These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb–V–IX complex.. To address these questi
Trang 1Biosynthesis of platelet glycoprotein V expressed as a single subunit
or in association with GPIb-IX
Catherine Strassel, Sylvie Moog, Marie-Jeanne Baas, Jean-Pierre Cazenave and Franc¸ois Lanza
INSERM U.311, Etablissement Franc¸ais du Sang-Alsace, Strasbourg, France
Glycoprotein (GP) V is noncovalently linked to GPIba,
GPIbb and GPIX within the platelet GPIb–V–IX complex,
a receptor for von Willebrand factor and thrombin Two
functions have been ascribed to GPV, namely, the
modula-tion of thrombin- and collagen-dependent platelet responses
The biosynthesis of this molecule was investigated in pulse–
chase metabolic labelling experiments performed in CHO
cell lines transfected with GPV, alone or in the presence of
GPIb–IX GPV could not be detected at the surface of cells
expressing the single subunit but was found instead as a
soluble form in the culture medium In pulse–chase studies,
an immature 70 kDa protein was detected in cell lysates,
whereas a fully processed 80–82 kDa form was only
observed in the culture supernatants at later chase times
Immature GPV was N-glycosylated and retained before the
medial Golgi while the secreted molecule contained complex
sialylated sugars The mature soluble form of GPV was produced by an enzymatic cleavage which was not affected
by inhibitors of proteasome, calpain or metalloproteinases When GPV was cotransfected with GPIb–IX, the former was no longer found in the culture supernatant but was retained in the cell membrane as shown by fluorescence-activated cell sorting and confocal microscopy analyses Surface expressed GPV was processed from an immature
70 kDa form to produce a mature 80 kDa protein, pro-cessing similar to the intracellular trafficking of GPIba These results indicate that correct biosynthesis and surface expression of GPV in platelets requires the presence of the other subunits of the GPIb–V–IX complex
Keywords: biosynthesis; CHO; glycoprotein; glycosylation; platelet
The platelet GPIb–V–IX complex plays an essential role in
the formation of the haemostatic plug following vessel wall
injury [1] This cell surface receptor is required above all
under the conditions of high shear stress encountered
in arteries and microvessels, where it mediates reversible
platelet adhesion by binding to von Willebrand factor
(vWF) exposed on the damaged vessel wall [2] The
functional importance of the GPIb–V–IX complex is
highlighted by the existence of the Bernard–Soulier bleeding
syndrome, in which the complex is present in strongly
decreased amounts or in more rare cases is not functional
GPIb–V–IX is composed of four different type I
glycopro-teins: GPIba, GPIbb, GPIX and GPV, all belonging to the
leucine-rich repeat family [3] GPIba (135 kDa) is
disul-phide-linked to GPIbb (25 kDa) and noncovalently
com-plexed with GPIX (20 kDa) and GPV (82 kDa) in a
2 : 2 : 2 : 1 stoichiometry [4,5]
Several functions have been proposed for glycoprotein
(GP) V It could act as a negative regulator of thrombin
activation [6], or as an accessory receptor for collagen
dependent platelet adhesion and activation [7] Although it
has not yet been demonstrated, GPV could potentially play
a role in platelet signalling during adhesion to vWF Thus, 14–3-3f [8,9] and the calcium dependent regulator calmo-dulin [10] have been shown to bind to the cytoplasmic domain of GPV A distinctive feature of this molecule is its extreme sensitivity to cleavage by proteases After throm-bin-induced platelet activation, cleavage of GPV at a specific site releases a soluble 69 kDa fragment (f1) [11], the physiological significance of which is still unknown
In addition, GPV can be cleaved by elastase to release a
75 kDa fragment and by calpain, at a site near the cell membrane, to generate an 82 kDa fragment [12] Despite its association with the other subunits of the GPIb–V–IX complex, GPV is more loosely attached [4] and does not appear to be essential for the normal expression of these subunits GPIb–IX is expressed efficiently in transfected cells in the absence of GPV and in the platelets of GPV knock-out mice [13–15] This is consistent with the obser-vation that all the reported Bernard–Soulier defects are restricted to the GPIba, GPIbb and GPIX subunits [16] Nevertheless, some studies in transfected cells have indicated that GPV could enhance levels of expression of the complex
at the platelet surface [17]
The available biosynthetic studies of receptors restricted
to platelets (GPIb, GPIIbIIIa) have essentially relied on their expression in heterologous cells Approaches of this type have improved our understanding of the requirements for normal biosynthesis of the GPIb–IX complex and of the consequences of mutations encountered in Bernard–Soulier patients [18] These studies were, however, performed mostly
in the absence GPV On the contrary, the biosynthesis of
Correspondence toF Lanza, INSERM U.311, Etablissement Franc¸ais
du Sang-Alsace, 10 rue Spielmann, BP 36, 67065 Strasbourg Cedex,
France Fax: +33 388 21 25 21, Tel.: +33 388 21 25 25,
E-mail: francois.lanza@efs-alsace.fr
Abbreviations: Endo-H, endoglycosidase-H; FACS,
fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; GP,
glycoprotein; SV40, simian virus 40; vWF, von Willebrand factor.
