(Received for publication, May 22, 1995; and in revised form, June 19, 1995 )
From the
Glycosylphosphatidylinositol (GPI) substitution is now recognized to be a ubiquitous method of anchoring a protein to membranes in eukaryotes. The structure of GPI and its biosynthetic pathways are known and the signals in a nascent protein for GPI addition have been elucidated. The enzyme(s) responsible for GPI addition with release of a COOH-terminal signal peptide has been considered to be a transamidase but has yet to be isolated, and evidence that it is a transamidase is indirect. The experiments reported here show that hydrazine and hydroxylamine, in the presence of rough microsomal membranes, catalyze the conversion of the pro form of the engineered protein miniplacental alkaline phosphatase (prominiPLAP) to mature forms from which the COOH-terminal signal peptide has been cleaved, apparently at the same site but without the addition of GPI. The products, presumably the hydrazide or hydroxamate of miniPLAP, have yet to be characterized definitively. However, our demonstration of enzyme-catalyzed cleavage of the signal peptide in the presence of the small nucleophiles, even in the absence of an energy source, is evidence of an activated carbonyl intermediate which is the hallmark of a transamidase.
Proteins that are anchored to membranes via a
glycosylphosphatidylinositol (GPI) ()linkage, although
discovered relatively recently(1, 2) , are now known
to be ubiquitous among eukaryotes where they serve a variety of
important
functions(3, 4, 5, 6, 7, 8, 9) .
Some GPI anchored proteins are known to be required for the survival of
yeast (10) and of human erythrocytes(11, 22) .
Much is already known about the structure and pathway of biosynthesis
of the GPI
moiety(13, 14, 15, 16, 17) .
A fair amount has also been learned about the structural requirements
of a protein in order for it to condense with
GPI(18, 19, 20, 21, 22, 23) .
However, the mechanism of the condensation and the enzyme(s) that
catalyzes it have yet to be elucidated.
A GPI-anchored protein is
coded for as a preproprotein containing both NH- and
COOH-terminal signal peptides (Fig. 1). As the nascent chain is
translocated into the endoplasmic reticulum the NH
-terminal
signal peptide is removed giving rise to the proprotein. Cleavage of
the COOH-terminal signal peptide from the proprotein occurs
concomitantly with the condensation of the ethanolamine group of GPI
with the COOH group of the newly formed COOH-terminal amino acid
(
site). Apparently both processes occur in a single step
catalyzed by what has been assumed to be a transamidase. We have
studied this process using rough microsomal membranes (RM) from various
cells coupled to a translational system with mRNA coding for an
engineered form of human placental alkaline phosphatase (PLAP) as a
template. In the latter, the catalytic domain and all the glycosylation
sites were removed to form a much smaller product (miniPLAP). With this
system, the translation of preprominiPLAP mRNA and the subsequent steps
in its processing to mature GPI-linked miniPLAP can be readily
monitored (Fig. 1) (24, 25) (
)Processing of GPI proteins by a
transamidase would involve a nucleophilic attack by the ethanolamine
group of GPI on an activated carbonyl on the
residue of the pro
form of a protein. In a previous report(26) , we presented
evidence that water, a highly abundant nucleophile, competes with GPI
and gives rise to a mature form of miniPLAP (free miniPLAP) that is
also cleaved at the
site but does not contain the GPI anchor. In
RM from most cells, although GPI-linked miniPLAP is the major product,
it is always accompanied by some free miniPLAP (Fig. 1). In RM
from GPI-deficient cells, free miniPLAP is formed in amounts comparable
to the GPI form(26) . Hydrazine and hydroxylamine are known to
be nucleophilic acceptors in transpeptidase (27) and
transamidase (28) reactions. To obtain additional evidence that
processing to GPI proteins involves a transamidase, we investigated
hydrazine and hydroxylamine as possible substrates in our cell-free
system.
Figure 1:
Schematic presentation of processing of
a proprotein to a GPI protein by RM. The NH-terminal signal
peptide is cleaved once the protein has been introduced into the ER.
Any preproprotein that is isolated with RM is protease sensitive and
does not exist in the context of the membrane. The proprotein
apparently requires energy, the chaperonin BiP and GTP for proper
folding and/or translocation to the site of the transamidase. The
latter presumably combines with the proprotein through the COOH group
of the
site amino acid to form an activated carbonyl group.
