(Received for publication, May 23, 1994; and in revised form, November 8, 1994)
From the
Glycosylphosphatidylinositol phospholipase C (GPI-PLC) from Trypanosoma brucei and phosphatidylinositol phospholipase C
(PI-PLC) from Bacillus sp. both cleave
glycosylphosphatidylinositols (GPIs). However, phosphatidylinositol,
which is efficiently cleaved by PI-PLC, is a very poor substrate for
GPI-PLC. We examined GPI-PLC substrate requirements using glycoinositol
analogs of GPI components as potential inhibitors.
Glucosaminyl(1
6)-D-myo-inositol
(GlcN(
1
6)Ins), GlcN(
1
6)Ins 1,2-cyclic phosphate,
GlcN(
1
6)-2-deoxy-Ins, and GlcN(
1
6)Ins 1-dodecyl
phosphonate inhibited GPI-PLC. GlcN(
1
6)Ins was as effective
as Man(
1
4)GlcN(
1
6)Ins; we surmise that
GlcN(
1
6)Ins is the crucial glycan motif for GPI-PLC
recognition. Inhibition by GlcN(
1
6)Ins 1,2-cyclic phosphate
suggests product inhibition since GPIs cleaved by GPI-PLC possess a
GlcN(
1
6)Ins 1,2-cyclic phosphate at the terminus of the
residual glycan. The effectiveness of GlcN(
1
6)-2-deoxy-Ins
indicates that the D-myo-inositol (Ins) 2-hydroxyl is
not required for substrate recognition, although it is probably
essential for catalysis.
GlcN(
1
6)-2-deoxy-L-myo-inositol, unlike
GlcN(
1
6)-2-deoxy-Ins, had no effect on GPI-PLC; hence,
GPI-PLC can distinguish between the two enantiomers of Ins.
Surprisingly, GlcN(
1
6)Ins 1,2-cyclic phosphate was not a
potent inhibitor of Bacillus cereus PI-PLC, and
GlcN(
1
6)Ins had no effect on the enzyme. However, both
GlcN(
1
6)Ins 1-phosphate and GlcN(
1
6)Ins 1-dodecyl
phosphonate were competitive inhibitors of PI-PLC. These observations
suggest an important role for a phosphoryl group at the Ins 1-position
in PI-PLC recognition of GPIs. Other studies indicate that abstraction
of a proton from the Ins 2-hydroxyl is not an early event in PI-PLC
cleavage of GPIs. Furthermore, both GlcN(
1
6)-2-deoxy-Ins
1-phosphate and
GlcN(
1
6)-2-deoxy-L-myo-inositol inhibited
PI-PLC without affecting GPI-PLC. Last, the aminoglycoside G418
stimulated PI-PLC, but had no effect on GPI-PLC. Thus, these enzymes
represent mechanistic subclasses of GPI phospholipases C,
distinguishable by their sensitivity to GlcN(
1
6)Ins
derivatives and aminoglycosides. Possible allosteric regulation of
PI-PLC by GlcN(
1
6)Ins analogs is discussed.
African trypanosomiasis is a human disease caused by the
protozoan parasite Trypanosoma brucei. In the mammalian host, T. brucei is protected by a surface coat composed of a variant
surface glycoprotein (VSG). ()VSG is
glycosylphosphatidylinositol (GPI)-anchored; its GPI contains
EtN-phospho-6Man(
1
2)Man(
1
6)Man(
1
4)GlcN(
1
6)-myo-Ins
1-phosphodimyristoylglycerol linked to the
-carboxyl of the
COOH-terminal residue of VSG through an amide bond with
EtN(1) . T. brucei contains a
glycosylphosphatidylinositol phospholipase C (GPI-PLC) that can cleave
dimyristoylglycerol from VSG GPI, leaving VSG containing a
GlcN(
1
6)Ins 1,2-cyclic phosphate attached to the residual
GPI glycan components(2) .
