Laboratory of Molecular Parasitology, The Rockefeller University, 1230 York Avenue, New York, NY 10021, USA
* Author for correspondence (e-mail: george.cross{at}rockefeller.edu )
Accepted 13 November 2001
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Glycosylphosphatidylinositol anchor, GPI, Mutation, Signal sequence, Trypanosoma brucei
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proteins destined to be GPI-anchored are translated with cleavable N- and
C-terminal signal peptides. The N-terminal signal peptide directs the nascent
polypeptide to the endoplasmic reticulum (ER)
(Walter et al., 1984). The
C-terminal signal peptide is replaced by a preformed GPI anchor in an
immediately post-translational transamidation reaction catalyzed by an ER
protein complex containing at least four distinct proteins: GPI8, GAA1,
GPI16/PIG-T and GPI17/PIG-S (Benghezal et
al., 1996
; Meyer et al.,
2000
; Ohishi et al.,
2000
; Sharma et al.,
2000
; Fraering et al.,
2001
; Ohishi et al.,
2001
; Vidugiriene et al.,
2001
). In all of the GPI-anchored proteins, the C-terminal signal
peptide has certain conserved features. It consists of a hydrophilic spacer
sequence of 8-12 amino acids, followed by a more hydrophobic region of 8-20
amino acids. Mutational analyses of the C-terminal GPI signal sequences of
placental alkaline phosphatase (Berger et
al., 1988
; Kodukula et al.,
1992
; Lowe, 1992
),
5'-nucleotidase (Furukawa et al.,
1994
; Furukawa et al.,
1997
), decay accelerating factor
(Caras, 1991
;
Moran and Caras, 1991a
;
Moran and Caras, 1991b
), CD46
(Coyne et al., 1993
),
acetylcholinesterase (Bucht and
Hjalmarsson, 1996
; Bucht et
al., 1999
) and other proteins have demonstrated that the length of
the hydrophobic region and the spacer sequence are important for GPI
anchoring. The site of GPI attachment is called the
site
(Micanovic et al., 1990
).
Comparison of known and predicted GPI addition sites suggest that the
site is restricted to six amino acids with small side chains, namely (in order
of predominance) Ser > Asn > Asp > Gly, Ala and Cys, whereas
+2 can be Ala > Gly > Ser, Thr and Val. The
+1 position,
where Ala > Ser > Asp > Thr, Arg, Cys, Met, Trp, is less restricted.
Mutational studies on several mammalian GPI signal sequences, especially on
placental alkaline phosphatase (Gerber et
al., 1992
; Kodukula et al.,
1992
; Kodukula et al.,
1993
; Udenfriend and Kodukula,
1995a
), have examined the consequences of substituting about 10 of
the possible 20 amino acids at the
+1 and
+2 sites. Only Ala or
Gly functioned efficiently at the
+2 position. Of the 10 amino acids
tested at the
+1 site, only Pro did not function. Whether a protein
will be GPI anchored can be predicted, with
80% accuracy, either by manual
inspection or by computer algorithms on the basis of whether an N-terminal
signal sequence can be predicted (von
Heijne, 1986
; Nielsen et al.,
1997
; Nielsen et al.,
1999
), then whether the C-terminus contains the expected sequence
motifs for GPI anchoring, as defined by `linear' sequence considerations
derived from experimental studies (Julien Kronegg internet site
http://dgpi.pathbot.com) or using a `knowledge-based algorithm' based on
sequence properties extracted from a set of known GPI-anchored proteins and
mutants thereof (Eisenhaber et al.,
1998
; Eisenhaber et al.,
1999
). Theoretical considerations also suggested
(Eisenhaber et al., 1998
) that
the region immediately upstream of the
site, which is not so conserved
but is generally unstructured and hydrophilic, could influence GPI addition, a
possibility that has not been experimentally tested. However, this region is
highly structured in many mature VSGs, and GPI anchoring probably precedes
folding and disulphide bonding of the region immediately upstream of the
site.
VSG comprises about 10% of total cellular protein, making T.
brucei an excellent model for studying GPI anchoring and a potential
expression system for high-value medically important mammalian GPI-anchored
proteins, which have sometimes proved difficult to express in more
conventional cell systems (Azzouz et al.,
2000). In comparison with other GPI-anchored proteins, the VSG GPI
signal sequence is remarkably conserved. The
position is always Ser,
Asp or Asn, and the length of the signal sequence is either 17 (
Ser)
or 23 (
Asp) amino acids. Two positions in the spacer sequence are also
remarkably conserved.
+2 is always Ser, and
+7 is almost always
Lys. This high conservation suggested that even a modest change in one of
these amino acids would have an impact on GPI anchoring. The development of
tools for reverse genetics in T. brucei
(Wirtz et al., 1998
;
Wirtz et al., 1999
) allowed us
to initiate mutational studies of the VSG GPI signal sequence. Attempts to use
various non-trypanosomal reporter proteins, including potentially GPI-anchored
forms of placental alkaline phosphatase, green fluorescent protein and
Saccharomyces cerevisiae prepro-
-factor, were frustrated by
extremely low levels of expression, which are at least partly attributable to
rapid degradation of these alien proteins (K. P. Davies, M. Engstler, U.B. and
G.A.M.C, unpublished; U. Böhme, Ph.D. Thesis,
Universität
Tübingen). We therefore turned to expressing
mutants of VSG itself, in cells that concomitantly expressed a different
wild-type VSG, as VSG expression is essential for T. brucei (V. B.