Nucleophilic attack by the ethanolamine component of GPI on the
carbonyl group combines GPI with the protein in amide linkage to yield
the GPI-protein with concomitant cleavage of the COOH-terminal signal
peptide. Water is also capable of attacking the
site carbonyl
group to yield the free protein with concomitant release of the
COOH-terminal signal peptide. The inset shows a typical gel
with the products (after immunoprecipitation and SDS-PAGE) obtained by
cotranslational processing of miniPLAP mRNA in the presence of RM from
cells that produce GPI.
We have used two types of experimental conditions to study GPI addition in cell-free systems, cotranslational and post-translational. In the latter the translational elements are separated from the RM after a defined period of translation(33) . We refer to these as preloaded RM. During short periods of incubation, RM from HeLa cells can be preloaded so that they contain an appreciable amount of substrate, prominiPLAP, but little mature GPI-linked or free miniPLAP. The post-translational system was chosen for initial studies because we felt that it provided a more direct study of COOH-terminal processing and were concerned that hydrazine and hydroxylamine might interfere with translation in our cotranslational system. What we were looking for was conversion of prominiPLAP to either the hydrazide or hydroxamate of miniPLAP. Since the latter substituents are fairly small (31 and 32 Da, respectively), the miniPLAP derivatives would be expected to migrate with free mature miniPLAP on SDS-PAGE.
Initial studies were carried out with HeLa RM preloaded for 20 min. Surprisingly, when these were incubated further, the amount of GPI-linked miniPLAP (24.7 kDa) initially present in the preloaded RM was reduced accompanied by an even larger increase in 23-kDa product(s) (data not shown). This unexpected finding could be explained by an exchange in which the GPI anchor was removed from miniPLAP and the corresponding hydrazide or hydroxamate formed. The following experiments were carried out to verify this.
Figure 2: Processing of prominiPLAP in preloaded HeLa RM. The RM were preloaded by incubating them in the presence of miniPLAP mRNA and the translational system for 60 min, following which they were isolated by differential centrifugation. Preloaded RM were then incubated for an additional 30 min then immunoprecipitated and subjected to SDS-PAGE as described under ``Experimental Procedures.'' Heated samples were incubated at 50 °C for 15 min prior to the 30 min incubation. Toppanel, images of the gels in the PhosphorImager. Bottom panel, the amount of each protein species was quantified on the PhosphorImager, and the values on the y axis represent relative density units. Preload indicates the composition at the end of the preloading period with no additional incubation. Control RM were incubated for 30 min in the presence of buffer alone; HDZ represents incubation in the presence of 10 mM hydrazine and HXL, incubation in the presence of 10 mM hydroxylamine. One of two duplicate gels are shown (top panel). The relative densities (bottom panel) represent the average (±10%) of the values obtained in each of the duplicate gels.
Figure 3: Effect of PI-PLC, hydrazine, or hydroxylamine on PLAP. Following treatment with each reagent, aliquots of the reaction mixtures were subjected to partitioning between 0.15 M NaCl and Triton X-114, and PLAP enzyme activity was measured in the aqueous phases. Each sample contained 20 units of PLAP activity. HDZ represents incubation in the presence of 100 mM hydrazine and HXL, incubation in the presence of 100 mM hydroxylamine.
To investigate the effects of hydrazine and hydroxylamine on another GPI protein, VSG, the glycolipoprotein, labeled with tritium in its myristic acid residues, was treated with either of the two nucleophiles or with PI-PLC. It is known that diacylglycerol, produced by treatment of VSG with PI-PLC and free GPI that may be formed, are extractable into butanol, whereas VSG is not(40) . As shown in Fig. 4only 3% of the radioactivity of untreated VSG was extracted. After PI-PLC treatment 97% of the radioactivity was extracted by butanol. In contrast, on treatment of VSG with hydrazine or hydroxylamine the radioactivity extractable by butanol was again only about 3%. As with PLAP, the nucleophilic agents, by themselves, did not cleave the GPI moiety from VSG. It should be noted that concentrations of the nucleophiles at least 10 times higher were used here than in the studies with RM. These findings are further evidence that the conversion of GPI miniPLAP (24.7 kDa) to a 23-kDa moiety in RM in the presence of 10 mM hydrazine or hydroxylamine at neutral pH and 30 °C is not due to chemical cleavage but is enzymatically catalyzed.