(Received 20 April 2004, revised 21 June 2004, accepted 26 July 2004)
Trang 2GPV has never been thoroughly investigated A better
knowledge of the processing of GPV is likewise necessary to
obtain greater insight into its structural and functional role
in platelets To address these questions, we have developed
two heterologous expression cell systems where GPV was
transfected in the presence or absence of the other three
subunits of the GPIb–V–IX complex, allowing study of its
biosynthesis and cellular distribution
Experimental procedures
Materials and cell lines
The mAbs V.1 and V.5 against GPV, ALMA.12 against
GPIba, ALMA.16 against GPIX and RAM.1 against
GPIbb, were developed in our laboratory [19] CHO cell
lines CHO-K1 and CHO-DUK (deficient in dihydrofolate
reductase) were purchased from ATCC (Rockville, MD,
USA) The CHO/GPIb–V–IX cell line was derived from a
clone expressing GPIbb and GPIX (gift from J A Lopez,
Baylor College of Medicine, Houston, TX, USA) by
transfection with cDNAs coding for GPIba inserted in the
pDX vector [20] and GPV inserted in the pZeoSV vector
(Invitrogen, San Diego, CA, USA) and selection in the
presence of zeocin These cells were grown in aMEM (Gibco
BRL, Cergy-Pontoise, France) supplemented with 10%(v/v)
fetal bovine serum, 200 lgÆmL)1zeocin and 400 lgÆmL)1
G418 (Boehringer–Mannheim, Germany) The CHO/GPV
cell line was obtained by transfection of CHO-DUK cells
with GPV cDNA inserted in the pTG2328 vector, selection in
the absence of nucleosides and amplification of GPV
expression by growing cells in the presence of step increased
concentrations of methotrexate [21] This clone was
main-tained in suspension in serum free CHO-S-SFMII medium
(Gibco BRL) supplemented with 1.2 lgÆmL)1methotrexate
(Calbiochem Novabiochem, La Jolla, CA, USA)
Expres-sion in pZeoSV and pTG2328 is driven by the simian virus 40
(SV40) early enhancer/promoter
Flow cytometry
CHO cells (2· 105in 100 lL) were incubated for 30 min at
4C with purified IgG (10 lgÆmL)1) in fluorescence
microfluorimetry buffer [RPMI medium, 5% (v/v) normal
goat serum, 0.2% (v/v) sodium azide] After centrifugation
at 270 g, the cells were resuspended in buffer containing a
100-fold dilution of fluorescein
isothiocyanate-(FITC)-con-jugated goat anti-(rat IgG) F(ab¢)2 or FITC-conjugated
goat anti-(mouse IgG) IgG (Jackson Immunoresearch,
West Baltimore, PA, USA) for 30 min at 4C Analyses
were performed on 10 000 cells in a FACS Calibur flow
cytometer (BD Biosciences, Rungis, France)
35
S metabolic labelling
CHO cells (2· 108ÆmL)1) were incubated twice in 50 mL of
cysteine- and methionine-free RPMI medium (ICN, Costa
Mesa, CA, USA) without serum for 30 min at 37C The
cells were then pulse-labelled for 15 min at 37C
in the same medium containing 1% (v/v)
penicillin–strep-tomycin–glutamine (Gibco), 20% (v/v) dialysed fetal bovine
serum (Biowest, Nuaille, France) and a mixture of
[35S]methionine and [35S]cysteine (Amersham Pharmacia Biotech, Uppsala, Sweden) The labelled cells were diluted
in 10 volumes of ice-cold NaCl/Pi, washed once and chased for different times at 37C At each chase time, cell samples were washed twice in ice-cold NaCl/Piand lysed in buffer I [20 mMTris (pH 7.5), 150 mMNaCl, 5 mMEDTA, 0.2% (w/v) BSA, 1% (v/v) Triton X-100, 1· complete protease inhibitor cocktail, 2 lgÆmL)1 calpain inhibitor (Roche Diagnostics GmbH, Mannheim, Germany)]
Immunoprecipitation and SDS/PAGE Lysates corresponding to 3· 107 cells from suspensions containing 5· 106 cellsÆmL)1 and 2 mL culture superna-tants corresponding to 107cells were clarified by incubating them twice for 1 h at 4C with 100 lL of protein G-Sepharose (Sigma, Saint Louis, MO, USA) The samples were then incubated overnight at 4C with 10 lg of the relevant mAbs and 100 lL of fresh protein G-Sepharose After centrifugation, the bead pellets were washed once in buffer I, twice in buffer II [0.5% (v/v) Triton X-100, 20 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.2% (w/v) BSA, 0.1% (w/v) SDS], three times in buffer III [0.5% (v/v) Triton X-100, 20 mMTris (pH 7.5), 500 mMNaCl, 5 mM EDTA, 0.2% (w/v) BSA] and twice in buffer IV [50 mM Tris (pH 7.5)] The beads were resuspended in Laemmli buffer containing 10 mM dithiothreitol and heated for
5 min at 95C Proteins were separated by 7.5–15% gradient SDS/PAGE and the gels were fixed and processed for autoradiography
