted by passage over
a column of AG 50-X12 (H+) over AG 3-X8 (OH)
for analysis by HPTLC (3).
Subcellular Fractionation--
L. mexicana
promastigotes (8 × 108) were harvested by
centrifugation (750 × g, 10 min), washed with
phosphate-buffered saline, and then resuspended (2 × 108 cells/ml) in assay buffer A supplemented with 0.25 M sucrose. The cell suspension was transferred to a
prechilled nitrogen cavitation bomb (Kontes Glass Co.) equilibrated at
450 p.s.i. N2 pressure for 15 min at 0 °C, and
cells were lysed by abruptly releasing the pressure. Equilibration and
expulsion of the suspension were repeated twice more to achieve
complete cell lysis. Cell debris and nuclei were removed by
centrifugation of the lysate at 3000 × g for 10 min,
and the supernatant was layered on top of a linear sucrose gradient.
The sucrose gradient (density = 1.05-1.27 g/ml) was prepared by
layering 10 0.8-ml fractions (0.25-2 M sucrose in 0.25 mM HEPES-NaOH, pH 7.4) over a sucrose cushion (2.5 M) in Ultraclear centrifugation tubes (Beckman Instruments)
and centrifuging at 218,000 × g for 1 h at
4 °C in a Beckman L-80 ultracentrifuge using an SW 41 rotor.
Organelles in the cell lysate were fractionated by equilibrium
centrifugation at 218,000 × g for 6 h at 4 °C. Fractions (0.5 ml) were collected from the bottom of the tube, and
densities were calculated by measuring refractive index. The distribution of leishmanial BiP and Rab1, markers for the bulk endoplasmic reticulum and cis-Golgi cisternae, respectively
(28, 29), were determined by immunoblotting. Proteins in each fraction were precipitated in biphasic mixtures of chloroform/methanol/water (32), measured by the BCA assay, separated by SDS-polyacrylamide (12%)
gel electrophoresis, and transferred to nitrocellulose by electroblotting. Strips of nitrocellulose corresponding to regions containing the BiP (70 kDa) and Rab1 (25 kDa) proteins were cut and
processed individually. For detection of BiP, the nitrocellulose was
blocked with 0.05% Tween 20 in Tris-buffered saline, incubated with
rabbit anti-BiP antibody (1 h), washed with 0.05% Tween 20 in
Tris-buffered saline, and then probed with horseradish
peroxidase-conjugated anti-rabbit antibody (1:10,000 dilution; Silenus
Laboratories, Pty., Ltd.) in 0.05% Tween 20 in Tris-buffered saline
containing 1% powdered skim milk (1 h) and visualized with the ECL
Western detection system (Amersham Pharmacia Biotech). Nitrocellulose strips containing Rab1 protein were probed in the same way, except that
the nitrocellulose was initially blocked with 0.05% Tween 20 in
Tris-buffered saline with 1% powdered skim milk, the rabbit anti-L. major Rab1 antibody was used at 1:500 dilution, and
incubation with the secondary antibody was done in 0.05% Tween 20 in
Tris-buffered saline.
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RESULTS |
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Phosphoglycosylation of Peptides Containing the Ser/Thr-rich Repeat
Sequences of L. mexicana sAP--
We have previously shown that the
major secretory proteins of L. mexicana promastigotes are
modified with phosphoglycan chains that are probably linked to the
polypeptide backbone via a Man1-PO4-Ser linkage (10). To
confirm this linkage by mass spectrometry and to develop an assay for
the initiating enzyme, a series of peptides containing the Ser/Thr-rich
peptide repeats from L. mexicana sAP-1 and sAP-2 (7) were
synthesized (sapp-1, -2, -3, and -4 in Table I) and incubated with L. mexicana promastigote membranes and GDP-[3H]Man as a
sugar donor. [3H]Man-labeled peptides were purified on a
Sep-Pak C18 cartridge and further analyzed by RP-HPLC. As
shown in Fig. 2, incubation of the
membranes with sapp-1 (containing two Ser-rich motifs) generated at
least five 3H-labeled peaks that eluted earlier than the
unmodified peptide on RP-HPLC. These peaks were not detected when
membranes were incubated in the absence of either the exogenous peptide
or GDP-Man (data not shown). Treatment of the total peptide mixture or
the individual HPLC-purified peaks with either jack bean
-mannosidase (data not shown) or mild trifluoroacetic acid (to
selectively cleave hexose 1-phosphate linkages) generated a single
labeled component in each case that comigrated with mannose on HPTLC
(Fig. 2C). These data suggested that sapp-1 was being
variably modified with one or more Man
1-PO4 residues.