Carruthers and G.A.M.C., unpublished)
(Nagamune et al., 2000
). We
introduced various mutations into the 23 amino-acid C-terminal GPI signal
peptide of VSG 117. In case of toxicity, the mutations were expressed under
tetracycline regulation (Wirtz et al.,
1998
; Wirtz et al.,
1999
) in cells expressing wild-type VSG 221.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
A series of deletions was also created by PCR. The forward primer for all
deletions (P4) was 5'-CGAAGCTTATGGACTGCCATACAA-3',
corresponding to the N-terminus of VSG 117. Specific reverse primers were used
for each truncation: 5'-CGGGATCCTTTATCTTTGCAAGCATTATTT-3' for
504-526; 5'-CGGGATCCTTATTTCTTGGTTACTAGAATA-3' for
512-526; 5'-CGGGATCCTTAAGCAGAAACCACGGTGAGG-3' for
519-526;
5'-CGGGATCCTTAAAAAAGCAAGGCCACAAATGCAGCAGAAACCACGGTGAGGGCGAATAGAATAGAGGAATC-3'
for
515-520;
5'-CGGGATCCTTAAAAAAGCAAGGCCACAAATGCAGCAGAAACCACGGTGAGGGCGAAATCTTTGCAAGCATT-3'
for
504-511. The PCR-products were cut with HindIII and
BamHI and inserted into HindIII- and BamHI-digested
pUB39.
To create VSG117TM, the transmembrane and cytoplasmic domain of a T.
brucei 65 kDa invariant surface glycoprotein (ISG65)
(Ziegelbauer et al., 1992) was
amplified with the forward primer
5'-CGGGATCCGCAATGATTATATTAGCAG-3' and the reverse
primer 5'-CGGGATCCTTACATTACCGCCTTTCCA-3'. We used plasmid
p3'CRAM-XTM.CD as template (Yang et
al., 2000
) (a gift of Mary G.-S. Lee, New York University Medical
School). The insert was cut with BamHI and inserted into pUB73pre,
which had been previously cut with BamHI and treated with alkaline
phosphatase. pUB73pre, which contains VSG 117 lacking the C-terminal
signal peptide, is a derivative of pLew82
(Wirtz et al., 1998
). For VSG
117Ty, we used as a template pUB39 and as forward primer
5'-CGAAGCTTATGGACTGCCATACAAA-3' and as reverse primer
5'-CGGGATCCTTAGTCAAGTGGGTCCTGGTTAGTATGGACTTCAAAAAGCAAGGCCACAAATGCA-3'.
The PCR product was cut with BamHI and HindIII and inserted
into pUB39, which was also cut with BamHI and HindIII.
Culture and transfection of trypanosomes
Bloodstream-form T. brucei were cultured in HMI-9 at 37°C
(Hirumi and Hirumi, 1989). The
T7-promoter-driven Tet-operator-regulated VSG 117 cassettes were
integrated into an rDNA spacer in trypanosome cell line 13-90, which expresses
wild-type VSG 221, T7 RNA polymerase and the Tet repressor
(Wirtz et al., 1999
). All
transfections were performed as described previously
(Navarro and Cross, 1998
).
Expression of the selectable marker was induced by adding 2.5 ng/ml
doxycycline to the medium. For maximum induction of mutant VSGs, doxycycline
was added at 100 ng/ml. Genomic DNA was isolated from a representative
selection of mutant cell lines, and the mutant sequences were amplified by PCR
to verify that there was no reversion of the mutation.
GPIPLC release as an assay for GPI anchoring
1x107 cells were resuspended in 200 µl ice-cold water
containing 0.1 mM TLCK and held on ice for 5 minutes. After centrifugation at
3,000 g for 5 minutes, the supernatant was discarded. The cell ghosts
were resuspended in 200 µl 10 mM sodium phosphate buffer, pH 8.0,
containing 0.1 mM TLCK. After incubation at 37°C for 15 minutes, the
sample was centrifuged at 16,000 g for 15 minutes and the
different steps of the protocol were analyzed by western blotting. This
protocol quantitatively releases cell surface GPI-anchored VSG, in the
37°C incubation, by activating an endogenous phospholipase C that is GPI
specific under these conditions (Butikofer
et al., 1996; Leal et al.,
2001
). Most cytoplasmic proteins are released in the initial
0°C lysis step (Cross,
1984
).
Western blot analysis
Crude lysates of 2x104 cells were loaded per lane in a 10%
SDS-PAGE and transferred onto nitrocellulose membranes (Amersham-Pharmacia).
Tris-buffered saline, pH 7.6, plus 0.1% Tween-20 (TBST), containing 5% non-fat
dry milk, was used as a blocking solution, for 1 hour at room temperature. The
membranes were subseqently incubated for 1 hour either with CRD-depleted
rabbit anti-native VSG 221 or rabbit anti-rVSG-117 antibodies
(Hoek et al., 1999) (dilution
1:10,000), washed in TBST, then incubated with the corresponding
horseradish-peroxidase-conjugated goat-anti-rabbit antibody for 1 hour
(dilution 1:10 000). Proteins were visualized with the supersignal Pico
chemiluminescence substrate (Pierce).
Northern blot analysis
Total RNA was isolated with RNA Stat-60 (Tel-Test Inc), electrophoresed on
2.2 M formaldehyde 1.5% agarose gels and transferred to nylon membranes.
Filters were hybridized with [-32P]dATP random-primed
full-length VSG 117. Final washings were performed at room
temperature for 15 minutes and at 65°C for another 15 minutes with
2xSSC, 0.1% SDS. The last wash was done at 65°C for 30 minutes with
0.1xSSC, 0.1% SDS.
Immunofluorescence
For immunofluorescence analysis, 5x106 cells were washed
twice in phosphate-buffered saline (PBS), fixed with 2% formaldehyde in PBS
for 10 minutes on ice. The cells were attached to glass cover slips by
centrifugation at 400 g for 5 minutes at 4°C. For
visualization of intracellular proteins, the attached cells were permeabilized
with 0.2% NP-40 in PBS for 5 minutes. The cells were blocked twice for 10
minutes in PBG (PBG with 0.1% cold-water-fish-skin gelatin from Sigma and 0.5%
BSA) and incubated overnight at 4°C with a 1:200 dilution of the primary
rabbit antibodies to recombinant VSG 117
(Hoek et al., 1999), to native
VSG 221 or mouse antibodies to T. cruzi BiP (a generous gift of D. M.
Engman, Northwestern University, Chicago, Il, USA) or a 1:1000 dilution of
mouse monoclonal antibody 280 to T. brucei p67 (a generous gift of J.