Carbohydrate analyses After the last washing step in buffer IV, the immunopre-cipitates were dissociated from the beads in 30 lL of 50 mM Tris (pH 7.5) containing 1% (w/v) SDS and 5 mM dithio-threitol and boiled for 5 min at 95C Sodium citrate (pH 5.5) was added to a final concentration of 0.1M The samples were then incubated with endoglycosidase-H (5 mU per sample) for 6 h at 37C in the presence of 1.7 lgÆmL)1 calpain inhibitor and 1· protease inhibitor cocktail In neuraminidase treatment, the beads were resuspended directly in 20 lL of 100 mM sodium acetate (pH 5) containing 1 mMCaCl2and 150 mMNaCl, before addition of 10 lL of neuraminidase (100 mUÆmL)1) (Roche) and incubation for 6 h at 37C
Western blotting Supernatants from CHO/GPV cell cultures were treated or not with thrombin (5 UÆmL)1) (Sigma) for 5 min at 37C, before separation by 4.5%)15% SDS/PAGE and immu-noblotting The blots were probed with the mAb V.5 followed by goat anti-(mouse IgG)–horseradish and protein bands were revealed by ECL (Amersham Biosciences, UK) Confocal microscopy
CHO cells seeded at 20.104cellsÆmL)1 in NaCl/Pi were allowed to adhere to polyL-lysine-coated coverslips in four-well plates for 2 h at 37C After washing, the cells were fixed with 3% (w/v) paraformaldehyde and permeabilized
Trang 3with 0.05% (w/v) saponin in blocking solution [NaCl/Pi
containing 0.2% (w/v) BSA and 1% (v/v) goat serum]
CHO/GPIb–V–IX cells were incubated for 45 min at room
temperature with the mAb V.1 in blocking solution, washed
and incubated with a GaM–Cy5 secondary antibody
(Jackson Immunoresearch) for 30 min These cells were
then incubated for a further 30 min with a 1 : 400 dilution
of a third antibody (ALMA.12 or ALMA.16) directly
coupled to Alexa-488 (Molecular Probes, Eugene, OR,
USA) CHO/GPV cells were incubated with V.1 coupled
directly to Cy3 for 45 min at room temperature The
coverslips were washed in NaCl/Pi, rinsed in water and
mounted in Mowiol 4-88 (Calbiochem Novabiochem) and
the labelled cells were examined under a Zeiss laser scanning
microscope (LSM 410 invert) equipped with a Planapo oil
immersion lens
Results
Transfection of GPV in the absence of GPIb–IX results
in its inefficient cell surface exposure and release
of a soluble form of GPV
The CHO/GPV cell line transfected with full length GPV
lacked surface expression of GPV as measured by flow
cytometry (Fig 1A), despite selection of a strongly
expressing clone by a series of amplifications in the
presence of increasing concentrations of methotrexate
Analysis of permeabilized cells by confocal microscopy revealed an intracellular pool of GPV with a granular appearance (Fig 1B) Analysis of culture supernatants by GPV ELISA showed the presence of high levels of soluble GPV [21], which was identified as a single 82 kDa band by Western blotting with the mAb V.5 (Fig 1C) The identity
of this band was further established by its cleavage to a
69 kDa species following thrombin treatment These properties of soluble recombinant GPV are identical to those reported for a calpain-derived fragment of platelet GPV [12]
Singly transfected GPV is retained intracellularly as an N-mannose rich 70 kDa form and is released into the cell supernatant as a sialylated 82 kDa polypeptide
Pulse–chase and immunoprecipitation experiments were performed on CHO/GPV cell lysates and culture super-natants (Fig 2A) A broad 70 kDa band corresponding to immature GPV was present in the cells at early time points and throughout 180 min of chase, while shorter chase times revealed that this band was composed of three or four different molecular mass forms Parallel analysis of the supernatants revealed a positive signal starting at 60 min of chase and having a molecular mass (82 kDa) consistent with
a fully mature form This band was not observed in the cell lysates at any chase time and conversely the 70 kDa band was absent from the supernatants
Fig 1 Distribution of GPV in CHO/GPV cells in the absence of GPIb–IX (A) GPV expression on the surface of platelets and CHO/GPV cells was analysed by flow cytometry using the mAb V.1 without permeabilization of the cells A positive signal was observed on platelets but not on CHO/ GPV cells (B) The subcellular distribution of GPV was examined in permeabilized CHO/GPV cells by confocal microscopy using Cy3-coupled V.1.
