(Received for publication, November 7, 1995; and in revised form, January 3, 1996)
From the
The biosynthesis of glycosylphosphatidylinositol (GPI)
precursors in Trypanosoma brucei involves the D-mannosylation of D-GlcN1-6-D-myo-inositol-1-PO
-sn-1,2-diacylglycerol
(GlcN-PI). An assay for the first mannosyltransferase of the pathway,
Dol-P-Man:GlcN-PI
1-4-mannosyltransferase, is described.
Analysis of the acceptor specificity revealed (a) that the
enzyme requires the myo-inositol residue of the GlcN-PI
substrate have the D configuration; (b) that the
enzyme requires the presence of the NH
group of the D-GlcN residue; (c) that GlcNAc-PI is more
efficiently presented to the enzyme than GlcN-PI, suggesting a degree
of substrate channelling via the preceding GlcNAc-PI
de-N-acetylase enzyme; (d) that the fatty acid and
phosphoglycerol components of the phosphatidyl moiety are important for
enhancing substrate presentation and substrate recognition,
respectively; and (e) that D-GlcN
1-6-D-myo-inositol is the
minimum structure that can support detectable acceptor activity.
Analysis of the donor specificity revealed that short chain (C
and C
) analogues of dolichol phosphate can act as
substrates for the trypanosomal dolichol-phosphomannose synthetase,
whereas the corresponding mannopyranosides cannot act as donors for the
Dol-P-Man:GlcN-PI
1-4-mannosyltransferase.
Glycosylphosphatidylinositol (GPI) ()membrane anchors
are widely distributed among the eukaryotes. They anchor proteins to
the outer leaflet of the plasma membrane and may be associated with
other functions, such as signal transduction and protein targeting. The
structure, biosynthesis, and function of GPI anchors have been
reviewed, most recently by Englund(1993), McConville and
Ferguson(1993), Stevens(1995), Udenfriend and Kodukula(1995), and
Takeda and Kinoshita(1995).
The tsetse fly-transmitted African
trypanosomes, which cause human sleeping sickness and a variety of
livestock diseases, are able to survive in the mammalian bloodstream by
virtue of their dense cell-surface coat. This coat consists of 10
million copies of a 55-kDa GPI-anchored glycoprotein called the variant
surface glycoprotein (VSG) (Cross, 1990). The relative abundance of the
VSG protein in Trypanosoma brucei renders this organism
extremely useful for the study of GPI anchor biosynthesis. The
structure of the VSG GPI anchor is known (Ferguson et al.,
1988), and the principal features of the GPI biosynthetic pathway in
trypanosomes were elucidated using a cell-free system based on washed
trypanosome membranes (Masterson et al., 1989, 1990; Menon et al., 1990a). The first step in the pathway involves the
transfer of GlcNAc from UDP-GlcNAc to endogenous phosphatidylinositol
(PI), via a sulfydryl-dependent GlcNAc transferase (Milne et
al., 1992) to form GlcNAc-PI, which is rapidly
de-N-acetylated (Doering et al., 1989) to give
glucosaminyl PI (GlcN-PI). Three -Man residues are sequentially
transferred onto GlcN-PI from dolichol phosphate mannose (Dol-P-Man)
(Menon et al., 1990b) to produce the intermediate
Man
1-2Man
1-6Man
1-4GlcN-PI
(Man
GlcN-PI). All of the mannosylated intermediates can be
found in both acylated and nonacylated inositol forms, and a
Man
GlcN-(acyl)PI species appears to be the preferred
substrate for ethanolamine phosphate (EtNP) addition
(Güther and Ferguson, 1995), the donor for which is
phosphatidylethanolamine (Menon and Stevens, 1992; Menon et
al., 1993). The EtNP-Man
GlcN-(acyl)PI (glycolipid C`)
species is then deacylated to form glycolipid A`, which undergoes a
series of fatty acid remodeling reactions (Masterson et al.,
1990) in which the fatty acids of the PI moiety are removed and
replaced with myristate to yield the mature GPI precursor glycolipid A.
This mature precursor is transferred to the VSG polypeptide in the
endoplasmic reticulum in exchange for a carboxyl-terminal GPI signal
peptide, reviewed by Udenfriend and Kodukula(1995).