D. Bangs, University of Wisconsin, Madison, WI, USA). After washing the cells
six times with PBG for 5 minutes, the cells were treated for 2 hours at room
temperature with fluorescein-conjugated goat or rhodamine-conjugated goat
anti-rabbit or anti-mouse antibodies (1:200 in PBG). After treatment of the
cells with DAPI to stain nuclear and kinetoplast DNA, cells were mounted with
alkaline glycerol (45% glycerol, 50 mM Tris/HCl, pH8.0) containing
p-phenylenediamine as anti-fade. Cells were observed with a Nikon
epifluorescence microscope using a 100x Fluor objective and the
appropriate fluorescein or rhodamine filters. Images were captured with a Sony
DKC5000 CCD or Spot 2.13 camera at ISO 100, using an 0.5 second integration
time, and imported directly to Adobe Photoshop 6.0.
Dimerization analysis
2x108 cells expressing mutations 504-526 and
512-526 were permeabilized with 500 µl 20 mM HEPES, 0.15 M NaCl, pH
7.6, containing 1% (v/v) Nonidet P40. Insoluble material was removed by
centrifugation (16 000 g for 15 minutes), and the supernatant
was loaded onto a Sephacryl S-200 column (equilibrated with 20 mM HEPES, 0.15
M NaCl). Standards (Sigma) were as follows (250 µg of each): alcohol
dehydrogenase, 150 kDa; BSA, 66 kDa; carbonic anhydrase, 31 kDa; cytochrome C,
12.4 kDa. 500 µl fractions were collected and VSG was detected in each
fraction by immunoblotting after TCA precipitation and SDS-PAGE.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
GPI modification of the mutated proteins was evaluated by measuring the
GPI-phospholipase C (GPIPLC) releasable VSG 117 on western blots. Over 90% of
the surface GPI-anchored VSG is released into the supernatant in this assay by
activation of an endogenous PIPLC that is specific for GPI cleavage under
these conditions (Butikofer et al.,
1996). All of the point-mutated proteins were detected in the
supernatant, suggesting that all VSGs with mutations at position
+2 and
+7 were GPI-anchored on the cell surface. To demonstrate that the
GPIPLC release assay works reliably, several controls were performed
(Fig. 2). Inhibition of GPIPLC
with p-chloromercuriphenylsulfonate (pCMPS)
(Butikofer et al., 1996
)
reduced VSG release by 90% (Fig.
2B). Since the 13-90 cell line is naturally expressing VSG 221, we
used antibodies to reveal this VSG as a second control
(Fig. 2C). Both the endogenous
VSG 221 and ectopically expressed VSG 117 were GPI anchored and releasable to
a similar degree. To determine whether this protocol releases lumenal
ER-associated proteins, we tested whether the ER chaperone BiP could be
detected in the supernatant after release with GPIPLC, which it was not
(Fig. 2D). This confirms that
the protocol specifically releases GPI-anchored surface proteins, as
previously shown (Ferguson et al.,
1985
; Bangs et al.,
1986
; Ferguson et al.,
1986
) and does not disrupt the ER membrane. Another control was
performed with a previously acquired GPIPLC null-mutant cell line
(Leal et al., 2001
). As shown
in Fig. 2E, no VSG was released
into the supernatant of this cell line, which expresses VSG 221. The surface
location of all the mutated VSGs was confirmed by indirect immunofluorescence
with anti- VSG 117 antibodies (Hoek et
al., 1999
) (selected examples are shown in
Fig. 3).
|
To see whether the and
+1 positions play a role in GPI
signal sequence recognition, we introduced several mutations at these two
positions (Table 1). The
position was mutated to Glu or Cys and the
+1 position was
mutated to Leu, His, Lys, Tyr or Phe. Surprisingly, with one exception, these
mutated proteins were expressed at similar levels to wild-type VSG 117, and
all were completely released by GPIPLC, indicating that they were anchored on
the cell surface (summarized in Table
1; data for D503C and D503E are shown in
Fig. 4A,B). Expression of the
position Cys mutant was reduced by about 90%
(Fig. 4A). Indirect
immunofluorescence confirmed cell surface expression of D503C
(Fig. 3D).
|
Combination of mutations in the GPI signal peptide
Since the point mutations showed no effect on GPI anchoring, we introduced
more extensive mutations (Table
1). +3,
+4 and
+5 were simultaneously
substituted with Thr (506-508T) and
+7 and
+8 with Leu
(510-511L). A stretch of eight Leu residues was introduced at the end of the
hydrophobic region (519-526L). Expression levels of all three mutations were
comparable to wild-type VSG 117, and all of them were GPI anchored on the cell
surface, as shown by GPIPLC release and indirect immunofluorescence with
anti-VSG 117 antibodies. A representative western blot for the GPIPLC release
of mutant 506-508T is shown in Fig.
4C.
Deletions in the GPI signal peptide
To investigate the length requirements of the C-terminal signal peptide,
several deletion mutants were constructed
(Table 1). The effect of the
mutations on the expression and location of VSG 117 was analyzed by western
blotting. As expected, when the entire C-terminal signal peptide was deleted
(504-526), VSG was not anchored on the cell surface, as shown by the
GPIPLC release assay (Fig. 4D).
Deleting four amino acids from the spacer sequence (
515-520) did not
affect release by GPIPLC, indicating that this mutant is GPI anchored. In
contrast, deleting the entire spacer sequence (
504-511) showed a
partial effect on anchoring: about 50% of the protein was found in the
supernatant after GPIPLC activation (Fig.
4E). Deleting the entire hydrophobic region (
512-526)
prevented GPI anchoring (Fig.
4F), whereas deleting part of the hydrophobic region
(
519-526) resulted in a partly anchored protein
(Fig. 4G). This result
indicates that the hydrophobic region has to be at least eight amino acids
long for anchoring to occur.
Other investigators have shown that proteins lacking the GPI signal peptide
are secreted into the medium (Furukawa et
al., 1994; Bucht and
Hjalmarsson, 1996
; McDowell et
al., 1998
). We therefore examined the fate of unanchored proteins.