GPI-PLC is a 39-kDa integral
membrane protein(3, 4, 5, 6) . It
efficiently cleaves VSG GPI (apparent K = 370 nM; k
=
2920 min
) (5, 6) and some GPI
biosynthetic intermediates(7) . Phosphatidylinositol (PI) is a
very poor substrate for the
enzyme(3, 4, 5, 6) . Although the
biological function is unclear (reviewed in (8) and (9) ), GPI-PLC activity is detectable in bloodstream-form T. brucei, where VSG is expressed, and down-regulated
1000-fold in procyclic (insect stage) T. brucei. Thus,
the enzyme could be involved in catabolism either of the VSG GPI anchor
or of GPI biosynthetic intermediates. Nothing has been reported on the
catalytic mechanism of GPI-PLC.
Phosphatidylinositol phospholipase C (PI-PLC) from Bacillus cereus cleaves GPIs(10) ; however, unlike GPI-PLC, it cleaves PI efficiently(6, 11, 12) . PI-PLC has a region of protein sequence similarity to GPI-PLC(11) ; 80 residues beginning at positions 69 and 70 for the T. brucei and B. cereus enzymes, respectively, can be aligned with 19 matches in a region that is 27.6% identical and 51.3% similar(8, 9, 13) .
Detailed information
on GPI recognition is not available either for GPI-PLC or PI-PLC.
Interestingly, when VSG and PI are present in the same reaction mixture
at identical concentrations, GPI-PLC selectively cleaves
VSG(6) , suggesting that glycan constituents of GPIs might be
important for substrate recognition. Accordingly, we tested synthetic
glycan components of
EtN-phospho-6Man(1
2)Man(
1
6)Man(
1
4)
GlcN(
1
6)-myo-Ins, the ``conserved protein-GPI
core,'' as potential inhibitors of GPI-PLC. We report that
GlcN(
1
6)Ins is probably the major glycan determinant of
GPI-PLC specificity. Similar studies with GlcN(
1
6)Ins and
its derivatives on B. cereus PI-PLC indicate that steps toward
cleavage of the identical GPI phosphodiester are different between
GPI-PLC and PI-PLC.
PI-PLC from B. cereus (600
units/mg) was obtained from Boehringer Mannheim.
[H]Myristate-labeled VSG was isolated from T.
brucei (ILTat 1.3)(3, 14) .
GPI-PLC (19 units, 1.19 ng) was first added to 20
µl of assay buffer (1 assay buffer = 50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1% Nonidet P-40), following
which inhibitors were added to their specified final concentrations
(defined for 30 µl), and the mixture was incubated on ice for 10
min. [
H]Myristate-labeled membrane-form VSG (2
µg in 10 µl of 1
assay buffer) was added, and the tubes
were incubated at 37 °C for 15 min. The reaction was terminated by
chilling the mixture on ice and vortex mixing with 500 µl of
water-saturated 1-butanol (at room temperature). Phases were separated
by centrifugation (12,000
g, 1 min, 25 °C). Enzyme
activity was quantified by measuring the amount of
[
H]dimyristoylglycerol released into the upper
butanol phase using a Beckman LS 6000TA scintillation
counter(3, 6) . Radioactivity from a mock digest (no
enzyme addition) of [
H]myristate-labeled VSG
using 30 µl of assay buffer was subtracted as background from all
counts obtained. Activity of GPI-PLC obtained without the addition of
potential inhibitors in a parallel assay was assigned a value of 100%.
Compounds that inhibited [
H]myristate-labeled
membrane-form VSG cleavage by >70% at 5 mM were
investigated further (see ``Interfacial Kinetic Analysis'').
Average values from duplicate determinations performed in several
independent experiments showed a variation of
10%.
PI-PLC was
diluted into 50 mM Tris-HCl, pH 8.0, and added (1
10
units, 0.17 ng) to 20 µl of PI-PLC buffer (25
mM HEPES/KOH, pH 7.5, 0.1% sodium deoxycholate) (10) with or without inhibitor on ice in a 1.5-ml
microcentrifuge tube. The enzyme assay protocol was similar to that
described above for GPI-PLC.