The GPI biosynthetic pathways in mammalian cells (Sugiyama et al., 1991; Lemansky et al., 1991, Hirose et al., 1991, 1992a, 1992b; Kamitani et al., 1992; Puoti et al., 1991; Puoti and Conzelmann, 1992, 1993; Mohney et al., 1994) and yeast (Costello and Orlean, 1992; Sipos et al., 1994), as well as in other protozoa, such as Toxoplasma (Tomavo et al., 1992a, 1992b) and Plasmodium falciparum (Gerold et al., 1994), appear to be broadly similar to that described above for the bloodstream form of T. brucei. Some notable differences among the trypanosomal, mammalian, and yeast GPI pathways include the almost quantitative acylation of all mammalian and yeast GPI intermediates from GlcN-PI onwards and the addition of extra ethanolamine phosphate groups to the mammalian intermediates. The fatty acid remodeling reactions, as described above, appear to be unique to the bloodstream form of African trypanosomes.
In this paper, we show
that exogenously added GlcN-PI and analogues thereof can prime the GPI
pathway in a trypanosome cell-free system, thereby providing a
convenient means for probing the substrate specificity of
Dol-P-Man:GlcN-PI 1-4-mannosyltransferase, the first
mannosyltransferase of the GPI biosynthetic pathway.
D-Glc1-6-D-myo-inositol-1-PO
-sn-1,2-dipalmitoylglycerol
(Glc-PI) and D-2-deoxy-Glc
1-6-D-myo-inositol-1-PO
-sn-1,2-dipalmitoylglycerol
(2-deoxy-Glc-PI) were synthesized according to Cottaz et al.
(1995b). The authenticity and purity of the synthetic compounds was
checked by electrospray mass spectrometry prior to use (see Fig. 1for the recorded pseudomolecular ions), and the
concentrations of stock solutions were measured by analyzing the myo-inositol content by gas chromatography/mass spectroscopy,
as described below. Isoamyl phosphate
(CH
-CH(CH
)-CH
-CH
-PO
H)
and didehydrofarnesol phosphate
(CH
-C(CH
)=CH-CH
-CH
-C(CH
)=CH-CH
-CH
-CH(CH
)-CH
-CH
-PO
H)
were synthesized. (
)Briefly, isoamyl alcohol was reacted
with 1-naphthalenemethyl phosphonic acid (Katritzky et al.,
1990) followed by oxidation with iodine and catalytic hydrogenation.
Farnesol was selectively hydrogenated in the
-isoprene unit using
NaBH
and a PtO
catalyst (Mankowski et
al., 1976), and the product was subsequently phosphorylated with
POCl
(Danilov and Chojnacki, 1981).
Figure 1:
Substrate
analogues of D-GlcN1-6-D-myo-inositol-1-PO
-sn-1,2-dipalmitoylglycerol
(GlcN-PI). The synthetic compounds are shown together with their
abbreviations and mass spectrometric data.
Water-soluble substrates were used at a final concentration of 0.5 mM, and the assays were terminated as described above. The products were recovered in the aqueous phase of a butan-1-ol/water partition and analyzed for glycan as described below.
Competition assays were carried out in the presence of either 35
µM GlcN-PI or 35 µM GlcNAc-PI as substrate
and with the substrate analogues at 0.5 mM. The lysate was
preincubated with GDP-[H]Man, and 20-µl
aliquots (1
10
cell equivalents) were added to the
reaction tubes containing a mixture of substrate and substrate analogue
and incubated at 30 °C for 1 h. Glycolipids were extracted and
analyzed by HPTLC. Mannosylation of the competing water-soluble glycans
was investigated by glycan analysis of the aqueous phase, as described
below.
Assays for donor specificity were performed as described
above, except that exogenous pig liver dolichol phosphate (25
µM) and didehyrofarnesol phosphate (50 µM) or
isoamyl phosphate (50 µM) were added with the
GDP-[H]Man.
N-Acetylation of glycans
was performed at 0 °C in 100 µl of saturated NaHCO by the addition of three aliquots (2.5 µl) of acetic
anhydride at 10-min intervals. The reaction mixture was allowed to come
to room temperature, and the solution of N-acetylated glycan
was desalted by passage through AG50X12(H
)
ion-exchange resin and evaporated, and residual acetic acid was removed
by coevaporation with toluene (2
50 µl).