Figure 4:
Effect of PI-PLC, hydrazine, and
hydroxylamine on VSG containing [H]myristic acid.
Total radioactivity was 5000 counts/min/sample. Following reaction,
each sample was extracted with n-butanol and an aliquot taken
for measurement of radioactivity. HDZ represents incubation in
the presence of 100 mM hydrazine and HXL, incubation
in the presence of 100 mM hydroxylamine.
Our original goal was to demonstrate an interaction of prominiPLAP with the nucleophilic agents that would yield a product of essentially the same mass as free mature miniPLAP. However, in RM from HeLa cells the relatively large amounts of free miniPLAP formed under preloading conditions (Fig. 2) and the contribution of 23-kDa material produced by the enzyme-catalyzed cleavage of GPI-linked miniPLAP by the nucleophilic agents, presumably the corresponding hydrazide or hydroxamate of miniPLAP, obscured any contribution of 23-kDa product(s) that might arise from a direct interaction of the nucleophiles with prominiPLAP. Nevertheless, we consistently observed 30-40% increases in the total amount of mature miniPLAP species (24.7 kDa plus 23 kDa) in the presence of the nucleophiles, all in the 23-kDa product(s) suggesting that some of the 23-kDa material arose by direct interaction of the nucleophilic agents with prominiPLAP (27 kDa). Cotranslational experiments with RM from various cells were carried out to explore this further.
Cotranslational processing by RM from HeLa cells in
the presence of hydrazine caused a reproducible (30%) increase in
the total amount of the mature forms of miniPLAP, (24.7 and 23 kDa) (Fig. 5, lanes A and B). Again such an
increase (all in the 23 kDa species) is consistent with the formation
of some miniPLAP hydrazide directly from prominiPLAP. We therefore
turned to RM from GPI-deficient Ltk
and M31/25-C1
cells because of their limited ability to produce GPI-linked proteins
while producing large amounts of the precursor prominiPLAP. Such RM
also produce much less free mature miniPLAP for reasons stated
previously (26) . If hydrazine could act as an effective
acceptor for an enzyme activated
carbonyl intermediate, its
addition to the cotranslational system should increase the amount of
23-kDa product(s) processed by the RM. As shown in Fig. 5,
cotranslational processing of miniPLAP mRNA in the presence of
hydrazine resulted in a
200% increase in the total amount of
mature products in Ltk
RM (lanes C and D) and a
110% increase in C1 RM (lanes E and F). Most importantly, in each case, all of the increase was in
the 23-kDa product(s), consistent with the formation of the hydrazide
of miniPLAP.
Figure 5:
Cotranslational processing of mRNAs of Ser and
Gly miniPLAP in the presence
of hydrazine. Following incubation in the presence of 10 mM hydrazine, samples were treated and assayed as described in the
legend to Fig. 2. Both gels were exposed on the same
PhosphorImager screen simultaneously. Because
Ser
miniPLAP is a much better substrate than
Gly miniPLAP,
the PhosphorImager contrast was increased for the latter, hence the
more intense image. This instrumental manipulation does not affect
quantitation. The wide range of densities made it necessary to expand
the scale to present the data more clearly to the reader. Group I represents the full scale between 0-400
10
relative density units; group II expands the range on
scale I from 0 to 100
10
; group III,
expands the scale from 0 to 20
10
. It should be
noted that the relative density of the 23 kDa band in lane L,
shown in group III is about 10
background. As in Fig. 2, one of two duplictae gels is shown (top panel)
and relative densities are the average (±10%) of the duplicates (bottom panel). HDZ represents incubation in the
presence of 10 mM hydrazine.