After induction of mutants
504-526 and
512-526 for 48 hours, we
resuspended the cells in culture medium containing 100 ng/ml doxycyline and
only 1% fetal calf serum for 8 hours. Proteins in the medium were
concentrated, electrophoresed, and VSG was revealed by western blotting. An
equivalent of 2x106 cells was loaded per lane. Although this
loading would permit the detection of 1% of the normal amount of cell-surface
VSG, neither mutant VSG could be detected in the medium (data not shown),
implying that they were entirely retained in the cell or extensively
degraded.
To more precisely determine the intracellular location of 504-526
and
512-526, fixed and permeabilized cells were stained simultaneously
with antibodies to VSG 117 and the ER-resident protein BiP. The pattern of
staining seen with anti-BiP (Fig.
5B,F) is characteristic of ER morphology in trypanosomes
(McDowell et al., 1998
). The
staining seen with anti-VSG 117 in the mutants
(Fig. 5A,E) exactly matches the
distribution of BiP. This colocalization can be seen best as a yellow
pseudo-color in the merged images (Fig.
5D,H). Thus, mutated VSGs that are not GPI-anchored accumulate in
the ER, the initial compartment of the secretory pathway. By contrast,
mutations that were GPI anchored resulted in a typical surface staining
(Fig. 3). As a control, we used
the wild-type cell line, MITat 1.2, expressing VSG 221. No staining was
observed with anti-VSG 117 antibodies (data not shown). In partly anchored
mutants, the surface staining obscured any intracellular staining.
|
VSG exists as a dimer on the membrane. To determine whether the failure of
504-526 and
512-526 to exit the ER was due to misfolding and a
lack of dimerization, we determined the oligomeric state by gel filtration on
a Sephacryl S-200 column. Data for mutation
512-526 are shown in
Fig. 6. The different fractions
were subjected to SDS-PAGE and western blotting. By comparison with the
molecular mass standards, VSG dimers should elute at fraction 19. As shown in
Fig. 6, this is true for the
co-expressed control (VSG 221) and mutant (VSG 117) proteins. This result
shows that the accumulated mutant VSG is not misfolded and ER retention is
only attributable to lack of a GPI anchor. The increased VSG degradation,
revealed in the gel analysis of the column fractions, is attributed to the
longer duration of the analysis, in which protease inhibitors were not
included, and overexposure of the peak lanes in the western blot.
|
Elongation of the GPI signal peptide
Two elongation mutants, 511FK and VSG117Ty, were constructed. Their
expression levels were comparable with wild-type VSG 117. As determined by the
GPIPLC release assay and indirect immunofluorescence, these two mutations were
anchored and expressed on the cell surface
(Fig. 7A,B). Although VSG117Ty
has a Ty epitope tag at the C-terminus, we were not able to detect this mutant
with the monoclonal antibody BB2 (Bastin et
al., 1996). This result confirms the cleavage of the GPI-anchor
signal peptide. This cell line could be useful for identifying the transient
precursor prior to exchange of the signal peptide for the GPI anchor.
|
To investigate the effect of a transmembrane anchor on VSG localization, we
replaced the GPI signal sequence with the C-terminal transmembrane plus
cytoplasmic domains of the invariant surface glycoprotein 65 (ISG65)
(Ziegelbauer et al., 1992)
(Table 1). The expression level
of this fusion protein (VSG117TM) was about 1% of wild-type GPI-anchored VSG
(Fig. 7C). By northern blot,
however, no difference was detected in the mRNA abundance between mutant and
wild type (data not shown). To examine the cellular localization, we performed
indirect immunofluorescence in trypanosomes expressing VSG117TM
(Fig. 8). Although the chimeric
protein contains a transmembrane domain, it was not found on the cell surface,
instead it showed a faint staining characteristic of the ER and larger
accumulations in compartments close to the flagellar pocket. Some of this
staining did not overlap with the regions reacting with a mouse monoclonal
antibody to the major lysosomal membrane protein, p67
(Kelley et al., 1999
),
suggesting that they might represent pre-lysosomal compartments. The staining
pattern is similar to that reported for endocytosed transferrin.
|
Effects of mutations on RNA and protein stability
To see whether mutations of the C-terminal signal sequence that showed
significant effects on VSG expression or anchoring had affected RNA stability,
we extracted RNA from these mutant cell lines at different times after
transcriptional shutoff by actinomycin D. Compared to wild-type VSG 117, none
of the mutations showed any effect on RNA stability (representative data for
mutant 519-526 are shown in Fig.
9A). We then checked whether the mutated VSGs were degraded faster
than wild-type VSG. A selection of cell lines expressing different mutated
VSGs were cultured for 8 hours in medium containing cycloheximide to terminate
protein synthesis. Samples were taken at intervals and analyzed on a western
blot. Mutations
504-526 and
512-526 were degraded rapidly
(Fig. 9B), compared with
wild-type VSG 117, whose half-life is
33 hours
(Seyfang et al., 1990
). Adding
lactacystin, a proteasome inhibitor that is known to work in T.
brucei (Mutomba et al.,
1997
; Fenteany and Schreiber,
1998
) did not prevent degradation
(Fig. 9C), suggesting that the
unanchored truncated mutants are targeted for lysosomal rather than
proteasomal degradation.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To address the minimum length of the spacer sequence and the hydrophobic
region, we constructed a series of deletion mutants. Just two severe changes,
which deleted the entire C-terminal signal peptide or the hydrophobic region,
prevented GPI addition. Deleting the entire spacer region, or eight amino
acids from the hydrophobic region, reduced the efficiency of anchoring.
Previous work showed that the length of both the spacer and hydrophobic
regions were important for efficient GPI anchoring
(Caras, 1991;
Moran and Caras, 1991b
;
Moran and Caras, 1991a
;
Lowe, 1992
;
Coyne et al., 1993
;
Furukawa et al., 1994
;
Bucht and Hjalmarsson, 1996
;
Furukawa et al., 1997
;
Bucht et al., 1999
), whereas we
found that a spacer sequence consisting of just four amino acids sufficed.