Dephosphorylation was performed with 50 µl of 48% aqueous HF (0 °C for 48-60 h). Saturated LiOH was used to neutralize HF, which precipitated as LiF (Ferguson, 1992).
(a) Dephosphorylated/N-acetylated glycans were
generated by N-acetylation, passage through
AG50X12(H) ion-exchange resin, aqueous HF
dephosphorylation, and re-N-acetylation (Ferguson, 1992).
(b) Dephosphorylated/deaminated glycans were generated by
deamination and subsequent NaBH reduction followed by
aqueous HF dephosphorylation and re-N-acetylation (Ferguson,
1992).
The neutral glycans from these procedures were dissolved in
water and mixed with oligomeric glucose internal standards, filtered
through a 0.2-µm membrane, and analyzed by Bio-Gel P4 gel
filtration using an Oxford Glycosystems GlycoMap. Fractions (250
µl) were collected and counted for radioactivity. The yields of
[H]Man-containing glycan products for each
substrate were normalized using the internal standard
Gal
Man
[1-
H]AHM.
Figure 2:
The effects of n-octyl
-D-glucopyranoside analogues on the GPI pathway. The
trypanosome cell-free system (1
10
cell
equivalents/sample) was incubated at 30 °C for 1 h with GlcN-PI and
GDP-[
H]Man in the presence of sub-CMC
concentrations of the following detergents: 10 mMn-octyl
-D-glucopyranoside (lane
1), 5 mMn-octyl-1-thio-
-D-glucopyranoside (lane
2), and 3 mM
-D-glucopyranosyl octyl
sulfone (lane 3). Radiolabeled glycolipids were extracted and
then separated by HPTLC with fluorographic detection. The identities of
the radiolabeled glycolipids were based on R
values and sensitivities to jack bean
-mannosidase,
PI-specific phospholipase C, and PI-specific phospholipase D (see Table 2).
In the system without added detergent, enzymatic activity was observed between pH 6.0 and 9.0, with a maximum at about pH 7.4 (data not shown). All subsequent assays were performed at pH 7.4.
The assay is dependent on endogenous Dol-P-Man synthetase, which
converts GDP-[H]Man to
Dol-P-[
H]Man in situ. The enzyme is
thermally unstable (Prado-Figueroa et al., 1994) so that the
temperature of the assay was limited to 30 °C. The loss in GPI
pathway activity using detergents above their CMCs was not associated
with the loss of Dol-P-Man sythetase activity, since the formation of
Dol-P-[
H]Man is usually stimulated under these
conditions (data not shown).
The data in Table 1show that
several detergents can support GPI biosynthesis below their CMCs, but
only CHAPS and n-octyl -D-glucopyranoside (and
certain derivatives thereof) were able to stimulate the pathway. The
significant (3.5-fold) stimulation of the pathway with 10 mM (0.3% (w/v)) n-octyl
-D-glucopyranoside was
exploited in all subsequent assays. Stimulation of the GPI pathway was
not due to stimulation of Dol-P-Man synthetase, since Triton X-100 was
at least as effective as n-octyl
-D-glucopyranoside in stimulating Dol-P-Man synthesis,
whereas it did not stimulate GPI biosynthesis (data not shown). The
sensitivity of the pathway to the detergent environment is illustrated
by the inhibitory effect of n-octyl
1-thio-
-D-glucopyranoside (Fig. 2, lane
2), which can be reversed by oxidation of the sulfur atom to the
more polar sulfone (Fig. 2, lane 3, and Table 1).
Figure 3:
Analysis of the products of various
lipid-containing GlcN-PI acceptor analogues. The trypanosome cell-free
system (1 10
cell equivalents/sample) was incubated
at 30 °C for 1 h with GDP-[
H]Man and 10
mMn-octyl
-D-glucopyranoside in the
presence of 35 µM GlcN-PI (lane 1), 35 µM GlcN-P[L]I (lane 2), no exogenous
acceptor (lane 3), 35 µM GlcNAc-PI (lane
4), 50 µM Glc-PI (lane 5), and 50 µM 2-deoxy-Glc-PI (lane 6). Glycolipids were extracted and
analyzed by HPTLC with fluorographic detection. The identities of the
radiolabeled glycolipids were based upon R
values and sensitivities to jack bean
-mannosidase,
PI-specific phospholipase C, and PI-specific phospholipase D (see Table 2).