This was confirmed by positive ion ESI-MS of the HPLC-purified
phosphomannosylated peaks 1 and 2 in Fig. 2. Multiply charged ions were
obtained that were in agreement with the calculated mass for sapp-1
modified with one or two hexose phosphate residues, respectively (Table
II). As the ionization of these
phosphomannosylated peptides was relatively poor, a smaller peptide
(sapp-2) containing a single Ser/Thr-rich repeat unit and a C-terminal
lysine residue was used as substrate. As with sapp-1, several
[3H]Man-labeled peaks were generated when this peptide
was incubated with sonicated membranes (Fig.
3A). Positive ion ESI-MS of
the two major peaks contained both [M + H]+ and [M + 2H]2+ ions corresponding to sapp-2 with either one (Fig.
3B) or two (Fig. 3C) hexose-PO4
residues (Table II). Fragment ions corresponding to the loss of one or
two hexose residues were also evident in these mass spectra, indicating
that the Man-PO4 linkage is relatively labile under the
ionization conditions employed. Taken together, these data indicate
that peptides containing the same Ser/Thr-rich domains as endogenous
polypeptides are modified with one or more single
Man
1-PO4 residues, providing the first evidence for the presence of a pep-MPT activity.
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Collision-induced dissociation of the monophosphomannosylated sapp-1
and sapp-2 peptides provided negligible or incomplete sequence
information on the site(s) of Man-PO4 substitution,
reflecting the relatively poor ionization of these peptides. However,
complete sequence information was obtained for monophosphomannosylated sapp-3, a peptide with a single serine-rich motif and two C-terminal lysine residues (Tables I and II). Tandem mass spectrometry of the
monosubstituted sapp-3 [M + 2H]2+ ion (m/z
995.4) resulted in the loss of mannose and fragmentation along the
peptide backbone to produce a series of N-terminal (b-type) and
C-terminal (y-type) ions (Fig. 4, A and
B). The y series of fragment
ions were particularly informative. As shown in Fig. 4, the
y1-8 fragment ions observed in the mass spectrum corresponded to the unmodified peptide, indicating that none of the
C-terminal residues in the peptide, including Ser15 and
Ser16, were phosphorylated. In contrast, two series of
y9-11 ions, differing in mass by 80 Da (corresponding to
both unmodified and phosphorylated forms of Ser8-10), and
one series of y12-16 ions corresponding to the
phosphorylated peptide were observed in the mass spectrum (Fig. 4,
A and B). Additional fragment ions corresponding
to loss of H3PO4 from the phosphorylated
peptides, diagnostic ions for serine- and threonine-phosphorylated peptides (37), were also present (yn' in Fig. 4, A and B). The progressive decrease in ion intensity of the
y9-12 ions for the unmodified peptide and the
corresponding increase in ion intensity of the y9-12 ions
for the phosphorylated peptide (Fig. 4C) suggest that all
four Ser residues in the central domain of this peptide were partially
phosphorylated. Whereas the b series of fragment ions was less
complete, the identification of the expected b2-5 series
for the unmodified peptide indicates that neither Ser1 nor
Ser2 is phosphorylated. These data indicate that sapp-3 is
heterogeneously modified with Man1-PO4 on
Ser7-10 and also suggest that none of the Thr residues are
modified.
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Properties of the pep-MPT Activity-- pep-MPT activity in detergent-solubilized microsomes was not affected by inclusion of amphomycin (0.6 mg/ml), a potent inhibitor of dolichol-phosphate mannose synthesis,2 indicating that the mannose phosphate residue was being directly transferred from GDP-Man (data not shown). Although pep-MPT activity was solubilized from microsomal membranes (>80%) with a range of non-ionic detergents (see "Experimental Procedures"), Triton X-100 was the most effective at retaining enzyme activity (Fig. 5A) (data not shown). However, enzyme activity was considerably more labile after detergent solubilization, as maximal activity occurred at 16 °C, rather than at 32 °C, when non-solubilized membranes were assayed (data not shown). The detergent-solubilized pep-MPT activity displayed a pH optimum at 7.4 (Fig. 5B) and had an apparent Km for GDP-Man of 180 µM (Fig. 5C). All activity was abolished when 5 mM EGTA was included in the reaction mixture in the absence of added divalent cations, but was restored to maximal levels with the addition of 10 mM MnCl2 (i.e. 5 mM free Mn2+) (Fig. 5D). MgCl2 also stimulated activity, but was not as effective as MnCl2 and did not significantly further stimulate the effect of adding MnCl2 alone (Fig. 5D). Addition of 5 mM free Ca2+ had no effect on the pep-MPT activity in the absence (Fig. 5D) or presence (data not shown) of MnCl2 or MgCl2. Interestingly, the pep-MPT activity varied markedly in a growth- and stage-dependent manner (Fig. 5A). Rapidly growing (procyclic) promastigotes contained 5- or 10-fold higher activity than either late stationary (metacyclic) promastigotes or freshly isolated lesion amastigotes, respectively.