GPI-PLC is presumed to be bound at the surface of Nonidet P-40
micelles with its active site facing bulk medium, a notion supported by
the enzyme's ability to cleave GPI biosynthetic intermediates in vivo on the cytoplasmic side of the endoplasmic
reticulum(22) . The micelle concentration of 1% Nonidet P-40 is
118.4 µM, while the concentrations of GPI-PLC and
[
H]myristate-labeled VSG are 1 nM and
1.2 µM, respectively, in the assay. Effective inhibitor
concentration at the micelle interface was expressed as a mole fraction (X
)(20, 21) . X
was determined as described above
for PI-PLC.
For both GPI-PLC and PI-PLC analysis, the concentration
of hydrophilic compounds was included in the denominator for
calculation of X for two reasons. 1) It emphasizes
the interfacial nature of the inhibition events under discussion.
Assuming that the interaction of hydrophilic compounds with detergent
micelles is transient, the compounds still have to bind
enzyme-micelle-VSG complexes to exert their inhibitory effects. 2) It
makes for consistency in comparison of data from Fig. 2with X
values presented in Table 1. The alternative approach of excluding the concentration
of hydrophilic compounds from the denominator in X
determinations produced similar conclusions, except that in
examining X
values, one was
restricted to comparing hydrophilic compounds with each other and,
likewise, amphipathic inhibitors with each other. The approach used
here eliminates this apparent limitation.
Figure 2:
Structures of glycans and their effects on
GPI-PLC activity. Compounds were tested as described under
``Experimental Procedures.'' Percent inhibition at 5 mM was determined by comparison of the amount of
[H]dimyristoylglycerol cleaved from
[
H]myristate-labeled VSG by GPI-PLC in the
presence or absence of inhibitor. Values varied <10% in two or more
independent duplicate determinations.
GPI-PLC assay conditions used for these inhibitor studies (see
``Experimental Procedures'') were empirically chosen to be
linear with respect to enzyme concentration (Fig. 1A)
and time (Fig. 1B), thereby ensuring that effects of
potential inhibitors were discernible, as illustrated for
GlcN(1
6)Ins 1,2-cyclic phosphate (compound VP-601L) (Fig. 1C). The glycan
6-O-(2-amino-2-deoxy-
-D-glucopyranosyl)-D-myo-inositol
(GlcN(
1
6)Ins; compound VP-606L) (Fig. 2) inhibited
GPI-PLC moderately (28.9%). (All percentage inhibitions are quoted at 5
mM glycan. Variability in sets of duplicate determinations
performed on different occasions was <10%.) We then explored whether
modifications of GlcN(
1
6)Ins could produce better
inhibitors.
Figure 1:
GPI-PLC inhibition conditions. A, the enzyme dose-response curve is shown. Duplicate sets of
a standard 30-µl reaction mixture containing 2 µg of
[H]myristate-labeled VSG (10,000 dpm, 1
µM final concentration) in 1
assay buffer were
assembled on ice. Varying amounts of GPI-PLC (1.6
10
units/mg) were added as indicated, and the reaction was incubated
at 37 °C for 15 min. [
H]Dimyristoylglycerol
cleaved from VSG was quantitated after extraction into 500 µl of
water-saturated 1-butanol as described under ``Experimental
Procedures.'' Data points are averages from duplicate reactions. A
background of 353 dpm from a blank reaction (i.e. no GPI-PLC
addition) has been subtracted from the data. The plot was generated
with ``UltraFit'' (BioSoft, Ferguson, MO). B, the
time course is shown. To a standard reaction mixture (as described for A) was added 19 units of GPI-PLC. The mixture was maintained
at 37 °C and terminated at the indicated intervals, followed by
quantitation of the released
[
H]dimyristoylglycerol as described for A. C, GlcN(
1
6)Ins 1,2-cyclic phosphate
(VP-601L) inhibits GPI-PLC cleavage of
[
H]myristate-labeled VSG. A reaction mixture
containing 19 units of GPI-PLC (as described for B) was
assembled on ice with or without GlcN(
1
6)Ins 1,2-cyclic
phosphate (VP-601L) as detailed under ``Experimental
Procedures.'' Following a 15-min incubation at 37 °C, released
[
H]dimyristoylglycerol was quantitated (see
``Experimental Procedures''). The mole fraction (X
) is the ratio of inhibitor
concentration to the sum of inhibitor and detergent concentrations (see
``Experimental Procedures''). A mole fraction of 0.3 is
equivalent to 5 mM VP-601L under these
conditions.