In order to compare
the abilities of glycolipid and water-soluble substrates to prime the
GPI pathway (see Fig. 4), they were incubated with the cell-free
system in the presence of n-octyl
-D-glucopyranoside (10 mM) and
GDP-[
H]Man, as described above. The glycan
components of the products were recovered following (a) N-acetylation, aqueous HF dephosphorylation and
re-N-acetylation or (b) deamination/reduction and
aqueous HF dephosphorylation. The radiolabeled neutral glycans so
produced were fractionated on a Bio-Gel P4 column. In all cases, a
substantial peak of [
H]Man was seen at 1.0
glucose units, which corresponds to the label carried over from
GDP-[
H]Man. All of the acceptors were shown by
method (a) to produce a labeled glycan at 5.7 glucose units,
which corresponds to
Man
1-2Man
1-6Man
1-4GlcNAc
1-6-myo-inositol
(Ralton et al., 1993). Minor products were detected at 4.7 and
3.8 glucose units, which correspond to
Man
1-6Man
1-4GlcNAc
1-6-myo-inositol and
Man
1-4GlcNAc
1-6-myoinositol,
respectively. The yields of these products were normalized by means of
the internal standard
Gal
Man
[1-
H]AHM (at 6.8
glucose units). Similar results were obtained using method (b)(data not shown). Since method (b) depends on
nitrous acid deamination of a GlcN residue, it showed that all of the N-acetylated substrates had been de-N-acetylated
prior to mannosylation.
Figure 4:
Comparison of the acceptor activities of
lipid-containing and water-soluble substrate analogues. The trypanosome
cell-free system (1 10
cell equivalents/sample) was
incubated at 30 °C for 1 h with GDP-[
H]Man
and 10 mMn-octyl
-D-glucopyranoside in
the presence of various acceptor substrate analogues (50
µM). Analysis of the radiolabeled GPI glycan products was
achieved by delipidation (if required), dephosphorylation, N-acetylation and desalting, followed by fractionation on a
Bio-Gel P4 column. After normalization, using the internal standard
Gal
Man
[1-
H]AHM, the total
radioactivity associated with the GPI glycan products was expressed as
a percentage relative to that obtained with GlcN-PI (100%). The values
shown are mean values of at least three determinations. The control
figure of 4% represents the low level of
[
H]mannosylation of endogenous
acceptors.
Figure 5:
Analysis of donor substrate specificity of
dolichol-P-Man:GlcN 1-4-mannosyltransferase. The trypanosome
cell-free system (1
10
cell equivalents/sample) was
incubated at 30 °C for 1 h with GDP-[
H]Man
and 10 mMn-octyl
-D-glucopyranoside in
the presence of 35 µM GlcN-PI without further additions (lane 1) or with added 25 µM dolichol phosphate (lane 2), 50 µM didehydrofarnesol phosphate (lane 3), and 50 µM isoamyl phosphate (lane 4). Glycolipids were extracted and analyzed by HPTLC with
fluorographic detection.
Protein-linked GPI anchors and GPI-related glycolipids, such
as the lipophosphoglycans and glycoinositol-phospholipids of the Leishmania, are particularly abundant in protozoa (McConville
and Ferguson, 1993). All of these molecules, several of which are
essential for parasite infectivity, share the structural motif
Man1-4GlcN
1-6-myo-inositol-1-PO
-lipid.