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The pep-MPT Activity Is Distinct from the LPG
Phosphoglycan-initiating and -elongating MPTs--
Although pep-MPT
utilizes exogenously added peptides, it was possible that this activity
was due to two previously characterized leishmanial MPT activities that
are thought to be required for initiation and elongation of the LPG
chain biosynthesis (20, 22). These oligosaccharide MPT activities add
Man-PO4 to terminal galactose residues on either the LPG
anchor precursor or phosphodisaccharide repeat units on the growing LPG
phosphoglycan chain, respectively (20, 22). A second assay was
developed to assess whether synthetic oligosaccharide acceptors for
these MPTs competed with the peptide substrates for pep-MPT activity.
In this assay, N-terminally 3H-labeled sapp-1 peptide (at a
concentration of 2 × Km) and unlabeled GDP-Man were
added to microsomal membranes in the absence or presence of unlabeled
oligosaccharide or peptide substrates. In the absence of competitive
substrate, ~30% of the 3H-labeled sapp-1 peptide was
modified with Man-PO4 residues over a 30-min incubation
period, as shown by the appearance of additional labeled bands on
thin-layer electrophoresis that migrated more rapidly toward the
cathode (Fig. 6A). The
additional bands corresponded to the sapp-1 peptide with one, two, or
three Man1-PO4 residues, as sequential treatment of the
peptide mixture with jack bean
-mannosidase and alkaline phosphatase
collapsed these bands back to a single labeled species that comigrated
with unmodified sapp-1 (Fig. 6B). As expected, the addition
of 1 mM unlabeled sapp-1 resulted in a 55% decrease in the
extent to which the reductively methylated 3H-labeled
sapp-1 peptide was modified (Fig. 6C). In contrast, the
oligosaccharide acceptors for the LPG-specific MPTs, L2
(Gal
1-4Man
1-PO4-(CH2)8CH=CH2) and stachyose (Gal
1-6Gal
1-6Glc
1-2Fru), did not inhibit the pep-MPT activity when present at a 6-30-fold excess over the
concentration of the sapp-1 peptide (Fig. 6C). The
concentration of L2 used in these experiments was higher than that used
previously (~600 µM) to achieve close to maximal
saturation of the putative elongating MPT (20). Moreover, in separate
experiments, unlabeled sapp-4 was found not to inhibit the
phosphomannosylation of L2. As shown in Fig.
7, incubation of detergent-solubilized
microsomal membranes with L2 (1 mM) and
GDP-[3H]Man generated a labeled product with a slower
HPTLC mobility compared with unmodified L2 (compare lanes 1 and 3). This product has previously been shown to correspond
to L2 modified with a single terminal Man-1-PO4 residue
(20). Significantly, the modification of L2 by the putative elongating
MPT was not affected by the inclusion of sapp-4 (up to 0.5 mM) in the assay (Fig. 7, compare lanes 1 and
2). As the apparent Km of pep-MPT
for sapp-4 is 0.05 mM (Table
III), these data support the notion that
the elongating MPT and pep-MPT are separate enzyme activities. Similar
experiments were not performed with substrates of the initiating MPT
(stachyose or LPG anchor) because high concentrations of GDP-Man (1 mM) (22) are needed to detect this activity,
precluding the use of GDP-[3H]Man.