An intracellular labelling was observed with a granular appearance (C) Soluble GPV in the supernatants of CHO/GPV cells was analysed by Western blotting with the mAb V.5 An 82 kDa band was detected in untreated samples, which was converted into a 69 kDa band by treatment with 5 UÆmL)1thrombin.
Trang 4Post-translational sugar modifications of the cellular and
soluble forms of GPV were studied by treatment with
endoglycosidase-H (Endo-H) (Fig 3A) or neuraminidase
(Fig 3B) The 70 kDa form was sensitive to Endo-H
treatment, which reduced it to a narrow 50 kDa band, but
was not cleaved by neuraminidase This indicated its
enrichment in N-mannose sugars added before the medial
Golgi compartment On the other hand, the 82 kDa soluble
molecule was resistant to Endo-H and cleaved by
neura-minidase to a 72 kDa form, indicating the presence of
terminal sialic acid residues added in the trans-Golgi These
experiments show that the secreted 82 kDa form of GPV is
fully processed during transit through the different Golgi
compartments but is not retained in the plasma membrane
In the presence of the GPIb–IX complex, GPV is fully
processed and targeted mainly to the cell surface
Contrary to the singly transfected subunit, GPV
cotrans-fected with GPIb–IX was efficiently expressed at the cell
surface together with the other subunits of the GPIb–V–IX
complex, as demonstrated by flow cytometry (Fig 4A)
Double-labelling confocal microscopy with antibodies
against GPV and GPIba or GPIX revealed colocalization
of these subunits with GPV primarily at the cell membrane
Hardly any labelling could be detected intracellularly, unlike
in CHO/GPV cells (Fig 4B) These results imply that
GPIb–IX is required for stable surface expression of GPV in
transfected cells Very comparable results were obtained
when GPV was cotransfected with GPIb–IX into leukaemic
human K562 cells This cell line was chosen for biosynthetic
studies in order to analyse the processing of GPV in a cell
system more closely resembling platelets Pulse–chase
experiments performed on cell extracts alone showed
immunoprecipitation at early times of the immature
70 kDa form of GPV, which progressively matured to a
cell associated 82 kDa protein Concomitantly, GPIba
progressed from an immature 85 kDa form to a mature
125 kDa molecule GPIbb and GPIX displayed more modest mass increases of 1–2 kDa and gradually reached their mature sizes of 25 and 20 kDa, respectively, over
30 min of chase This maturation time-course is very similar
to that reported previously for CHO/GPIb–IX cells [18,22] (Fig 4C) Analysis of the supernatants revealed no secreted forms of GPV (data not shown), while the mature GPV in K562/GPIb–V–IX cells was sensitive to neuraminidase treatment but resistant to Endo-H (data not shown)
Discussion
Using stably transfected cell lines, we examined the biosyn-thesis of platelet GPV, a subunit of the GPIb–V–IX
Fig 2 Biosynthesis of GPV in CHO/GPV cells in the absence of
GPIb–IX CHO/GPV cells were pulse-labelled with [35S]Cys and
[35S]Met and chased for various periods of time At different chase
times, the culture supernatants were collected, the cells were lysed in
Triton X-100 buffer and the cell lysates and supernatants were
ana-lysed by immunoprecipitation with the mAb V.1 At T 0 , the cells
contained an immature 70 kDa form of GPV which did not appear to
progress to a higher molecular mass form during the 180 min of chase.