Thus the enzymes responsible for the synthesis of this conserved unit
represent attractive targets for the development of anti-parasite
agents. In this paper we disclose the substrate specificity of one of
these enzymes, the trypanosomal Dol-P-Man:GlcN-PI
1-4-mannosyltransferase
A previous report by DeLuca et al.(1994) describes an assay for a comparable mammalian
Dol-P-Man:GlcN-(acyl)PI 1-4-mannosyltransferase, based on
the microsomes of mutant CHO-K1 cells that were unable to synthesize or
utilize endogenous Dol-P-Man and which therefore accumulated endogenous
GlcN-(acyl)PI. Using this assay, they were able to study the donor
substrate specificity of the enzyme, which exhibited strict specificity
for the
-anomer of Dol-P-Man, although the dolichol moiety could
be replaced by a similar polyisoprenyl moiety without abolishing
enzymatic activity. The assay described here for the trypanosomal
Dol-P-Man:GlcN-PI
1-4-mannosyltransferase differs from that
of DeLuca et al.(1994) in that it relies on the addition of
exogenous (synthetic) acceptors and can be used to determine both donor
and acceptor specificities.
The trypanosomal dolichols are unusually
short -unsaturated polyisoprenes (C
) (Low et al., 1991). We have investigated the possibility that the
dolichol phosphate moiety of the Dol-P-Man donor might be replaced by
even shorter analogues, namely didehydrofarnesol phosphate (an
-unsaturated C
tri-isoprene) or isoamyl phosphate (a
saturated C
isoprene). Both of these analogues were
mannosylated by the trypanosomal Dol-P-Man synthetase, but the products
(didehydrofarnesol-P-[
H]Man and
isoamyl-P-[
H]Man) were unable to act as donors
for the trypanosomal Dol-P-Man:GlcN-PI
1-4-mannosyltransferase. Although the addition of exogenous
dolichol phosphate increased the amount of
Dol-P-[
H]Man, the synthesis of
[
H]Man-labeled GPI intermediates was not
affected. Thus, under the conditions of the assay, the levels of
Dol-P-[
H]Man generated in the cell-free system
appear to exceed the requirements of the GPI biosynthetic pathway, and
the addition of exogenous dolichol phosphate is not required.
Several synthetic compounds were tested for their ability to act as
acceptors for the Dol-P-Man:GlcN-PI 1-4-mannosyltransferase.
As expected, synthetic GlcN-PI (i.e. the natural acceptor) was
a good acceptor, and the enzyme showed selectivity for the D-myo-inositol component ( Fig. 3and Fig. 4). The selectivity of the Dol-P-Man:GlcN-PI
1-4-mannosyltransferase toward the D-myo-inositol component suggests that the
orientation of one or more hydroxyl groups and/or the spatial
orientation of the phosphatidyl moiety (relative to
-D-GlcN) are crucial for acceptor substrate recognition.
Such substrate specificity suggests that the Dol-P-Man:GlcN-PI
1-4-mannosyltransferase activity is truly specific for the
GPI pathway.
The 6-fold increase in acceptor activity of GlcNAc-PI
over GlcN-PI was unexpected, since GlcNAc-PI is thought to require
prior de-N-acetylation to GlcN-PI in order to function as a
substrate for the first mannosyltransferase (Englund, 1993; Stevens,
1995; Takeda and Kinoshita, 1995). The fact that all of the GPI
products from Man-GlcN-PI were sensitive to nitrous acid deamination (a
reaction that requires a free NH group on the GlcN residue)
indicates that GlcNAc-PI was either de-N-acetylated before
mannosylation or quantitatively de-N-acetylated immediately
afterwards. The latter course seems unlikely since exogenous GlcNAc-PI
does not require prior mannosylation to be a substrate for the
de-N-acetylase (Doering et al., 1989), and the in
situ generation of Dol-P-Man does not stimulate the
de-N-acetylation of exogenous GlcNAc-PI (Sharma and Ferguson,
unpublished data). Assuming that de-N-acetylation of GlcNAc-PI
normally precedes mannosylation, the enhanced acceptor activity of
GlcNAc-PI over GlcN-PI suggests that the GlcNAc-PI
de-N-acetylase and the Dol-P-Man:GlcN-PI
1-4-mannosyltransferase are associated in a complex that
allows a degree of substrate channelling from the
de-N-acetylase to the acceptor site of the
mannosyltransferase. The fact that the product
(Man
1-4GlcN-PI) of Dol-P-Man:GlcN-PI
1-4-mannosyltransferase activity is always further processed
to later GPI intermediates (see Fig. 3and Table 2)
suggests that other enzymes of the pathway are also physically
associated in the endoplasmic reticulum membrane with these enzymes.