The addition of a phosphate group to the Ins 1-hydroxyl
of VP-606L, resulting in GlcN(1
6)Ins 1-phosphate (VP-600L),
did not increase inhibitory potency (Fig. 2). However,
cyclization of the Ins 1-phosphate to the Ins 2-hydroxyl, forming
GlcN(
1
6)Ins 1,2-cyclic phosphate (VP-601L), increased
potency 2.3-fold (over that observed for GlcN(
1
6)Ins
1-phosphate) to 74.6% (Fig. 2). The glycan O-(
-D-mannopyranosyl)-(1,4)-O-(2-amino-2-deoxy-
-D-glucopyranosyl)-(1,6)-D-myo-Ins
(Man(
1
4)GlcN(
1
6)Ins), which extends the conserved
protein-GPI glycan core components to three, was only as inhibitory as
GlcN(
1
6)Ins.
Replacing the Ins 1-phosphate of VP-600L
(see above) with Ins 1-dodecyl phosphonate increased inhibition of
GPI-PLC. GlcN(1
6)Ins 1-dodecyl phosphonate (VP-604L) was
2.6-fold more inhibitory (81.1%) than GlcN(
1
6)Ins
1-phosphate. However, GlcN(
1
6)Ins 1-hexyl phosphonate
(VFT-2) was not significantly better than GlcN(
1
6)Ins
1-phosphate (Fig. 2). Interestingly, Ins 1-dodecyl phosphonate
(VP-602L) inhibited GPI-PLC (60.3%) (Fig. 2), even though Ins
1-phosphate had no effect on the enzyme (data not shown).
Modifications of the amino group of GlcN(1
6)Ins affected
inhibitory properties. Acetylation to GlcNAc(
1
6)Ins
(VC-105B) caused a 4.5-fold drop in the inhibition originally observed
with GlcN(
1
6)Ins (6.4%) (Fig. 2). Inhibitory activity
was partially restored when the methyl group of
GlcNAc(
1
6)Ins was replaced by the bulkier N,N-dimethylamino group to produce N-(N,N-dimethylcarbamyl)-GlcN(
1
6)Ins
(VC-109B) (19.3%) (Fig. 2). Nevertheless, N-(N,N-dimethylcarbamyl)-GlcN(
1
6)Ins
was less inhibitory than GlcN(
1
6) Ins.
Inositol ring
modifications were also examined. Elimination of the hydroxyl group at
the Ins 2-position of GlcN(1
6)Ins 1-phosphate (VP-600L) to
form GlcN(
1
6)-2-deoxy-Ins 1-phosphate (VP-612L) abolished
inhibitory activity (Fig. 2). Interestingly, removal of the
phosphate group from VP-612L, forming GlcN(
1
6)-2-deoxy-Ins
(VP-615L), re-established inhibitory activity (80.5% inhibition) (Fig. 2). This 2-deoxyIns analog is more potent than the parent
compound, GlcN(
1
6)Ins (VP-606L).
GlcN(
1
6)-2-deoxy-L-myo-inositol (VP-614L),
in contrast to GlcN(
1
6)-2-deoxy-Ins, had little effect on
GPI-PLC (1.6%) (Fig. 2).