The studies above were all carried out with Ser miniPLAP mRNA. While prominiPLAP with serine at
the
site is by far the best precursor for GPI
addition(41) , it also yields the largest amount of free mature
miniPLAP, presumably because the putative
carbonyl form of
serine reacts to an inordinate extent with water compared to other
amino acids at that site(26) . To minimize the amount of free
mature miniPLAP formed due to hydrolysis of an intermediate, we
utilized
Gly miniPLAP mRNA. As shown in Fig. 5(lane G), the proportion of prominiPLAP converted
to mature GPI-linked miniPLAP (24.7 kDa) in HeLa RM was markedly
reduced and, as we expected(26) , smaller amounts of the
hydrolysis product (23 kDa) appeared. Addition of hydrazine (lane
H) during cotranslational processing by HeLa RM increased the
amount of 23-kDa product(s) with a small increase in total products
(24.7 and 23 kDa). Again we found it necessary to use RM from the
mutant cells Ltk
and M31/25-C1 to minimize the
production of GPI-linked proteins. The findings clearly indicate that
in the presence of hydrazine, processing to mature products is
increased; 114% for Ltk
RM (Fig. 5, lanes
I and J) and 44% for RM from M31/25-C1 cells (lanes K and L). Furthermore, in each case all the increase was in
a product that migrated with a mass of 23 kDa. This is again consistent
with the formation of the hydrazide of mature miniPLAP concomitant with
cleavage of the signal peptide. Observations similar to the ones with
glycine were also obtained with constructs containing aspartic acid,
alanine, and cysteine at their
sites (data not shown).
Figure 6:
Processing of prominiPLAP in preloaded RM
from GPI-deficient cells. RM from Ltk cells were
preloaded for 20 min. After an additional incubation of 60 min, samples
were treated and assayed as described under the legend to Fig. 2. Data are presented as in the legend to Fig. 2. HDZ represents incubation in the presence of 10 mM hydrazine and HXL, incubation in the presence of 10
mM hydroxylamine.
While the overall biosynthetic pathway of GPI proteins has
been deduced and has been demonstrated in cell-free systems, one of the
key components of the overall reaction, the enzyme(s) that catalyzes
condensation of the ethanolamine of GPI with the COOH at the
site of a proprotein along with the elimination of a signal peptide
remains elusive. In fact, evidence that the condensing enzyme is a
transamidase is still only indirect. Ferguson and Williams (14) concluded that the reaction is so rapid that condensation
and signal peptide elimination must be concomitant reactions as would
be catalyzed by a transamidase. Mayor et al.(42) have
shown that membrane fractions from trypanosomes incorporate GPI into
VSG (i.e. condense the ethanolamine of GPI with the
carboxyl of pro-VSG) in the absence of an energy source. Production of
an amide or a peptide bond requires energy unless it is catalyzed by a
transamidase or transpeptidase. Amthauer et al.(33) showed that while ATP increased conversion of
prominiPLAP to GPI-linked miniPLAP in RM, the energy was required to
release the chaperonin BiP and not for formation of the amide bond of
the GPI protein. More recently, Maxwell et al.(26) demonstrated that during the cell-free processing of
prominiPLAP in RM from GPI-deficient cells, while the major product was
GPI linked, as much as 40% was cleaved at the correct
site, but
with the addition of the elements of water instead of GPI. Evidence was
presented that the latter product arose through a competition of water,
an abundant nucleophile, with the ethanolamine of GPI, for condensation
with an active carbonyl moiety at the
site of prominiPLAP.
Activation of a carboxyl group in the absence of an energy source so
that it can condense with an appropriate nucleophile is the hallmark of
a transamidase.
The present studies with hydrazine and hydroxylamine
give further credence to the transamidase nature of the condensing
enzyme. Nevertheless, these experiments, too, are not as conclusive as
they could be because the amounts of product formed were too small
(10-100 fmol) to permit chemical identification. There is a
colorimetric assay for hydroxamates(43) , but it requires far
greater amounts of material than were formed in these studies. Another
factor that makes clear interpretation difficult is that the reaction
products of the nucleophiles with prominiPLAP, the corresponding
hydrazide or hydroxamate of miniPLAP, are of about the same size as
free miniPLAP, 23 kDa. The latter, which is formed by the reaction of
prominiPLAP with water, always appears as a side product during the
processing of prominiPLAP. It would, therefore, be expected to
comigrate with any hydrazide or hydroxamate of miniPLAP that was
formed. At present we can only surmise that the 23-kDa products formed
in the presence of the nucleophilic agents are the corresponding
hydrazides or hydroxamates. On the positive side, hydrazine, in the
cotranslational system, increased the amount of 23-kDa product(s)
2-3-fold when added to RM from GPI deficient Ltk
and M31/25-C1 cells. These large increases did not occur when the
RM were heated prior to treatment with hydrazine or when incubations
were carried out at 4 °C. In addition, reaction with hydrazine or
hydroxylamine was found to be concentration- and time-dependent. All of
the above are clear indicators of enzyme catalysis. The findings with
preloaded GPI-deficient RM (Fig. 6) are most cogent. They show
that GPI-linked miniPLAP is not an intermediate in the conversion of
prominiPLAP to 23-kDa product(s) in the presence of hydrazine and
hydroxylamine. There remains the remote possibility that for some
reason GPI-linked miniPLAP (24.7 kDa) is formed but does not accumulate
in Ltk
RM as it does in RM of cells that produce
normal amounts of GPI.