However, inspection of the GPI signal sequence of VSG 117, coupled with our
observation that KK in the spacer can be replaced by LL, suggests that there
may be less distinction, in the VSG signal sequence, between the
hydrophilicity/hydrophobicity of what have been designated as spacer and
hydrophobic domains. In addition, it should be kept in mind that the VSG 117
wild-type signal sequence contains 23 amino acids, whereas the other major VSG
subclass (represented by VSG 221, which is coexpressed in the cell line used
for the current experiments) has only a 17 amino-acid signal, suggesting that
up to six amino acids might be dispensed with, from VSG 117, without affecting
anchoring. In conclusion, our analysis suggests that the requirements for VSG,
despite initial appearances, might be less stringent than for mammalian
GPI-anchored proteins.
Our results, together with others
(Engstler et al., 2000),
suggest that there is a stringent quality control for VSG secretion in
bloodstream-form T. brucei and that the structure of the VSG itself,
and the presence of a GPI anchor, are both required for efficient secretion.
Failure to add a GPI anchor to VSG results in ER retention followed by
degradation. This result indicates that the GPI anchor is a strong facilitator
of VSG secretion. This was suggested by earlier studies of a VSG 117
504-526 mutation expressed in procyclic forms of T. brucei
(McDowell et al., 1998
), where
the GPI-anchored wildtype VSG 117, although expressed at far lower levels than
in bloodstream forms, was transported to the surface, but the
504-526
mutation was retarded in the ER and secreted five-fold more slowly than the
wild type. A similar result was subsequently reported for bloodstream forms,
where degradation of
504-526 following its retention in the ER was
sensitive to inhibitors of lysosomal proteases
(Triggs and Bangs, 1999
).
Something about VSG structure, other than the presence of a GPI anchor, is
also important for efficient secretion in T. brucei. Several
investigators have failed to efficiently express alternative GPI-anchored
reporters with VSG signal sequences or chimeras consisting of VSG or its
subdomains fused to different reporter proteins, for example, placental
alkaline phosphatase, Saccharomyces cerevisiae prepro-
factor
and green fluorescent protein. None of these fusions accumulated on the cell
surface (M. Engstler et al.,
2000
) (M. Engstler, unpublished; K. Davis, U.B., J. Wang and
G.A.M.C, unpublished).
Degradation of proteins that fail ER quality-control tests has been
described in a variety of situations and has been ascribed to
retrotanslocation from the ER and proteasomal degradation
(Lippincott-Schwartz et al.,
1988; Bonifacino et al.,
1990
; Pilon et al.,
1997
; Plemper et al.,
1997
; Zhang et al.,
1997
; Ellgaard et al.,
1999
). There are several examples where misfolded proteins fail to
dimerize and are eliminated by this route. In our experiments, truncated VSGs
were dimerized, and degradation did not appear to occur via the proteasomal
pathway. Rather, by considering all of our observations of non-GPI-anchored
VSG mutants, we speculate that degradation may take place either via
inefficient secretion followed by rapid endocytosis and lysosomal degradation
or by ejection from a later stage of the secretory pathway (Golgi or TGN).
Recent experiments suggest that the ER retention or retardation of mutant
proteins that are unable to receive a GPI anchor may be due to a sustained
interaction with a component of the transamidase complex
(Spurway et al., 2001
;
Vidugiriene et al., 2001
).
The different results obtained with the transmembrane (VSG117TM) versus the
VSG117Ty mutants are interesting. The ,
+1,
+2 positions,
and the length and composition of the spacer and hydrophobic domains of these
two constructs, are similar. The main difference is in the degree to which the
two proteins are extended, to form potential cytoplasmic domains. The Ty
extension of 15 amino acids did not prevent its anchoring, whereas the
transmembrane extension of 30 amino acids, onto what looks as if it would
otherwise be an acceptable GPI signal sequence, prevents GPI anchoring. This
transmembrane domain was also included in a mutational study of the C-terminal
targeting sequence of the T. brucei flagellar-pocket protein CRAM
(Yang et al., 2000
).
Interestingly, when the native CRAM sequence was truncated by 40 amino acids,
CRAM was partly distributed over the trypanosome surface. Inspection of the
truncated C-terminal sequence suggests the presence of several very probable
GPI-attachment sites, between 20 and 38 amino acids upstream of the truncated
C-terminus, suggesting that truncation revealed a latent signal sequence to
the GPI machinery. These observations highlight the unanswered question of how
the machinery for GPI addition distinguishes between internal and terminal
hydrophobic sequences, as it must, if GPI addition at fortuitous internal
signal sequences is to be avoided.
Variations in the efficiency with which different GPI signal sequences are
recognized could contribute to the regulation of surface protein abundance.
The ER quality-control mechanisms can destroy evidence of GPI anchoring
failures, rather than allow aberrant versions of these proteins to proceed
through the secretory pathway. Signal sequence requirements are similar but
not identical in trypanosomes and mammalian cells, where the VSG GPI signal
sequence functioned poorly in some studies
(Moran and Caras, 1994) but
better in others (White et al.,
2000
), suggesting that the reporter protein, or just the region
immediately upstream of the
site
(Eisenhaber et al., 1998
),
might influence the results.
GPI-anchored proteins may follow a novel secretory pathway, and the
presence of a GPI anchor may influence endocytic and recycling pathways. Not
all GPI-anchored proteins traverse the same recycling pathway
(Nichols et al., 2001).
Sphingolipid-rich microdomains (`lipid rafts') are probably important for the
secretion and sorting of GPI-anchored proteins
(Bagnat et al., 2000
;
Muniz and Riezman, 2000
), and
a recent study shows that, in yeast, GPI-anchored proteins exit the ER in
different vesicles from other secretory proteins
(Muniz et al., 2001
). The
existence of alternative secretory pathways remains to be investigated in
T. brucei.
In conclusion, our results indicate that the high conservation of naturally occurring VSG GPI signal sequences is unnecessary for efficient VSG synthesis and anchoring, so the question of why these sequences are so conserved remains unanswered. It is possible that smaller changes than we could detect in GPI anchoring efficiency have played a subtle role in trypanosome virulence and have led to the evolutionary optimization of VSG GPI signals.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Azzouz, N., Kedees, M. H., Gerold, P., Becker, S., Dubremetz, J.