Compounds that inhibited GPI-PLC by
>70% at 5 mM (except GlcN(1
6)Ins and Ins
1-dodecyl phosphonate (VP-602L)) were analyzed further to determine
their inhibitory potency (X
) (see
``Experimental Procedures'' for rationale and
approach)(21, 23) . GlcN(
1
6)Ins 1,2-cyclic
phosphate (VP-601L) and GlcN(
1
6)-2-deoxy-Ins (VP-615L) had X
values of 0.16 and 0.11,
respectively (Table 1). The X
of GlcN(
1
6)Ins (VP-606L) was not approached under the
conditions of our assay. GlcN(
1
6)Ins 1-dodecyl phosphonate
(VP-604L) had an X
of 0.14 (Table 1). Thus, the inhibitory potency of GlcN(
1
6)Ins
1-dodecyl phosphonate (VP-604L), which is amphipathic, is comparable to
that of GlcN(
1
6)-2-deoxy-Ins (VP-615L), a hydrophilic
compound.
In contrast to its moderate inhibition of GPI-PLC,
GlcN(1
6)Ins (VP-606L) had no effect on PI-PLC (Table 1). However, GlcN(
1
6)Ins 1-phosphate (VP-600L)
inhibited PI-PLC competitively (66.9%) (Fig. 3A).
Remarkably, cyclization of the Ins 1-phosphate to the Ins 2-hydroxyl
reduced inhibitory potency 3.8-fold; GlcN(
1
6)Ins 1,2-cyclic
phosphate (VP-601L) inhibited PI-PLC by only 17.3% (Table 1).
GlcN(
1
6)Ins 1-dodecyl phosphonate (VP-604L) was 44.6%
inhibitory (Table 1); the inhibition was competitive (Fig. 3B) with an X
of 0.69. On the contrary, GlcN(
1
6)Ins 1-hexyl
phosphonate (VFT-2) did not inhibit PI-PLC (data not shown). Ins
1-dodecyl phosphonate (VP-602L) inhibited PI-PLC (66.9%).
Figure 3:
Nature of PI-PLC inhibition by hydrophilic (A) and (B) amphipathic GlcN(1
6)Ins
derivatives. Shown is the ratio of the initial PI-PLC reaction rate in
the absence of inhibitors (V
) to the
initial reaction rate in the presence of inhibitors (V
) plotted against X
/(1 - X
); see ``Experimental
Procedures'' for details. A:
,
GlcN(
1
6)-2-deoxy-Ins 1-phosphate (VP-612L);
,
GlcN(
1
6)-2-deoxy-L-myo-inositol (VP-614L);
, GlcN(
1
6)-2-deoxy-Ins (VP-615L);
,
GlcN(
1
6)Ins 1-phosphate (VP-600L). B:
,
GlcN(
1
6)Ins 1-dodecyl phosphonate (VP-604L). The coefficient
of determination and yaxis intercept for a linear
regression are as follows: VP-612L, 0.914 and -0.49; VP-614L,
0.968 and -0.027; VP-615L, 0.982 and 0.787; VP-600L, 0.998 and
1.357; and VP-604L, 0.978 and 0.769. If the coefficient of
determination was >0.90 and the intercept on the yaxis was 1 ± 0.5, then data were fit with a straight line in A and B.
Although
GlcN(1
6)Ins (VP-606L) did not inhibit PI-PLC, modifications
of the Ins and GlcN moieties produced better inhibitors, some of which
were highly specific for PI-PLC. The enzyme was competitively inhibited
by GlcN(
1
6)-2-deoxy-Ins (VP-615L) (X
= 0.44) (Fig. 3A). PI-PLC was inhibited 91.7% by
GlcN(
1
6)-2-deoxy-Ins 1-phosphate (VP-612L) (Table 1).
The X
of
GlcN(
1
6)-2-deoxy-Ins 1-phosphate was 0.39, although the
inhibition was not competitive (Fig. 3A). Intriguingly,
GlcN(
1
6)-2-deoxy-L-myo-inositol (VP-614L)
was nearly as effective as GlcN(
1
6)-2-deoxy-Ins 1-phosphate
(VP-612L) (89.1%) (Table 1). Similarly, the inhibition was not
competitive, and the X
was 0.46 (Fig. 3A and Table 1). Modification of the amino
group of GlcN(
1
6)Ins influenced inhibitory activity;
GlcNAc(
1
6)Ins (VC-105B) inhibited PI-PLC (60%) (Table 1).