The finding that both hydrazine and
hydroxylamine can convert preformed GPI miniPLAP (24.7 kDa) to 23-kDa
product(s) in the presence of RM was at first disturbing. However, this
reaction too is enzyme catalyzed since heated or disaggregated RM were
inactive. Furthermore, hydroxylamine and hydrazine, at concentrations
far higher than were used in the experiments with RM, did not cleave
GPI from two isolated GPI proteins, PLAP and VSG. In addition,
GPI-linked miniPLAP, isolated from HeLa RM that had been detergent
extracted and heated, was not cleaved by the nucleophilic agents (data
not shown). The best explanation for a catalytic conversion of
GPI-miniPLAP to the putative hydrazide or hydroxamate with the
elimination of GPI is the following. In our in vitro system,
at least, the GPI protein remains in contact with the transamidase. As
in prominiPLAP, the carboxyl of GPI-miniPLAP could then be
activated to a carbonyl moiety which in turn could be attacked by
potent or more abundant nucleophiles such as hydrazine or
hydroxylamine. In the process the GPI moiety would be cleaved and the
nucleophile elements added.
There are precedents for such an
enzymatically catalyzed attack of hydrazine or hydroxylamine on the end
product of a transamidation reaction. For example, -glutamyl
transpeptidase (
GT) normally catalyzes the transfer of a glutamate
residue of glutathione to cysteine to form
-glutamyl-cysteine(27) . The latter, in the presence of
GT and hydrazine or hydroxylamine can be converted to the
-glutamyl hydrazide or hydroxamate with the release of cysteine.
In this case, cysteine in the dipeptide may be considered to be the
equivalent of GPI. Another example of a nucleophilic attack of
hydrazine on an enzymatic product of a transpeptidase in the presence
of the enzyme is provided by glutaminase(28) . In accord with
the above, the enzyme-catalyzed exchange of hydrazine or hydroxylamine
for GPI on mature miniPLAP does not require the addition of ATP or GTP
to the preloaded RM and occurs even in the presence of nonhydrolyzable
analogs of ATP or GTP (data not shown).
The nucleophiles
unquestionably cleave the GPI moiety from miniPLAP and apparently form
the corresponding hydrazide or hydroxamate. This raises the question
whether the 23-kDa material that is formed in the presence of hydrazine
or hydroxylamine arises by a direct nucleophilic attack on prominiPLAP
as in Fig. 7(c and d), or in two steps with
GPI miniPLAP as an intermediate (Fig. 7, a and e or a and f). The large increases in 23-kDa
product(s) formed cotranslationally in RM, particularly from
Ltk and M31/25-C1 cells, unquestionably indicate that
the reaction with hydrazine by whichever pathway is enzyme catalyzed.
Since even in the presence of hydrazine some GPI miniPLAP appears, it
is likely that under cotranslational conditions miniPLAP hydrazide (23
kDa) arises via both pathways. However, the findings with preloaded RM
from Ltk
cells clearly show that 23-kDa product(s)
can also be formed directly from prominiPLAP without GPI miniPLAP
appearing as an intermediate (Fig. 7, c and d). Thus, it may be possible to monitor GPI transamidase
activity in the absence of GPI, unless, as has been
suggested(44) , enzymatically active transamidase exists as a
GPI
enzyme complex.
Figure 7: Summary of the processing of prominiPLAP in RM in the presence of different nucleophiles.