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J Biol Chem, Vol. 274, Issue 45, 32153-32158, November 5, 1999
From the Department of Neurology, Hadassah University Hospital,
Jerusalem, 91120, Israel
PrPC, the normal isoform of the
prion component PrPSc, is a 33-35-kDa
glycophosphatidylinositol-anchored glycoprotein expressed in the plasma
membrane of many cells and especially in the brain. The specific role
of PrPC is unknown, although lately it has been shown to
bind copper specifically. We show here that PrPC is present
even in mature sperm cells, a polarized cell that retains only the
minimal components required for DNA delivery, movement, and energy
production. As opposed to PrPC in other cells, PrP in
ejaculated sperm cells was truncated in its C terminus in the vicinity
of residue 200. Sperm PrP, although membrane-bound, was not released by
phosphatidylinositol phospholipase C as well as not localized in
cholesterol-rich microdomains (rafts). Although no infertility was
reported for PrP-ablated mice in normal situations, our results suggest
that sperm cells originating from PrP-ablated mice were significantly
more susceptible to high copper concentrations than sperm from wild
type mice, allocating a protective role for PrP in specific stress
situations related to copper toxicity. Since the functions performed by
proteins in sperm cells are limited, these cells may constitute an
ideal system to elucidate the function of PrPC.
PrPC is the normal isoform of PrPSc, the
protein that is believed to be the major if not the only component of
the prion (1). Prions cause transmissible neurodegenerative diseases
such as scrapie in sheep and bovine spongiform encephalopathy
(BSE)1 in cattle, which may
have transmitted to humans as VCJD (2). Both PrP isoforms share the
same amino acid sequence but, in contrast to PrPC,
PrPSc is relatively resistant to digestion by proteases and
contains a considerable amount of The function of PrPC, a glycophosphatidylinositol (GPI)
anchored glycoprotein, remains elusive albeit the creation and
investigation of several lines of PrP0/0 mice (5, 6). These
mice are immune to infection by prions, and none of them develop
prion-like disease (7, 8). However, each line of PrP0/0
mice displays diverse phenotypes that, although not severe or fatal,
are reminiscent of prion disease symptoms (9, 10). Lately, PrP was
shown to bind specifically to copper (11-13), but the significance of
this finding remains to be established. As most GPI-anchored proteins,
both PrP isoforms are associated with cholesterol-rich membranal
microdomains, denominated rafts (14). In an effort to elucidate the
function of PrPC, we decided to look for its expression in
cells that perform only limited functions, such as sperm cells.
During spermiogenesis, spermatocytes differentiate into sperm in a
process in which the Golgi develops into the large acrosome and
mitochondria migrate from the rest of the cytoplasm to form a tightly
wrapped sheet around the upper part of the flagellum (15, 16). After
the excess cell cytoplasm is pinched off, the sperm cell only carries
with it an acrosome containing enzymes required for the penetration of
the membranes covering the egg, as well as those other cellular
components essential to provide energy for its movement. Sperm cell
become independently motile following an additional maturation step
that occurs during their passage through the epididymis and are
considered fully mature in ejaculates (17). There is no de
novo protein synthesis in mature sperm cells, and therefore
changes occurring in protein patterns are due either to protein
degradation or protein processing during sperm maturation.
In this work we studied the properties of PrP in sperm cells from
several species during different stages of maturation. For practical
reasons, we only obtained sperm ejaculates containing mature sperm
cells from cattle and human, whereas sperm cells isolated from the
epididymis (semi-mature) were available from cattle and rodents. Our
results show that during the process of sperm maturation,
PrPC changes from its regular rafts-associated,
GPI-anchored structure to a unique C-terminal-truncated peptide devoid
of a GPI group and, as a result of that, is not released from the cell
surface by phosphatidylinositol phospholipase C (PIPLC). In addition, mature sperm PrP was not longer associated with membranal rafts. Our
results also suggest that sperm cells from PrP-ablated mice were more
susceptible to copper-induced toxicity than sperm cells from wt mice,
although no difference in the motility or survival could be identified
when comparing these cells at control conditions. Sperm cells may be
the ideal system to investigate whether a protective role against
copper toxicity-induced stress situations can be attributed to
PrPC.
Tissue Samples--
Human ejaculated semen samples were obtained
from the laboratory for human fertility in the Hadassah University
Hospital in Jerusalem. The samples received for our experiments tested
in the fertility laboratory as normal regarding morphology and
motility. Bovine ejaculated semen samples were received from an Israeli company for artificial insemination of cattle.