PI was a weak inhibitor of PI-PLC (15.6%) (Table 1). Similarly, PG, PS, PE, and PC had little effect on PI-PLC (Table 1).
Figure 4:
G418 stimulates PI-PLC. G418 was incubated
with PI-PLC (0.017 ng, 1 10
units) (
)
or GPI-PLC (1.19 ng, 4 units) (
) and analyzed as described for the
glycans (see ``Experimental Procedures''). The ratio of
reaction velocity in the presence or absence of G418 is
presented.
Susceptibility of polysaccharide-GPIs (glycoinositol
phospholipids) to GPI-PLC provided clues to the substrate requirements
of the enzyme. Glycoinositol phospholipids found in protozoan parasites
of Leishmania sp. have a GPI with a conserved
``tetrasaccharide glycan core'' of
galactofuranosyl(1
3)Man(
1
3)Man(
1
4)GlcN
(reviewed in (26) and (29) ) and are cleaved by
GPI-PLC(22) . This observation and the knowledge that PI is a
very poor substrate suggested that GPI-PLC recognizes a glycan motif
consisting minimally of Man(
1
4)GlcN(
1
6)Ins. We
therefore focused on Man(
1
4)GlcN(
1
6)Ins as
potentially having the requisites for GPI-PLC binding. Individual
components of the protein-GPI core (Man, GlcN, Ins, and EtN, in all
possible combinations) did not inhibit GPI-PLC, indicating that
specific glycosidic bonds between the GPI components might be necessary
for GPI-PLC recognition.
Glucosaminylinositol and its analogs
inhibited GPI-PLC. GlcN(1
6)Ins (VP-606) and
GlcN(
1
6)Ins 1-phosphate (VP-600L) were about equally
inhibitory (Fig. 2), suggesting that 1) GlcN(
1
6)Ins
is the major glycan determinant of GPI-PLC specificity, and 2)
recognition of the phosphoryl group at the Ins 1-position is not
critical for substrate binding. Interestingly, GlcN(
1
6)Ins
1,2-cyclic phosphate (VP-601L) was a better inhibitor than
GlcN(
1
6)Ins 1-phosphate (VP-600L) ( Fig. 2and Table 1). GlcN(
1
6)Ins 1,2-cyclic phosphate is found at
the terminus of the EtN-phospho-6Man(
1
2)
Man(
1
6)Man(
1
4)GlcN(
1
6)-myo-Ins
1,2-cyclic phosphate group that is covalently linked to a cleaved
protein after GPI-PLC action(2) . Thus, GlcN(
1
6)Ins
1,2-cyclic phosphate (VP-601L) might be a product analog. Last, the
innermost mannosyl residue of the conserved glycan core of protein-GPIs
does not appear to play a critical role in GPI-PLC substrate
recognition since Man(
1
4)GlcN(
1
6)Ins was only as
effective as GlcN(
1
6)Ins.
Phosphonate derivatives of
GlcN(1
6)Ins 1-phosphate (VP-600L) were more potent
inhibitors, most likely because they are noncleavable substrate
analogs. GlcN(
1
6)Ins 1-dodecyl phosphonate (VP-604L) was
more inhibitory than Ins 1-dodecyl phosphonate (VP-602L) ( Fig. 2and Table 1), attesting to the importance of the
GlcN moiety in substrate recognition. Additionally, since GPI-PLC is
associated with Nonidet P-40 micelles in our assays, one would predict
that hydrophobic (and amphipathic) compounds would be better inhibitors
because they would gain access to the enzyme more easily by associating
initially with Nonidet P-40 micelles. If the phosphonate alkyl chain
length is used as an indicator of hydrophobicity, the prediction is
borne out. GlcN(
1
6)Ins 1-dodecyl phosphonate (VP-604L),
whose alkyl chain length is twice that of GlcN(
1
6)Ins
1-hexyl phosphonate (VFT-2), is more inhibitory than VFT-2 (Fig. 2). Nevertheless, since GlcN(
1
6)-2-deoxy-Ins
(VP-615L) was as effective as GlcN(
1
6)Ins 1-dodecyl
phosphonate (VP-604L), a compound need not have a hydrophobic moiety to
be a good inhibitor.
