Biosynthesis 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 p resence o f
GPIb–IX. GPV could not be detected at the surface of cells
expressing the single subunit but was f ound instead as a
soluble f orm i n the culture m edium. In pulse–chase studies,
an immature 70 kDa protein was detected in cell l ysates,
whereas a fully processed 80–82 kDa form was only
observed in the culture supernatants at later chase times.
Immature GPV w as N-glycosylated and r etained before the
medial Golgi while the secreted molecule contained c omplex
sialylated sugars. The mature soluble form of GPV was
produced by an enzymatic cleavage wh ich w as not affected
by inhibitors of proteasome, calpain or metalloproteinases.
When GPV was cotransfected with GPIb–IX, the former
was no longer found in the c ulture supernatant but was
retained in the cell m embrane 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 t o the intracellular trafficking of GPIb a.
These results indicate that correct biosynthesis and surface

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 k Da 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 o f these
subunits. GPIb–IX is expressed e fficiently 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 al l the reported B ernard–Soulier defects are
restricted to the GPIba,GPIbb and GPIX subunits [16].
Nevertheless, s ome 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. A pproaches 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]. T hese studies were, however, performed mostly
in the absence GPV. On the contrary, the biosynthesis of
Correspondence to F. 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:

zeocin 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 a mplification of GPV
expression by growing c ells in the presence of step i ncreased
concentrations of methotrexate [21]. This clone was main-
tained in suspension in s erum fre e CHO-S-SFMII mediu m
(Gibco BRL) supplemented with 1.2 lgÆmL
)1
methotrexate
(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 · 10
5
in 100 lL) were incubated for 30 min at
4 °C 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 g oat a nti-(rat I gG) F( ab¢)
2
or FITC-conjugated
goat anti-(mouse IgG) IgG (Jackson Immunoresearch,

M
Tris (pH 7.5), 150 m
M
NaCl, 5 m
M
EDTA, 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 · 10
7
cells from suspensions
containing 5 · 10
6
cellsÆmL
)1
and 2 mL culture superna-
tants corresponding to 10
7
cells were clarified by incubating
them twice for 1 h at 4 °Cwith100lLofprotein
G-Sepharose (Sigma, Saint L ouis, MO, USA). The s amples
were then in cubated overnight at 4 °Cwith10lgofthe
relevant mAbs and 100 lL of fresh protein G-Sepharose.
After centrifugation, the bead pellets were washed once in
buffer I, t wice in buff er II [ 0.5% (v/v) T riton X-100, 20 m
M

M
.The
samples were then incubated with endoglycosidase-H
(5 mU per sample) for 6 h at 37 °C in t he presence of
1.7 lgÆmL
)1
calpain i nhibitor and 1· protease inhibito r
cocktail. In neuraminidase treatment, the beads were
resuspended directly in 20 lL of 100 m
M
sodium acetate
(pH 5) containing 1 m
M
CaCl
2
and 150 m
M
NaCl, before
addition of 10 lL of n euraminidase (100 mUÆmL
)1
)
(Roche) and incubation for 6 h at 37 °C.
Western blotting
Supernatants from CHO/GPV cell cultures w ere treated o r
not with thrombin (5 UÆmL
)1
) (Sigma) for 5 min at 3 7 °C,
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 p rotein

mounted in Mowiol 4-88 (Calbiochem Novabiochem) and
the labelled cells were examined under a Zeiss laser scanning
microscope (LSM 410 invert) equipped w ith 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 m easured by flow
cytometry (Fig. 1A), despite selection of a strongly
expressing clone by a series of amplifications in the
presence of increasing concentr ations of metho trexate.
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 o f soluble
GPV [21], which was identified a s a single 82 kDa band by
Western blotting with t he mAb V.5 (Fig. 1C). The identity
of t his band was f urther established by its cleavage to a
69 kDa species following thrombin treatment. These
properties of s oluble recombinant GPV are identical t o
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