Hamster, mouse, or bovine epididymal sperm samples were obtained by
mincing epididymis in saline and subsequently collecting floating cells
from the saline suspension as described (18). Samples were looked upon
by light microscopy to ensure that most cells are indeed differentiated
sperm cells. Hamster nondifferentiated spermatocytes were obtained by a
similar procedure, using testis instead of epididymis.
Preparation of Cells for Immunoblotting--
107
N2a-C10 cells (an N2a clone expressing a chimeric mouse/hamster PrP or
sperm cells reacting with Flotation Assay--
Flotation of detergent-insoluble complexes
was performed as described by Naslavsky et al. (19).
Briefly, 100-150 mg of cerebellum or appropriate cells were
homogenized in 700 µl of an ice-cold buffer containing 150 mM NaCl, 25 mM Tris-HCl, pH 7.5, 5 mM EDTA, 1% Triton X-100. Insoluble particles were spun
down for 5 min at 2000 rpm, 4 °C, and the lysate was loaded at the
bottom of Beckman Instruments TLS-55 ultracentrifuge tubes. An equal
volume of ice-cold 70% Nycodenz in TNE (25 mM Tris-HCl, pH
7.5, 150 mM NaCl, 5 mM EDTA) was added and
mixed with the lysate. A 8-35% Nycodenz linear step gradient in TNE
was then overlaid above the lysate. The tubes were spun at 55,000 rpm
for 4 h at 4 °C in a TLS-55 rotor (200,000 × g). Fractions of 180 µl each were collected from the top
to the bottom of the tube and immunoblotted with the appropriate Copper Sensitivity Assay--
FVB or PrP0/0
epididymal sperm cells were resuspended in activation buffer as
described (18). After a 10-min incubation in the activation buffer,
sperm cells were separated into 3 wells, to which 0, 50, or 100 µg/ml
CuCl2 was added. Every 5 min the percentage of motile to
nonmotile cells were established in each of the samples. 100% motile
cells represent the percentage of motile cells at time zero for the
sample without copper.
Fig. 1 shows anti PrP immunoblots of
sperm cells extracts, either mature (human, bovine) or from the
epididymis (hamster, mouse, and bovine), as compared with brain
membranes of the same species. The antibodies used were 3F4 (directed
against residues 108-111) for human and hamsters (20), 6H4
("Prionics," directed against residues 144-152) for bovine
samples, 1E (against residue 200 in humans and mice) (21), and R073
(polyclonal anti-PrP antisera reacting mostly against mouse and hamster
PrP (22)). The molecular weight of PrP in semi-mature (epididymis)
sperm cells was similar to that of the brain, whereas in mature sperm cells obtained from ejaculates, it appears as if the apparent Mr of PrP was reduced to 24,000. The fact that
such a large panel of polyclonal and monoclonal To test whether the reduced molecular weight of PrP in mature sperm, as
compared with epididymal sperm cells and brain samples, is due to a
different N-glycosylation pattern of the PrP protein in
these cells, human and hamster brain and sperm samples were deglycosylated by endoglycosidase F (Fig.
2). The deglycosylated and control
samples were subsequently immunoblotted with mAb 3F4. The full-size
mature PrP peptide (residues 23-231) encloses two N-glycosylation sites at residues 181 and 197 and a GPI
anchor at its C-terminal (23, 24). Endoglycosidase F deglycosylation of
brain PrPC resulted in a major band of an apparent
Mr of about 26,000, whereas faint additional
bands representing either partial deglycosylation or degradation
products of PrPC could also be observed (25, 26).
Deglycosylation of PrP from hamster epididymis sperm cells produced a
major band of the same apparent molecular weight as in brain PrP. These
results show that sperm and brain PrP share a similar polypeptide
backbone and probably also a similar GPI (glycophosphatidylinositol)
anchor, since otherwise their deglycosylation products would not be
identical in molecular weight.
A C-terminal-truncated PrP Isoform Is Present in Mature
Sperm*
,,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheet structures insoluble in
nondenaturing detergents (3, 4). It has been postulated that
PrPSc is produced from PrPC by a conformational
conversion process that is not fully understood.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
PrP mAb 3F4(19)) were extracted in 1 ml of
lysis buffer containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, and 1% Nonident P-40. The
samples were centrifuged at 3000 rpm for 15 min at 4 °C. The
supernatants were concentrated by methanol precipitation.
PrP antibody.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PrP antisera
reacted with the same bands confirms that this is indeed PrP.