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Substrate Specificity of pep-MPT-- The minimal requirements for pep-MPT activity were investigated using a number of peptide substrates. As shown in Table III, peptides with two Ser/Thr-rich repeats (i.e. sapp-1) were slightly better substrates than peptides with one Ser/Thr-rich repeat (i.e. sapp-4) (Km = 20 and 50 µM, respectively). Replacement of the four Ser residues in sapp-4 with Thr (sapp-5) virtually abrogated all substrate potential, confirming the specificity of MPT for Ser residues (Table III). Interestingly, replacement of the two acidic amino acids in the sequences flanking the serines (sapp-6) resulted in a 17-fold increase in the Km of the pep-MPT activity, suggesting that these residues may contribute to MPT substrate recognition (Table III). In the competition assay, unlabeled sapp-4 (1 mM) was slightly less effective than unlabeled sapp-1 (50% versus 55%) at reducing the rate of phosphoglycosylation of 3H-labeled sapp-1 (Fig. 6C). However, both sapp-5 and sapp-6 and a peptide containing 11 serine residues (Ser11 peptide) inhibited this reaction by <10%. These data suggest that replacement of Ser residues with Thr or removal of the acidic flanking residues in the repeat sequences abrogates recognition of the sAP-based peptides by pep-MPT.
Subcellular Distribution of pep-MPT-- Initial experiments showed that pep-MPT activity was sedimented in a 100,000 × g microsomal pellet and that activity was detected only when intact microsomal membranes were disrupted by sonication or detergent lysis, indicating that pep-MPT is a membrane-associated enzyme and that the catalytic domain has a luminal orientation. To investigate the subcellular location of this enzyme and thus the site at which protein phosphoglycosylation is initiated, L. mexicana microsomal membranes were prepared by nitrogen cavitation and fractionated by equilibrium velocity sucrose gradient centrifugation. Previously characterized markers for the endoplasmic reticulum (BiP) (29) and cis-Golgi apparatus (Rab1) (28) were separated on this sucrose gradient (Fig. 8), whereas analysis for other organelle-specific markers (i.e. hexose kinase (glycosomes), acid phosphatase (plasma membrane), and HSP60 (mitochondria)) showed that organelle separation had been achieved (data not shown). The pep-MPT activity displayed an overlapping (but not coincident) sedimentation pattern with Rab1 and was reproducibly separated by two fractions from the endoplasmic reticulum marker BiP. These data suggest that the phosphomannosylation of polypeptide acceptors is probably initiated in a post-endoplasmic reticulum compartment.
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Differential Phosphoglycosylation of Exogenous Peptides and
Endogenous Acceptors in Cell-free Systems--
Although the exogenous
peptides are utilized by pep-MPT in in vitro assays, the
addition of either unlabeled GDP-Man (data not shown) or UDP-Gal (Fig.
9, lane 1) to these assays did
not result in further extension of the peptide-linked
Man-PO4 residues with either 1-2-linked mannose
oligosaccharides or the phosphorylated Gal
1-4Man repeat units as
occurs on endogenous sAP polypeptide acceptors (10). This was not due
to disruption of the enzymatic machinery for phosphoglycan
biosynthesis, as endogenous membrane-bound LPG acceptors were
elaborated with at least one phosphodiester-linked disaccharide unit
(Fig. 9, lane 3). This repeat unit probably corresponds to
the backbone repeat unit PO4-6Gal
1-4Man, as the dephosphorylated species comigrated with authentic Gal
1-4Man on the
HPTLC (Fig. 9, lane 3), and the synthesis of this
oligosaccharide was dependent on the inclusion of UDP-Gal in the assay
mixture (data not shown). Interestingly, the Triton X-100-insoluble
pellet retained a significant amount of [3H]Man label
(~30% of the non-peptide-associated activity), which could be
released by mild acid treatment (Fig. 9, lane 2). As Triton
X-100 quantitatively extracts both LPG and hydrophilic (non-lipid-linked)
phosphoglycans,3 it is likely
that most of the labeled material in the Triton X-100 pellet
corresponds to tightly bound polypeptide acceptors. Unlike the
endogenous LPG acceptors, the Triton X-100-insoluble acceptors were
modified primarily with Man-PO4 and only to a small extent
with the Gal
1-4Man disaccharide (Fig. 9, lane 2). Thus, this second pool of acceptors may have limited access to the enzymes involved in the synthesis of the disaccharide repeat units.
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DISCUSSION |
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We have characterized a novel pep-MPT activity from L. mexicana promastigotes that is likely to be responsible for
initiating the phosphoglycosylation of a major class of secreted
glycoproteins and mucin-like molecules. The phosphoglycans may account
for 20-90% of these secreted molecules and are structurally variable,
ranging from single Man-PO4 residues to exceedingly complex
and highly branched phosphoglycans, depending on the nature of the
polypeptide backbone and parasite developmental stage (10, 14, 15). However, all these glycans are thought to be linked to the polypeptide backbone by a common Man1-PO4-Ser linker sequence (10,
14, 15). This linkage was indicated by the detection of
O-phosphoserine in sAPs and PPGs and the resistance of these
residues to alkaline phosphatase unless the masking glycans were
removed with
-mannosidase or mild acid hydrolysis (10, 14, 15).