fully processed during transit through the different Golgi
compartments b ut is not retained in the p lasma 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 t ogether with the other subunits of the G PIb–V–IX
complex, as demonstrated by flow cytometry (Fig. 4A).
Double-labelling confocal m icroscopy with antibodie s
against GPV and GPIba or GPIX revealed colocalization
of these s ubunits with G PV primarily at the cell m embrane.
Hardly any labelling c ould be detected intracellularly, unlike
in CHO/GPV cells (Fig. 4B). These results imply that
GPIb–IX is required for stable surface expression o f GPV in
transfected cells. Very comparable results were obtained
when GPV was cotransfected with GPIb–IX i nto 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. C oncomitantly, GPIba
progressed from an immature 85 kDa form to a mature
125 kDa mo le cule. GPIbb and GPIX displayed more
modest mass increases of 1–2 kDa and gradually reached
their mature sizes of 25 and 20 kDa, respectively, ove r
30 min of chase. This maturation time-course is very similar
to that reported p reviously for C HO/GPIb–IX cells [18,22]
(Fig. 4 C). Analysis of t he supernatants revealed no secreted

cipitated as described in Fig. 2 and th e immunoprecipitates from the
cell lysates or supernatants were analysed for Endo-H (A) or neura-
minidase (B) sensitivity. (A) The band at  70 kD a corresponding to
immature cell associated GPV was converted into a 50 kDa band (the
expected siz e of deglycosylated G PV) b y End o-H treatment. In con-
trast, the 80 kDa secreted molecule was i nsensitive to Endo-H. (B) The
cell associated 70 kDa form o f GPV was resistant to neuraminidase,
whereas th e solu ble 80 kDa fo rm was reduced to 70 kD a t hrough loss
of terminal s ialic acid res idues.
3674 C. Strassel et al.(Eur. J. Biochem. 271) Ó FEBS 2004
complex, and the influence on its pro cessing 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 t ransfected GPV w as secreted
as a soluble m olecule, 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 e ncoding 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 aft er transfec-
tion but we were never a ble to obtain a stable surface

soluble 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 u sing methotrexate amplification in CHO cells found
inefficient membrane insertion of GPIba transfected as a
single-chain and secretion of a glycocalicin-like s oluble
form.
The exact m echanism l eading to release of GPV into t he
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). T he
soluble form probably r esulted from e nzymatic cleavage of
GPV above or near the point of membrane insertion. Th is
would resemble the reported cleavage of GPV from the
platelet surface by calpain an d possibly matrix metallopro-
teinases, which releases a soluble 82 kDa fragment. How-
ever, attempts to p revent GPV cleavage through incubation
of CHO/GPV cells in the presence of a Ca
2+
chelator,
impermeable or permeable forms of c alpain 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 o f mouse strains mutated in the GPIb–V–IX
complex and the availability of efficient culture systems for
megakaryocyte precursors [24,25].

of a s oluble protein as reported here. Surprisingly, t hese
same cells displayed surface expression of GPV in flow
cytometric experiments, suggesting membrane targeting of
an incompletely processed form. Immunoprecipitation o f
surface l abelled proteins would however, be required to f ully
confirm this hypothesis.
In the presence of GPIb–IX, t he cell associated immature
70 kDa protein progressed to a more mature s ialylated
82 kDa species with kinetics comparable to those of the full
maturation of GPIbab and GPIX. This mole cule was able
to reach a nd remai n at the cell membrane as d emonstrated
by surface b iotinylation 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 t hrough the Golgi a pparatus followed by i ts rapid
cleavage. SDS/PAGE and glycosidase analyses indicated
that singly expressed GPV had similar properties to the
complex a ssociated form in CHO cells and p latelets. Despite
these similarities, the nature of the s ugars could differ in
CHO and platelet GPV, as observed previously for the
GPIba subunit.
Availability of a recombinant form of soluble G PV with
biochemical and functional properties [7] similar to those o f
the native platelet species will be important in the perspec-
tive of determination of its 3-D s tructure, the mapping of

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