View larger version (47K):
[in a new window]
Fig. 1.
PrP is expressed in sperm cells. Brain
(b) and sperm, either epididymal cells (s) or
ejaculated cells (s*) were immunoblotted with diverse PrP antibodies. The specificities of the antibodies are described under
"Experimental Procedures."
View larger version (37K):
[in a new window]
Fig. 2.
Deglycosylation of sperm PrP. Brain and
sperm samples either epididymal (s) or from ejaculates
samples (s*) were digested in the presence or absence of
endoglycosidase F as described (25). Samples were subsequently
immunoblotted with mAb 3F4.
As opposed to the results with brain and with semi-mature sperm cells,
the apparent molecular weight of deglycosylated PrP in mature human
sperm from ejaculates, as detected by immunoblotting with mAb 3F4, was
of about 17,000. These results raise several interesting points. 1) The
lower molecular weight of PrP in untreated mature sperm (24,000) could
not be only a result of PrP deglycosylation occurring during sperm
maturation, since there is a further reduction in
Mr of sperm PrP following endoglycosidase F
treatment. 2) PrP mAb 3F4 reacts with a site composed of residues
108-111 in the human or hamster PrP sequence; therefore it is unlikely
that a 17-kDa peptide that still reacts with this antibody can be
produced by proteolysis of only the N-terminal part of PrP. Indeed,
during the normal degradation pathway of PrPC, a 17-kDa
peptide, which does not react with mAb 3F4, is produced (14, 27). These
results suggest the possibility that PrP in mature sperm is truncated
in its C terminus.
To further delineate the site of such a putative C-terminal trimming,
human mature sperm samples were immunoblotted with an antiserum raised
against a synthetic peptide comprising residues 195-213 of the human
and mouse PrP sequence (1E) and that has been shown to preferentially
recognize the glutamate residue at position 200 (21). As can be seen in
Fig. 3, although human brain PrP reacted
readily with both the 1E antisera and the mAb 3F4, sperm PrP reacted
only with mAb 3F4. That the 1E antisera can react in principle with
sperm PrP can be seen from the fact that PrP from mouse epididymis
samples (semi-mature sperm cells) did react with this antiserum (Fig.
1). These results show that mature sperm carries a C-terminal-truncated
PrP isoform, unlike the previously characterized PrPC
degradation products, that are truncated in the N-terminal part of the
protein. Since bovine mature sperm PrP reacted with PrP mAb 6H4
that is directed against residues 144-152 and, we must assume from the
endoglycosidase F digestion results that at least one if not both
mature sperm PrP N-glycosylation sites are occupied, it is conceivable
that mature sperm PrP is cleaved between residues 181 to 200. The
molecular weight of the deglycosylated peptide, which is probably
devoided of the GPI anchor attached to the PrP C-terminal, is
consistent with such a scenario. To explore whether and how the
C-truncated PrP in mature sperm cells is attached to the cell membrane,
we tested 1) whether sperm PrP could be released by PIPLC; 2) whether
it is located in rafts membranes; 3) or whether it is attached to
membranes at all.
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In brain samples as well as in cultured cells, PrPC is
linked to the cell membrane through a GPI group attached to its C
terminus (28). As other GPI-anchored proteins, PrPC can be
released from cell membranes by the enzyme PIPLC (29). After cleavage
of the lipid group, the remaining protein is converted into a soluble
peptide. We therefore tested whether PIPLC can release PrP from sperm
cells. Most of the PrP is released by PIPLC from either N2a C10 cells
as well as from early, predifferentiated spermatocytes (from the
testis). This was not the case for PrP from sperm cells, although, at
least in semi mature sperm cells, the molecular weight of PrP suggests
the presence of the GPI group on PrP (Fig.
4). These results suggest either (i) that
the GPI anchor became inaccessible to the enzyme, (ii) that the GPI
anchor has been chemically modified to resist PIPLC hydrolysis, or
(iii) that PrP, in addition to its GPI-membrane anchoring, is attached to the membrane by an additional mechanism. The fact that PrP in testis
spermatocytes is releasable by PIPLC but that this property is lost
during cell maturation suggests the appearance of an additional binding
site for PrP in the membrane during cell processing. This conclusion
was reinforced by the presence of the C-terminal-truncated PrP isoform
in ejaculation samples, since this isoform is probably devoid
altogether of the GPI anchor and, therefore, must be attached to the
sperm outer membrane via a different mechanism. Interestingly, it has
been shown lately that also PrP in platelets, another example of cells
that do not synthesize new proteins and perform only specific
functions, cannot be released from the cell membrane by PIPLC
(30). As opposed to PrP, other GPI-anchored proteins are normally
processed in sperm cells, and some of them are known to play a
crucial role in cell maturation (31, 32).