An unmodified amino group on
GlcN(1
6)Ins is optimal for substrate recognition.
Acetylation of the GlcN(
1
6)Ins amino group abolishes
recognition because GlcNAc(
1
6)Ins (VC-105B) did not inhibit
GPI-PLC (Fig. 2). This result is consistent with
GlcNAc(
1
6)Ins 1-phosphodiacylglycerol being a poor substrate
for the enzyme(27) . We rule out steric hindrance at the
GPI-PLC active site, due to replacement of an amino hydrogen of
GlcN(
1
6)Ins (VP-606) with an acetyl group, as the cause of
GlcNAc(
1
6)Ins loss of inhibitory activity because VC-109B,
which contains an N,N-dimethylcarbamyl group instead
of the hydrogen on the amino group of GlcN, inhibits GPI-PLC better
than GlcNAc(
1
6)Ins (Fig. 2). We do not have a simple
structure-activity explanation for the VC-109B results.
Lack of
inhibition by GlcN(1
6)-2-deoxy-Ins 1-phosphate (VP-612L) as
compared with GlcN(
1
6)Ins 1-phosphate (VP-600L) raises the
possibility that inhibition by GlcN(
1
6)Ins 1-phosphate is
enzyme-mediated. Possibly, GlcN(
1
6)Ins 1-phosphate inhibits
only after enzyme-catalyzed cyclization to GlcN(
1
6)Ins
1,2-cyclic phosphate, the GPI-PLC product analog. In the absence of the
hydroxyl at the Ins 2-position, cyclization is impossible; hence,
GlcN(
1
6)-2-deoxy-Ins 1-phosphate is ineffective. Cyclization
(possibly needed to eliminate the negative charge on the phosphate
group) is not essential for inhibition when the phosphate at the Ins
1-position is removed from GlcN(
1
6)-2-deoxy-Ins 1-phosphate.
Hence, GlcN(
1
6)-2-deoxy-Ins (VP-615L) is a potent inhibitor
( Fig. 2and Table 1). Specificity of these compounds is
underscored by the lack of inhibition by
GlcN(
1
6)-2-deoxy-L-myo-inositol (VP-614L),
the enantiomer of GlcN(
1
6)-2-deoxy-Ins (VP-615L) (Fig. 2).
The role of the Ins 2-hydroxyl and a requirement
for the D-myo-inositol enantiomer in GPI-PLC
substrate recognition are addressed by the effects of
GlcN(1
6)-2-deoxyIns and
GlcN(
1
6)-2-deoxy-L-myo-inositol,
respectively. GlcN(
1
6)-2-deoxy-Ins (VP-615L) inhibited
GPI-PLC (Table 1), but
GlcN(
1
6)-2-deoxy-L-myo-inositol (VP-614L)
had no effect. These results indicate that the Ins 2-hydroxyl is not
required for substrate recognition, even though it is presumably needed
for catalysis. The ineffectiveness of
GlcN(
1
6)-2-deoxy-L-myo-inositol (VP-614L)
as compared with GlcN(
1
6)-2-deoxy-Ins establishes the
ability of GPI-PLC to distinguish between L-myo-inositol and D-myo-inositol.
Inhibitory constants obtained for the Ins 1-phosphonates in this
study were slightly better than, but in the same range as, those
obtained in the inhibition of B. cereus PI-PLC by Ins
1-palmitoyl phosphonate(30) . To our knowledge, however, our
work represents the first use of GlcN(1
6)Ins and its
derivatives as inhibitors of GPI phospholipases. If tested against
other membrane-bound GPI-specific enzymes (e.g. phospholipases
and glycosyltransferases), the inhibitory constants obtained are likely
to be in the same range as reported here, mainly because the inhibition
occurs at the interface between a micelle-bound enzyme and an aqueous
soluble inhibitor.