F., Klenk, H. D., Eckert, V. and Schwarz, R. T. (2000). An
early step of glycosylphosphatidyl-inositol anchor biosynthesis is abolished
in lepidopteran insect cells following baculovirus infection.
Glycobiology 10,177
-183.
Bagnat, M., Keranen, S., Shevchenko, A. and Simons, K.
(2000). Lipid rafts function in biosynthetic delivery of proteins
to the cell surface in yeast. Proc. Natl. Acad. Sci.
USA 97,3254
-3259.
Bangs, J. D., Andrews, N. W., Hart, G. W. and Englund, P. T. (1986). Posttranslational modification and intracellular transport of a trypanosome variant surface glycoprotein. J. Cell Biol. 103,255 -263.[Abstract]
Bastin, P., Bagherzadeh, A., Matthews, K. R. and Gull, K. (1996). A novel epitope tag system to study protein targeting and organelle biogenesis in Trypanosoma brucei. Mol. Biochem. Parasitol. 77,235 -239.[Medline]
Benghezal, M., Benachour, A., Rusconi, S., Aebi, M. and Conzelmann, A. (1996). Yeast Gpi8p is essential for GPI anchor attachment onto proteins. EMBO J. 15,6575 -6583.[Abstract]
Berger, J., Howard, A. D., Brink, L., Gerber, L., Haubert, J.,
Cullen, B. R. and Udenfriend, S. (1988). COOH-terminal
requirements for the correct processing of a phosphatidylinositol-glycan
anchored membrane protein. J. Biol. Chem.
263,10016
-10021.
Bonifacino, J. S., Suzuki, C. K. and Klausner, R. D. (1990). A peptide sequence confers retention and rapid degradation in the endoplasmic reticulum. Science 247, 79-82.[Medline]
Bucht, G. and Hjalmarsson, K. (1996). Residues in Torpedo californica acetylcholinesterase necessary for processing to a glycosyl phosphatidylinositol-anchored form. Biochim. Biophys. Acta 1292,223 -232.[Medline]
Bucht, G., Wikstrom, P. and Hjalmarsson, K. (1999). Optimising the signal peptide for glycosyl phosphatidylinositol modification of human acetylcholinesterase using mutational analysis and peptide-quantitative structure-activity relationships. Biochim. Biophys. Acta 1431,471 -482.[Medline]
Butikofer, P., Boschung, M., Brodbeck, U. and Menon, A. K.
(1996). Phosphatidylinositol hydrolysis by Trypanosoma
brucei glycosylphosphatidylinositol phospholipase C. J. Biol.
Chem. 271,15533
-15541.
Caras, I. W. (1991). An internally positioned signal can direct attachment of a glycophospholipid membrane anchor. J. Cell Biol. 113,77 -85.[Abstract]
Caras, I. W., Weddell, G. N. and Williams, S. R. (1989). Analysis of the signal for attachment of a glycophospholipid membrane anchor. J. Cell Biol. 108,1387 -1396.[Abstract]
Coyne, K. E., Crisci, A. and Lublin, D. M.
(1993). Construction of synthetic signals for
glycosyl-phosphatidylinositol anchor attachment. Analysis of amino acid
sequence requirements for anchoring. J. Biol. Chem.
268,6689
-6693.
Cross, G. A. M. (1984). Release and purification of Trypanosoma brucei variant surface glycoprotein. J. Cell. Biochem. 24,79 -90.[Medline]
Cross, G. A. M. (1990). Glycolipid anchoring of plasma membrane proteins. Annu. Rev. Cell Biol. 6, 1-39.
Eisenhaber, B., Bork, P. and Eisenhaber, F.
(1998). Sequence properties of GPI-anchored proteins near the
-site: constraints for the polypeptide binding site of the putative
transamidase. Protein Eng.
11,1155
-1161.[Abstract]
Eisenhaber, B., Bork, P. and Eisenhaber, F. (1999). Prediction of potential GPI-modification sites in proprotein sequences. J. Mol. Biol. 292,741 -758.[Medline]
Ellgaard, L., Molinari, M. and Helenius, A.
(1999). Setting the standards: quality control in the secretory
pathway. Science 286,1882
-1888.
Englund, P. T. (1993). The structure and biosynthesis of glycosyl phosphatidylinositol protein anchors. Annu. Rev. Biochem. 62,121 -138.[Medline]
Engstler, M, Guenzel, M. and Boshart, M. (2000). 11th Molecular Parasitology Meeting, abstract 110.
Fenteany, G. and Schreiber, S. L. (1998).
Lactacystin, proteasome function and cell fate. J. Biol.
Chem. 273,8545
-8548.
Ferguson, M. A. J. (1999). The structure,
biosynthesis and functions of glycosylphosphatidylinositol anchors, and the
contributions of trypanosome research. J. Cell Sci.
112,2799
-809.
Ferguson, M. A. J., Low, M. G. and Cross, G. A. M.
(1985). Glycosyl-sn-1,2-dimyristylphosphatidylinositol is
covalently linked to Trypanosoma brucei variant surface glycoprotein.
J. Biol. Chem. 260,14547
-14555.
Ferguson, M. A. J., Duszenko, M., Lamont, G. S., Overath, P. and
Cross, G. A. M. (1986). Biosynthesis of Trypanosoma
brucei variant surface glycoproteins: N-glycosylation and addition of a
phosphatidylinositol membrane anchor. J. Biol. Chem.
261,356
-362.
Ferguson, M. A. J., Homans, S. W., Dwek, R. A. and Rademacher, T. W. (1988). Glycosyl-phosphatidylinositol moiety that anchors Trypanosoma brucei variant surface glycoprotein to the membrane. Science 239,753 -759.[Medline]
Fraering, P., Imhof, I., Meyer, U., Strub, J. M., van
Dorsselaer, A., Vionnet, C. and Conzelmann, A. (2001). The
GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p,
Gpi8p, and Gpi16p. Mol. Biol. Cell
12,3295
-3306.