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Most GPI-anchored proteins have been shown to be targeted to special cholesterol-rich membranal microdomains denominated rafts (33). Rafts are insoluble in cold Triton X-100, and as such, proteins inserted in them can be separated from other membrane proteins as well as from soluble proteins by an assay in which the insoluble rafts will float to the top of density gradients and the soluble or detergent-solubilized proteins will remain in the lower fractions of these gradients (19). PrP is also a raft protein. In fact, since both prion proteins (PrPC and PrPSc) are colocalized in those membranal microdomains, it has been speculated that the conversion of PrPC to PrPSc occurs in these rafts (19, 34). Transfection of genetically engineered transmembrane PrPC into ScN2a cells (that are not targeted to rafts) has shown that the membrane localization of PrPC is essential for the conversion process, since transmembrane PrPC did not convert into PrPSc (14, 35).
When brain membranes, early spermatocytes (from testis), and
semi-mature sperm cells (from epididymis) were subjected to a flotation
assay, PrP in sperm, as opposed to PrP from spermatocytes and brain,
could not be found in the buoyant fractions where rafts migrate (Fig.
5). It is possible that rafts are
altogether absent from mature sperm cells as a result of changes in
lipid membrane composition, which are known to occur during the
different stages of sperm maturation (36, 37). This speculation is
reinforced by the fact that ESA, a transmembrane raft protein (38),
also changed its membrane association during sperm maturation. It is, however, still conceivable that this is a unique effect for a small
group of sperm proteins that includes PrP.
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To investigate whether PrP is exposed to the outer plasma membrane, we
digested intact semi-mature hamster and mouse (full-length PrP) as well
as human cells (C-terminal-truncated PrP) with a low concentration of
proteinase K and compared the digestion pattern with sperm cells
digested in the presence of detergents (not shown). The intact cell
samples digested with proteinase K were immunoblotted with the
appropriate anti-PrP antibodies. Sperm PrP was completely accessible to
low proteinase K concentrations in all the species studied (Fig.
6A), as can be seen from the
fact that after proteinase K digestion, no PrP could be detected with
diverse antibodies. The association of sperm PrP with the cell membrane
is probably extremely strong, since it is resistant to incubation with
100 mM Na2CO3, a reagent that is
known to dismantle protein-protein interactions and that has been used
operationally to define integral membrane proteins (Fig.
6B).
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During prion disease, most of the conversion of PrPC to
PrPSc, the prion component, occurs in the brain of the
infected animal and subsequently causes neurodegeneration. The question
of whether sperm PrP can be converted into PrPSc may have
important implications for the possible transmission routes of prion
diseases. However, as can be seen in Fig.
7, PrP in sperm cells of scrapie-infected
hamsters is not more resistant to protease digestion than PrP from
normal sperm. These results are consistent with the general notion that
prion diseases are not transmitted sexually. Interestingly, it has been
shown that male mice inoculated with prions loose their fertility
before clinical signs of the disease are noticeable (39).
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PrP has been shown lately to bind copper specifically. Although a
specific role in copper metabolism as not yet been attributed to PrP,
it has been shown that cerebellar cells from PrP-ablated mice are more
susceptible to copper toxicity and oxidative stress than cerebellar
cells from control mice (40). Interestingly, sperm cells are notorious
for their sensitivity to high copper concentrations, as can be
appreciated from the fact that the intrauterine device contraception
principle is based on copper toxicity (41). We therefore tested the
motility of wt FVB mice sperm cells as compared with the motility of
sperm cells of PrP-ablated mice in the presence and absence of copper
(PrP0/0 mice contain 94% of the FVB genotype) (5).
Motility of sperm cells is considered as a good criteria for cell
viability (18, 41, 42). Epididymal sperm cells from wt or
PrP0/0 mice were placed in a buffer described previously
(18). At time point zero, CuCl2 was added at either 50 or
100 µg/ml to the appropriate wt or PrP0/0 sperm samples,
and subsequently the motile sperm cells were counted every 5 min. At
both copper concentrations, sperm cells from PrP0/0 mice
lost their motility faster than sperm cells from wt mice; however,
since 100 µg/ml CuCl2 was very toxic to both types of sperm cells, the difference between 50% survival was only 5 min between wt and PrP0/0 cells. At 50 µg/ml
CuCl22 the different in survival between both types of
sperm cells was significantly larger (Fig. 9). Although no motile cells
remained in the ablated samples after 30 min of incubation with 50 µg/ml CuCl2, about 50% control cells were still motile
at that time point. These results were extremely reproducible. Fig.