However, it has been difficult to confirm the nature of this linkage by
mass spectrometry or NMR, as the heavily glycosylated Ser/Thr-rich
domains are essentially resistant to proteolysis, preventing the
generation of small well defined glycopeptides that are amenable to
such analyses. In this context, the synthesis of
Man
1-PO4-modified peptides by L. mexicana membranes and the characterization of these peptides by electrospray collision-induced dissociation mass spectrometry and chemical and
enzymatic treatments have provided unequivocal confirmation of this
novel linkage. Although the Man
1-PO4-Ser linkage is
apparently unique to leishmanial glycoconjugates, it is related to
phosphodiester-linked glycans from other lower eukaryotic
glycoconjugates. For example, the cysteine proteases of
Dictyostelium discoideum are modified with
GlcNAc
1-PO4 residues linked to Ser, whereas secreted
glycoproteins from Trypanosoma cruzi may be modified with
structurally complex oligosaccharides containing a putative
Xyl-1-PO4-Thr (Ser) linker sequence (11, 12). The abundance
of this type of modification in several lower eukaryotes suggests that
the glycan-PO4-Ser (Thr) motif may be more common than
previously suspected.
We have provided evidence that L. mexicana pep-MPT transfers
Man1-PO4 from GDP-Man to Ser-rich peptide sequences that
are found in L. mexicana sAP. The transfer of
Man
1-PO4 from GDP-Man was supported by the finding that
product formation was not inhibited by amphomycin, suggesting that
dolichol-phosphate mannose is not utilized as a sugar donor by this
enzyme. In addition, there was no evidence for the modification of the
peptide in the absence of GDP-Man3 indicating that this
linkage is not being assembled by the sequential action of a serine
kinase followed by a novel mannosyltransferase. Based on ESI-MS product
analysis and the use of various peptide acceptors, the following
conclusions were made concerning the substrate specificity of pep-MPT.
First, this enzyme only adds Man
1-PO4 to Ser residues; a
peptide containing Thr instead of Ser was neither a substrate nor an
inhibitor of the enzyme. Second, the ESI-MS sequence analysis indicated
that the Man-PO4 residues are added to several Ser residues
within the Ser-rich repeats. Moreover, the partial characterization of
peptides with up to five Man-PO4 residues shows that at
least three of the four contiguous Ser residues in a single Ser-rich
repeat sequence can be modified, consistent with the very high degree
of substitution (~80%) observed on endogenous sAPs and PPGs (10,
14). Third, amino acids flanking the contiguous Ser sequences may be
important for pep-MPT recognition and influence the pattern of
substitution. For example, sAP peptides lacking the flanking Glu or Asp
residues (i.e. sapp-6) were poorly utilized by pep-MPT in
direct assays and were poor inhibitors in competition assays. It is
possible that these residues may be required to maintain a suitable
conformation or the solubility of the peptide, as polyserine peptides
become increasingly insoluble above six residues in length.
The pep-MPT characterized here appears to be distinct from the two
oligosaccharide-specific MPTs that are involved in adding Man-PO4 to the LPG anchor precursor (initiating MPT) or
preformed phosphoglycan chains on lipid or peptide acceptors
(elongating MPT) (20, 22). The initiating MPT can be assayed using
either dephosphorylated LPG core or the structurally related
oligosaccharide, stachyose, as acceptor (22). In contrast, the
elongating MPT adds Man1-PO4 to the minimal disaccharide
unit Gal
1-4Man
1-PO4 that is present in the synthetic
hydrophobic oligosaccharide acceptor L2 (20). Neither stachyose nor L2
inhibited the pep-MPT activity when present at 6-30-fold excess over
the peptide acceptors (Fig. 6C). Moreover, unlabeled sapp-4
peptide did not inhibit the phosphomannosylation of L2 when present at
concentrations that inhibit the phosphomannosylation of the
3H-labeled sapp-1 peptide (Fig. 7). pep-MPT could also be
distinguished from the other MPTs by its solubility and stability in
various detergents (20, 22). On the other hand, all three MPTs appear to utilize GDP-Man as the sugar donor and have a similar pH optimum and
requirement for divalent cations. Although the substrate specificities of these MPTs appear to be distinct, it is notable that both pep-MPT and elongating MPTs are maximally active against substrates that contain negatively charged groups near the aglycon acceptor
(i.e. acidic amino acids or phosphate, respectively) (this
study and Ref. 20), suggesting that these activities may have arisen
from a common progenitor enzyme.