Furukawa, Y., Tamura, H. and Ikezawa, H. (1994). Mutational analysis of the COOH-terminal hydrophobic domain of bovine liver 5'-nucleotidase as a signal for glycosylphosphatidylinositol (GPI) anchor attachment. Biochim. Biophys. Acta 1190,273 -278.[Medline]
Furukawa, Y., Tsukamoto, K. and Ikezawa, H. (1997). Mutational analysis of the C-terminal signal peptide of bovine liver 5'-nucleotidase for GPI anchoring: a study on the significance of the hydrophilic spacer region. Biochim. Biophys. Acta 1328,185 -196.[Medline]
Gerber, L. D., Kodukula, K. and Udenfriend, S.
(1992). Phosphatidylinositol glycan (PI-G) anchored membrane
proteins. Amino acid requirements adjacent to the site of cleavage and PI-G
attachment in the COOH-terminal signal peptide. J. Biol.
Chem. 267,12168
-12173.
Hirumi, H. and Hirumi, K. (1989). Continuous cultivation of Trypanosoma brucei bloodstream forms in a medium containing a low concentration of serum protein without feeder cell layers. J. Parasitol. 75,985 -989.[Medline]
Hoek, M., Xu, H. and Cross, G. A. M. (1999). Trypanosoma brucei: generation of specific antisera to recombinant variant surface glycoproteins. Exp. Parasitol. 91,199 -202.[Medline]
Holder, A. A. (1983). Carbohydrate is linked through ethanolamine to the C-terminal amino acid of Trypanosoma brucei variant surface glycoprotein. Biochem. J. 209,261 -262.[Medline]
Holder, A. A. and Cross, G. A. M. (1981). Glycopeptides from variant surface glycoproteins of Trypanosoma brucei: C-terminal location of antigenically crossreacting carbohydrate moieties. Mol. Biochem. Parasitol. 2, 135-150.[Medline]
Johnson, J. G. and Cross, G. A. M. (1979). Selective cleavage of variant surface glycoproteins from Trypanosoma brucei. Biochem. J. 178,689 -697.[Medline]
Kelley, R. J., Alexander, D. L., Cowan, C., Balber, A. E. and Bangs, J. D. (1999). Molecular cloning of p67, a lysosomal membrane glycoprotein from Trypanosoma brucei. Mol. Biochem. Parasitol. 98,17 -28.[Medline]
Kodukula, K., Cines, D., Amthauer, R., Gerber, L. and Udenfriend, S. (1992). Biosynthesis of phosphatidylinositol-glycan (PI-G)-anchored membrane proteins in cell-free systems: Cleavage of the nascent protein and addition of the PI-G moiety depend on the size of the COOH-terminal signal peptide. Proc. Natl. Acad. Sci. USA 89,1350 -1353.[Abstract]
Kodukula, K., Gerber, L. D., Amthauer, R., Brink, L. and Udenfriend, S. (1993). Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: Specific amino acid requirements adjacent to the site of cleavage and GPI attachment. J. Cell Biol. 120,657 -664.[Abstract]
Leal, S., Acosta-Serrano, A., Morita, Y. S., Englund, P., Böhme, U. and Cross, G. A. M. (2001). Virulence of Trypanosoma brucei strain 427 is not affected by the absence of glycosylphosphatidylinositol phospholipase C. Mol. Biochem. Parasitol. 114,245 -247.[Medline]
Lippincott-Schwartz, J., Bonifacino, J. S., Yuan, L. C. and Klausner, R. D. (1988). Degradation from the endoplasmic reticulum: disposing of newly synthesized proteins. Cell 54,209 -220.[Medline]
Lowe, M. E. (1992). Site-specific mutations in the COOH-terminus of placental alkaline phosphatase: A single amino acid change converts a phosphatidylinositol-glycan- anchored protein to a secreted protein. J. Cell Biol. 116,799 -807.[Abstract]
McDowell, M. A., Ransom, D. M. and Bangs, J. D. (1998). Glycosylphosphatidylinositol-dependent secretory transport in Trypanosoma brucei. Biochem. J. 335,681 -689.[Medline]
Metcalf, P., Down, J. A., Turner, M. J. and Wiley, D. C.
(1988). Crystallization of amino-terminal domains and domain
fragments of variant surface glycoproteins from Trypanosoma brucei brucei.J. Biol. Chem. 263,17030
-17033.
Meyer, U., Benghezal, M., Imhof, I. and Conzelmann, A. (2000). Active site determination of Gpi8p, a caspase-related enzyme required for glycosylphosphatidylinositol anchor addition to proteins. Biochemistry 39,3461 -3471.[Medline]
Micanovic, R., Kodukula, K., Gerber, L. D. and Udenfriend, S. (1990). Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined. Proc. Natl. Acad. Sci. USA 87,7939 -7943.[Abstract]
Moller, L. B., Ploug, M. and Blasi, F. (1992). Structural requirements for glycosyl-phosphatidylinositol-anchor attachment in the cellular receptor for urokinase plasminogen activator. Eur. J. Biochem. 208,493 -500.[Abstract]
Moran, P. and Caras, I. W. (1991a). Fusion of sequence elements from non-anchored proteins to generate a fully functional signal for glycophosphatidylinositol membrane anchor attachment. J. Cell Biol. 115,1595 -1600.[Abstract]
Moran, P. and Caras, I. W. (1991b). A nonfunctional sequence converted to a signal for glycophosphatidylinositol membrane anchor attachment. J. Cell Biol. 115,329 -336.[Abstract]
Moran, P. and Caras, I. W. (1994). Requirements for glycosylphosphatidylinositol attachment are similar but not identical in mammalian cells and parasitic protozoa. J. Cell Biol. 125,333 -343.[Abstract]
Moran, P., Raab, H., Kohr, W. J. and Caras, I. W.
(1991). Glycophospholipid membrane anchor attachment. Molecular
analysis of the cleavage/attachment site. J. Biol.