8 represents the average of four similar
experiments. In each experiment, sperm samples from 3-5 animals of
each kind (wt or PrP0/0) were tested individually in
triplicates. The results were not dependent of the animals ages, since
copper sensitivity was higher for PrP-ablated sperm that for wt sperm
in animals 6, 12, or 18 month old (not shown).
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The finding of substantial amounts of PrP in sperm cells provides us with a new tool to be used in the quest for the elusive function of this interesting protein. As stated above, mature sperm cells retain only the minimal components required for their unique function.
In addition, the presence of this unusual PrP isoform in sperm cells (for summary see Table I) may help us elucidate both the sites in the PrP sequence that are essential for the normal function of PrPC as well as to the molecules or cofactors required to accomplish such function. These sites in the PrP sequence seem to be different from the ones fundamental for the conversion of PrPC into PrPSc, since that process is independent of the presence of the N-terminal PrP sequence but does require a GPI anchor and a raft membrane location for both prion isoforms participating in the conversion process (14, 43). As we have shown here, this is not the case for sperm PrP, which is devoid both of the PrP C terminus containing the GPI anchor and of its raft location, but even so, is probably as active as PrPC. The mechanism by which PrPC is truncated during the sperm maturation process and thereafter remains attached to the sperm plasma membrane remains to be established.
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PrPC has been shown lately to bind copper specifically (12, 13, 44, 45). The binding site for copper is believed to be in the octa- repeats present in the N-terminal section of PrP (44), which are probably intact even in mature sperm PrP. This finding suggests a possible role for PrP in copper metabolism. Copper ions serve as cofactors of many enzymes and, in particular, of energy-associated systems (46, 47). In addition, copper has also been shown to be toxic when present in excess, and therefore, fine balance of copper concentration is required for good health (41, 48, 49). The putative role of PrP in copper metabolism can vary from a copper transporter to a suppressor of copper-induced cell damage.
The possibility that PrP protects against copper damage was recently suggested from experiments showing that brain cells from PrP knock-out mice were more susceptible to copper toxicity than brains cells from wt mice (50). The results of the experiments described in Fig. 8 are also consistent with such a protective role for PrP against copper induced damage. Although no difference in motility was observed between wt mice sperm and PrP knock-out mice sperm at control conditions, the addition of copper was remarkable more toxic to PrP knock-out mice sperm cells than to wt sperm cells. Interestingly, a group of markers present in CSF and serum during prion disease (S100 proteins) (51) have been shown to perform as copper-binding proteins and thereby protect against copper-induced cellular damage (52), suggesting a possible compensatory effect, which may be required during the last stages of prion infection.
A putative role for PrP as a protector against specific stress situations is also consistent with the fact that the absence of PrP in ablated mice did not cause any mayor apparent damage (5). If indeed the function of PrP is to protect cells during a specific stress situation such as a toxic copper concentration, no apparent damage should occur in the absence of such an insult.
The new PrP isoform described in this manuscript may play an important
role in the identification of PrPC metabolism and function.
The relative simplicity of sperm cells, as compared with nerve cells,
may shorten the pathway leading to the complete elucidation of
PrPC function. Once a reliable assay is developed for the
function of PrPC, it will be possible to establish whether
the loss of such function plays any role in the pathogenesis of prion diseases.
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FOOTNOTES |
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* This work was supported by grants from the Israeli Academy of Science and the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
These authors contributed equally to this work.
§ To whom correspondence should be addressed. Fax: 972-2-6429441; E-mail: gabizonr@hadassah.org.il.
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ABBREVIATIONS |
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The abbreviations used are: BSE, bovine spongiform encephalopathy; GPI, glycophosphatidylinositol; PIPLC, phosphatidylinositol phospholipase C; wt, wild type; mAb, monoclonal antibody.
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REFERENCES |
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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, FVB mouse (with 50 µg/ml CuCl2); 
, FVB
mouse (no CuCl2); 
, PrP0/0 mouse (with 50 µg/ml CuCl2); 
, PrP0/0 mouse (with 50 µg/ml CuCl2). The results represent the average of four
experiments. In each experiment, sperm was obtained from a group of
3-5 mice of each kind. Sperm cells from each mouse were tested for
copper sensitivity individually.