The existence of such a complex could explain the lability of the
pathway to detergents above their CMC values. The possibility that the
first step of the GPI pathway (i.e. the transfer of GlcNAc to
PI) in mammalian (Takeda and Kinoshita, 1995) and yeast (Leidich et
al., 1995) cells also involves a complex is supported by the fact
that at least three separate gene products are required for this step.
The inability of Glc-PI and 2-deoxy-Glc-PI to act as acceptors
suggests that the NH group of the GlcN residue is necessary
for acceptor activity. Neither of these compounds inhibited the
mannosylation of GlcN-PI, suggesting that they do not bind
significantly to the active site of Dol-P-Man:GlcN-PI
1-4-mannosyltransferase. It is possible that the NH
group of GlcN-PI is required to form either a salt bridge to a
negatively charged residue on the enzyme or a hydrogen bond to a
residue in or near the active site. Such a model would explain the
requirement for de-N-acetylation prior to mannosylation and
also suggests that de-N-acetylation of GlcNAc-PI may be an
important control point in GPI biosynthesis. Such control might be
exercised in the mammalian GPI pathway, where stimulation of a
comparable enzyme by GTP has been demonstrated (Stevens, 1993). We find
no such GTP effect for the trypanosomal de-N-acetylase (Milne et al., 1994). (
)However, these parasites
synthesize a significant excess of GPI precursors over that required
for protein anchorage (Masterson and Ferguson, 1991; Ralton et
al., 1993) and may regulate the GPI pathway via catabolism
(Güther et al., 1994).
GlcN-Ino-P-glycerol, which lacks the two fatty-acid components of
the natural substrate, was a much less efficient acceptor than GlcN-PI (Fig. 4). This suggests that the lipid moiety has a role either
in substrate recognition or, more likely, in presenting the substrate
to the membrane-bound enzyme. The removal of the glycerol component (as
in GlcN-Ino-P) or the glycerol-phosphate component (as in GlcN-Ino)
further reduced the acceptor activity, whereas GlcN-Ino-1,2-P had
hardly any detectable acceptor activity. Thus, a phospho group at C-1
of the D-myo-inositol appears to play a role in
substrate recognition, unless it involves the C-2 hydroxyl group in the
formation of a cyclic phosphate, as is the case with GlcN-Ino-1,2-P.
The notion that the C-2 hydroxyl group of myo-inositol is
involved in substrate recognition is supported by the observation that
trypanosomes do not acylate this position until after the first Man
residue has been added (Güther and Feguson, 1995).
This contrasts with observations made with mammalian and yeast cells
where acylation of the C-2 hydroxyl group of myo-inositol
appears to precede all mannosylations (Stevens, 1995). Thus, the
parasite Dol-P-Man:GlcN-PI 1-4-mannosyltransferase may have
a different, and potentially exploitable, acceptor substrate
specificity to those of the comparable mammalian and yeast enzymes.
The ability of GlcN-Ino and GlcNAc-Ino (but not GlcN or GlcNAc) to
act as weak acceptors for the Dol-P-Man:GlcN-PI
1-4-mannosyltransferase suggests that the minimum structural
features for acceptor substrate recognition are present within the
structure GlcN-Ino. Furthermore, the products generated from these
acceptors by the cell-free system included substantial quantities of
Man
-GlcN-Ino, suggesting that Man-GlcN-Ino and
Man
-GlcN-Ino contain sufficient structural information for
recognition by the second and third
-mannosyltransferases,
respectively, of the GPI pathway. The salient features recognized
within GlcN-Ino will be investigated using synthetic analogues having
hydroxyl group deletions or substitutions on the D-GlcN and D-myo-inositol residues. The 5-fold decrease in
acceptor activity of GlcN-Ino-P-glycerol, compared with GlcN-PI,
suggests that the presence of a hydrophobic group in a similar location
to the diacylglycerol group of GlcN-PI might usefully increase the
presentation of potential inhibitors to the enzyme. The inclusion of a
phosphodiester group at C-1 of the myo-inositol ring might
also be advantageous with regard to binding potential inhibitors to the
enzyme, although it might be difficult for such compounds to cross the
cell membrane.