Chem. 266,1250
-1257.
Muniz, M., Morsomme, P. and Riezman, H. (2001). Protein sorting upon exit from the endoplasmic reticulum. Cell 104,313 -320.[Medline]
Muniz, M. and Riezman, H. (2000). Intracellular
transport of GPI-anchored proteins. EMBO J.
19, 10-15.
Mutomba, M. C., To, W. Y., Hyun, W. C. and Wang, C. C. (1997). Inhibition of proteasome activity blocks cell cycle progression at specific phase boundaries in African trypanosomes. Mol. Biochem. Parasitol. 90,491 -504.[Medline]
Nagamune, K., Nozaki, T., Maeda, Y., Ohishi, K., Fukuma, T.,
Hara, T., Schwarz, R. T., Sutterlin, C., Brun, R., Riezman, H. and Kinoshita,
T. (2000). Critical roles of glycosylphosphatidylinositol for
Trypanosoma brucei. Proc. Natl. Acad. Sci. USA
97,10336
-10341.
Navarro, M. and Cross, G. A. M. (1998). In situ analysis of a variant surface glycoprotein expression-site promoter region in Trypanosoma brucei. Mol. Biochem. Parasitol. 94, 53-66.[Medline]
Nichols, B. J., Kenworthy, A. K., Polishchuk, R. S., Lodge, R.,
Roberts, T. H., Hirschberg, K., Phair, R. D. and Lippincott-Schwartz, J.
(2001). Rapid cycling of lipid raft markers between the cell
surface and Golgi complex. J. Cell Biol.
153,529
-541.
Nielsen, H., Brunak, S. and von Heijne, G.
(1999). Machine learning approaches for the prediction of signal
peptides and other protein sorting signals. Protein.
Eng. 12,3
-9.
Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein. Eng. 10,1 -6.[Abstract]
Ohishi, K., Inoue, N., Maeda, Y., Takeda, J., Riezman, H. and
Kinoshita, T. (2000). Gaa1p and gpi8p are components of a
glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of
GPI to proteins. Mol Biol Cell
11,1523
-1533.
Ohishi, K., Inoue, N. and Kinoshita, T. (2001).
PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a
complex with GAA1 and GPI8. EMBO J.
20,4088
-4098.
Pilon, M., Schekman, R. and Romisch, K. (1997).
Sec61p mediates export of a misfolded secretory protein from the endoplasmic
reticulum to the cytosol for degradation. EMBO J.
16,4540
-4548.
Plemper, R. K., Bohmler, S., Bordallo, J., Sommer, T. and Wolf, D. H. (1997). Mutant analysis links the translocon and BIP to retrograde protein transport for ER degradation. Nature 388,891 -895.[Medline]
Seyfang, A., Mecke, D. and Duszenko, M. (1990). Degradation, recycling, and shedding of Trypanosoma brucei variant surface glycoprotein. J. Protozool. 37,546 -552.[Medline]
Sharma, D. K., Hilley, J. D., Bangs, J. D., Coombs, G. H., Mottram, J. C. and Menon, A. K. (2000). Soluble GP18 restores glycosylphosphatidylinositol anchoring in a trypanosome cell-free system depleted of lumenal endoplasmic reticulum proteins. Biochem. J. 351,717 -722.[Medline]
Spurway, T. D., Dalley, J. A., High, S. and Bulleid, N. J.
(2001). Early events in glycosylphosphatidylinositol anchor
addition: substrate proteins associate with the transamidase subunit Gpi8p.
J. Biol. Chem. 276,15975
-15982.
Triggs, V. P. and Bangs, J. D. (1999).10th Molecular Parasitology Meeting, abstract 27 .
Udenfriend, S. and Kodukula, K. (1995a). How glycosylphosphatidylinositol-anchored membrane proteins are made. Annu. Rev. Biochem. 64,563 -591.[Medline]
Udenfriend, S. and Kodukula, K. (1995b).
Prediction of site in nascent precursor of
glycosylphosphatidylinositol protein. Methods Enzymol.
250,571
-582.[Medline]
Vidugiriene, J., Vainauskas, S., Johnson, A. E. and Menon, A.
K. (2001). Endoplasmic reticulum proteins involved in
glycosylphosphatidylinositol-anchor attachment: photocrosslinking studies in a
cell-free system. Eur. J. Biochem.
268,2290
-2300.
von Heijne, G. (1986). A new method for predicting signal sequence cleavage sites. Nucleic Acids Res. 14,4683 -4690.[Abstract]
Walter, P., Gilmore, R. and Blobel, G. (1984). Protein translocation across the endoplasmic reticulum. Cell 38,5 -8.[Medline]
White, I. J., Souabni, A. and Hooper, N. M.
(2000). Comparison of the glycosyl-phosphatidylinositol
cleavage/attachment site between mammalian cells and parasitic protozoa.
J. Cell Sci. 113,721
-727.
Wirtz, E., Hoek, M. and Cross, G. A. M. (1998).
Regulated processive transcription of chromatin by T7 RNA polymerase in
Trypanosoma brucei. Nucleic Acids Res.
26,4626
-4634.
Wirtz, E., Leal, S., Ochatt, C. and Cross, G. A. (1999). A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol. Biochem. Parasitol. 99, 89-101.[Medline]
Yang, H., Russell, D. G., Zheng, B. J., Eiki, M. and Lee, M. G.
S. (2000). Sequence requirements for trafficking of the CRAM
transmembrane protein to the flagellar pocket of African trypanosomes.
Mol. Cell. Biol. 20,5149
-5163.
Zhang, J. X., Braakman, I., Matlack, K. E. S. and Helenius,
A. (1997). Quality control in the secretory pathway: the role
calreticulin, calnexin and BIP in the retention of glycoproteins with
C-terminal truncations. Mol. Biol. Cell
8,1943
-1954.
Ziegelbauer, K., Multhaup, G. and Overath, P.
(1992). Molecular characterization of two invariant surface
glycoproteins specific for the bloodstream stage of Trypanosoma brucei.J. Biol. Chem. 267,10797
-10803.