The Ins 1-phosphoryl group is
very important for glycan recognition by PI-PLC. This inference is
backed by the observation that GlcN(1
6)Ins 1-phosphate
(VP-600L) competitively inhibits PI-PLC, while GlcN(
1
6)Ins
(VP-606L) is completely ineffective (Table 1). (The identical
phosphoryl group is not critical for GPI-PLC substrate recognition (see
discussion above).) Possibly, abstraction of a proton from the Ins
2-hydroxyl is not an early event in PI-PLC cleavage of GPIs. Instead,
attack on the phosphoryl group by an active-site nucleophile might lead
to formation of a pentacoordinate enzyme-phosphoinositol glycan
intermediate, which collapses subsequently to GlcN(
1
6)Ins
1,2-cyclic phosphate and finally to GlcN(
1
6)Ins 1-phosphate.
It has been suggested that one of the paths toward cleavage of PI by
mammalian PI phospholipases C involves formation of an
enzyme-phosphoinositol intermediate(33) , similar to that
proposed here for PI-PLC.
Other differences in the effects of the
GlcN(1
6)Ins derivatives include the following. 1)
GlcN(
1
6)-2-deoxy-L-myo-inositol (VP-614L)
inhibits PI-PLC, but has little effect against GPI-PLC (Table 1).
2) GlcNAc(
1
6)Ins (VC-105B) is effective only against PI-PLC (Table 1). 3) G418 stimulates PI-PLC only (Fig. 4). 4)
Acidic phospholipids inhibit GPI-PLC without exerting a significant
effect on PI-PLC.
We conclude that PI-PLC binds glycans containing
either D-myo-Ins or L-myo-Ins,
unlike GPI-PLC, which appears to bind glycans containing D-myo-Ins only. Furthermore, since the inhibition of
PI-PLC by GlcN(1
6)-2-deoxy-Ins 1-phosphate (VP-612L) and
GlcN(
1
6)-2-deoxy-L-myo-inositol (VP-614L)
was not competitive, our data raise the possibility of allosteric
regulation of PI-PLC at a novel carbohydrate-binding site. In this
regard, the aminoglycosides G418 and gentamicin could stimulate PI-PLC
by binding to a regulatory site analogous to the proposed allosteric
site occupied by hydrophilic glycans. Consistent with this latter
hypothesis, G418 reverses a GPI-negative phenotype in some mammalian
cells by uncharacterized mechanisms(34) . Our data on PI-PLC
suggest that G418 could bind an enzyme in the GPI
biosynthesis/regulatory pathway and cause reversal of the GPI-negative
phenotype. Inhibition of PI-PLC by GlcNAc(
1
6)Ins is
consistent with GlcNAc(
1
6)Ins 1-phosphodiacylglycerol being
a substrate for the enzyme(35) .
Regulation of GPI-PLC by acidic phospholipids in vivo is an intriguing possibility. GPI-PLC is a cytoplasmic membrane protein that cleaves GPI precursors in vitro. Yet, in T. brucei, where the enzyme is endogenous, GPI-PLC does not cause a depletion of GPI biosynthetic intermediates, even though it appears to colocalize with GPI intermediates on the cytoplasmic side of intracellular membranes, where GPI biosynthesis is initiated(22, 36) . How could GPI-PLC be prevented from catabolizing GPI intermediates in T. brucei? If inhibition by PI occurred in vivo (presumably at a lower concentration of PI since detergent is absent), GPI-PLC might be prevented from cleaving GPI intermediates in PI-enriched intracellular membrane microdomains where GPI biosynthesis might be initiated.
In summary, details of GPI recognition differ between
GPI-PLC and PI-PLC. The remarkable contrasts in sensitivity of the two
enzymes to some GlcN(1
6)Ins derivatives and gentamicins
during cleavage of the identical GPI phosphodiester suggest the
existence of mechanistic subclasses of GPI phospholipases C, of which
GPI-PLC and PI-PLC might be prototypes. Hence, GlcN(
1
6)Ins
derivatives seem likely to be powerful tools for analyzing the mode of
action of GPI phospholipases, and possibly GPI glycosyltransferases.