On the other hand, an 80 kDa mature form progressively appeared
and accumulated in the supernatants at chase times of 60–120 min.
Because at T 0 only the 70 kDa form is present it must be the
prede-cessor of the 80 kDa soluble form The 70 kDa form therefore matures
but probably becomes quickly secreted and does not accumulate in
sufficient amounts to allow detection in the cell lysate.
Fig 3 Sugar processing of cell associated and secreted forms of GPV in CHO/GPV cells Cells were pulse-labelled, chased and immunopre-cipitated as described in Fig 2 and the immunoprecipitates from the cell lysates or supernatants were analysed for Endo-H (A) or neura-minidase (B) sensitivity (A) The band at 70 kDa corresponding to
immature cell associated GPV was converted into a 50 kDa band (the expected size of deglycosylated GPV) by Endo-H treatment In con-trast, the 80 kDa secreted molecule was insensitive to Endo-H (B) The cell associated 70 kDa form of GPV was resistant to neuraminidase, whereas the soluble 80 kDa form was reduced to 70 kDa through loss
of terminal sialic acid residues.
Trang 5complex, and the influence on its processing and
expres-sion of the presence or absence of the other subunits of
the complex In both cases, GPV was processed from
an immature mannose-rich intracellular 70 kDa form to
a mature sialic acid-rich 82 kDa species Mature GPV
was expressed efficiently at the cell surface in the presence
of GPIb–IX, whereas singly transfected GPV was secreted
as a soluble molecule, presumably following enzymatic
cleavage
A lack of surface expression of GPV after single-chain
transfection was unexpected in view of the presence of a
transmembrane region in the construct encoding the entire
protein and from previous reports of its surface expression
in human melanoma cells and mouse L-cells stably
trans-fected with GPV alone [17] Cell membrane expression of
GPV has also been observed in CHO cells in transient
expression experiments [14] In our studies, using CHO cells
or a human K562 leukaemic cell line, low levels of GPV were detected at the cell membrane 48–72 h after transfec-tion but we were never able to obtain a stable surface expression Lack of expression in CHO/GPV cells is probably not related to differences in expression vectors compared to CHO/GPIb–V–IX, as both vectors contain the same SV40 promoter This was also not due to inefficient biosynthesis of GPV as CHO/GPV cells were submitted to extensive gene amplification with methotrexate resulting in a 80-fold increase in concentrations of soluble GPV Ampli-fication of GPV expression probably explains part of the intracellular accumulation observed in CHO/GPV cell lines
in confocal microscopy and metabolic labelling experi-ments Although differences with respect to a few studies could be related to the cell types used, in general the single subunit is not (or only very weakly) retained at the cell surface Consistent with this hypothesis, significant levels of
Fig 4 Surface expression and intracellular processing of GPV in CHO and K562 cells cotransfected with GPV and GPIb-IX (A) CHO cells stably transfected with GPV, GPIba, GPIbß and GPIX were analysed for surface expression of GPV by flow cytometry A positive GPV signal was observed with approximately half the intensity of those of the other subunits (B) CHO/GPIb–V–IX cells adherent to poly L -lysine were permeabilized, double-labelled with mAbs against GPV (pink) and GPIba (green) (left panel) or GPV (pink) and GPIX (green) (right panel) and analysed by confocal microscopy Co-localization at the cell membrane (white) was observed under both conditions (C) K562/GPIb–V–IX cells were pulsed-labelled with
35 S and chased as described in Fig 2 At different chase times, the cells were lysed in 1% (v/v) Triton in buffer I and the GPIb–IX complex was immunoprecipitated with ALMA.12 and GPV with V.1 At T 0 , the cells contained an immature 70 kDa form of GPV which progressed within 60 min
to an 80 kDa mature protein GPIba progressively evolved from an early immature 85 kDa form to a mature 125 kDa molecule and this process, first detected at 15 min, was completed within about 30 min The molecular masses of GPIbß and GPIX increased slowly to reach 25 and 20 kDa, respectively.