Marked differences were observed in the levels of pep-MPT activity during promastigote growth, with 5- or 10-fold higher levels of activity being observed in rapidly dividing procyclic promastigotes compared with late stationary phase promastigotes and lesion-derived amastigotes, respectively. There is no evidence that the low pep-MPT activity of the metacyclic promastigotes or the intracellular amastigotes is associated with a reduced level of phosphoglycosylation of secreted polypeptides. Indeed, aPPGs are much more extensively modified with phosphoglycan chains than promastigote sAPs or promastigote PPGs (15). It is thus possible that the low pep-MPT activity in non-dividing promastigotes and the intracellular amastigotes may reflect a low level of secretory activity in these developmental stages or the presence of other pep-MPT activities that preferentially recognize distinct peptide sequences in aPPGs (14, 15).
Subcellular fractionation studies suggested that pep-MPT was localized in a post-endoplasmic reticulum compartment and that it had an overlapping (but not coincident) distribution with the Golgi marker Rab1. Rab1 has been localized primarily to cis-Golgi cisternae in L. major (28), but may also be localized to endoplasmic reticulum-Golgi transport vesicles, where it is thought to be involved in regulating the organization of vesicle proteins (38). The broader distribution of the Rab1 marker may reflect the partial distribution of this protein in lighter transport vesicles from which pep-MPT is excluded. Localization of pep-MPT to the Golgi apparatus is consistent with the recent demonstration that the leishmanial Golgi apparatus contains a GDP-Man transporter that is required for phosphoglycan biosynthesis (24). Similarly, the initiating and elongating MPTs involved in LPG biosynthesis are also thought to be localized to the Golgi apparatus based on the finding that one of the enzymes involved in LPG anchor biosynthesis has been localized to the Golgi apparatus by electron microscopy (23). In this respect, it is of interest that Man-PO4 residues on either the peptide or a pool of endogenous polypeptide acceptors were negligibly or poorly elaborated with phosphoglycan repeat units. In contrast, Man-PO4 residues on the endogenous LPG acceptors were quantitatively modified with at least one repeat unit. Although it is possible that the Man-PO4-modified peptide is a poor substrate for these elongating enzymes, the low rate of elongation of Man-PO4 on the endogenous polypeptide pool suggests that pep-MPT and elongating enzymes (including the elongating MPT) are localized to functionally distinct subcompartments of the Golgi apparatus. Compartmentalization of these enzymes may provide a mechanism for generating phosphoglycan structures with markedly different chain lengths on different classes of glycosylphosphatidylinositol or polypeptide acceptors (see Fig. 1).
In summary, we have confirmed the nature of the peptide linkage of the
unusual phosphoglycans that are added to the major secretory proteins
of L. mexicana and identified a novel pep-MPT activity.
Similar types of phosphoglycosylation occur in all developmental stages
of the parasite, including the amastigotes that perpetuate disease in
the mammalian host and that have been shown to be functionally important. Consequently, it is likely that pep-MPT and possibly other
MPTs are potential targets for new anti-leishmanial drugs.
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ACKNOWLEDGEMENTS |
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We thank Professor Michael Ferguson for providing the synthetic L2 acceptor, Drs. Emanuela Handman and James Bangs for antibodies, and Professor Tony Bacic for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by the Australian National Health and Medical Research Council and the Wellcome Trust.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Wellcome Trust Senior Research Fellow and to whom correspondence should be addressed. Tel.: 61-3-9344-5681; Fax: 61-3-9347-7730; E-mail: m.mcconville{at}biochemistry.unimelb.edu.au.
2 J. E. Ralton and M. J. McConville, unpublished data.
3 M. J. McConville, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: LPG, lipophosphoglycan; PPG, proteophosphoglycan; aPPG, amastigote proteophosphoglycan; sAP, secreted acid phosphatase; MPT, mannose-1-phosphotransferase; pep-MPT, peptide-specific mannose-1-phosphotransferase; CHAPS, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonic acid; RP-HPLC, reversed-phase high performance liquid chromatography; HPTLC, high performance thin-layer chromatography; ESI-MS, electrospray ionization mass spectrometry.
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REFERENCES |
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