Trang 6soluble GPV were found in the culture supernatants of GPV
transfected melanoma cells [17] A similar phenomenon has
been reported for the GPIba subunit by Meyer et al [23],
who using methotrexate amplification in CHO cells found
inefficient membrane insertion of GPIba transfected as a
single-chain and secretion of a glycocalicin-like soluble
form
The exact mechanism leading to release of GPV into the
culture medium is still unknown GPV was not derived from
membrane fragments or microvesicles as it was not found in
a 100 000 g centrifugation pellet (data not shown) The
soluble form probably resulted from enzymatic cleavage of
GPV above or near the point of membrane insertion This
would resemble the reported cleavage of GPV from the
platelet surface by calpain and possibly matrix
metallopro-teinases, which releases a soluble 82 kDa fragment
How-ever, attempts to prevent GPV cleavage through incubation
of CHO/GPV cells in the presence of a Ca2+ chelator,
impermeable or permeable forms of calpain inhibitors
(calpastatin, lactacystin), were unsuccessful and did not
restore surface expression of the mature protein The similar
possibility that GPV is cleaved in platelet precursors in the
absence of the other GPIb–V–IX subunits cannot be readily
assessed for example in Bernard–Soulier patients Such
studies should be facilitated by the recent and future
development of mouse strains mutated in the GPIb–V–IX
complex and the availability of efficient culture systems for
megakaryocyte precursors [24,25]
The requirement for the other subunits of GPIb–V–IX
for correct surface expression of GPV is illustrated in the
present work and has also been documented in other cell
types [17,26] Studies of cells transfected with partial
complexes indicated that GPIba was a key subunit for
efficient membrane expression of GPV, a finding which
remains to be confirmed in platelets Conversely, addition of
GPV to cells already expressing GPIb–IX did not increase
surface expression of the complex [14,15], except in GPV
transfected megakaryocytic human erythro-leukemia
(HEL) cells [26] These discrepancies could be due to
different levels of GPIba in the various cell systems The
relevance of these findings for platelet biosynthesis is
unknown It is nevertheless clear that mice deficient in
GPV express normal levels of GPIb–IX on the surface of
platelets, suggesting that in these megakaryocytes the other
subunits are produced at sufficiently high levels [6,13]
Metabolic labelling experiments showed similar
process-ing of GPV in the presence or absence of GPIb–IX and in
both cases an immature cell associated 70 kDa form was
detected at early chase times In cells expressing only GPV,
this form was still present at later chase times and was
localized in a granular compartment Its sensitivity to
Endo-H indicated the presence of high mannose sugars which are
typically added before the cis-Golgi A 70 kDa band has
been reported by others in cells expressing GPV or GPIb–
V–IX [14,22] These biosynthetic studies in cell lysates did
not reveal any further sugar maturation, in agreement with
our findings in CHO/GPV cells, while their failure to detect
a mature form of GPV could have been due to rapid release
of a soluble protein as reported here Surprisingly, these
same cells displayed surface expression of GPV in flow
cytometric experiments, suggesting membrane targeting of
an incompletely processed form Immunoprecipitation of
surface labelled proteins would however, be required to fully confirm this hypothesis
In the presence of GPIb–IX, the cell associated immature
70 kDa protein progressed to a more mature sialylated
82 kDa species with kinetics comparable to those of the full maturation of GPIbab and GPIX This molecule was able
to reach and remain at the cell membrane as demonstrated
by surface biotinylation studies (data not shown) Both immature and mature GPV appeared as broad bands on SDS/PAGE gels, which is also a characteristic of platelet GPV and an indication of heterogeneity of the sugar content at the eight consensus N-glycosylation sites and putative O-glycosylation sites [11] The appearance of the cell attached and soluble mature forms of GPV with comparable kinetics would suggest a normal progression of the latter through the Golgi apparatus followed by its rapid cleavage SDS/PAGE and glycosidase analyses indicated that singly expressed GPV had similar properties to the complex associated form in CHO cells and platelets Despite these similarities, the nature of the sugars could differ in CHO and platelet GPV, as observed previously for the GPIba subunit
Availability of a recombinant form of soluble GPV with biochemical and functional properties [7] similar to those of the native platelet species will be important in the perspec-tive of determination of its 3-D structure, the mapping of ligand domains such as the sites interacting with collagen and the mechanisms of cleavage by proteases
Acknowledgements
We thank Juliette Mulvihill for reviewing the English of the manuscript, Martine Santer for her help in the development of the CHO cell lines and Sylvette Chasserot for help in confocal microscopy studies at Plateforme d’Imagerie In vitro IFR des Neurosciences (Strasbourg) Catherine Strassel is supported by a grant for ARMESA (Association d’Recherche et de De´veloppement en Me´decine et en Sante´ Publique) and from ARC (Association de Recherche contre le Cancer).
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