Fax: + 386 2 22 96 071, Tel.: + 386 2 25 05 800 Abbreviations: IFN-c, interferon-gamma; TrIFN-c, trophoblastic interferon-gamma; rGIFN-c, glycosylated recombinant IFN-c; LeIFN-c, leucocy
Trang 1The porcine trophoblastic interferon-c, secreted by a polarized
epithelium, has specific structural and biochemical properties
Avrelija Cencicˇ1,2, Ce´line Henry3, Franc¸ois Lefe`vre1, Jean-Claude Huet3, Srecko Koren4and Claude La Bonnardie`re1
1
Unite´ de Virologie et d’Immunologie Mole´culaires, INRA, Jouy en Josas, France;2Faculty of Agriculture, University of Maribor, Slovenia;3Unite´ de Biochimie des Prote´ines, INRA, Jouy-en-Josas, France;4Institute of Microbiology and Immunology, Medical Faculty, University of Ljubljana, Slovenia
At the time of implantation in the maternal uterus, the
trophectoderm of the pig blastocyst is the source of a massive
secretion of interferon-gamma (IFN-c), together with lesser
amounts of IFN-d, a unique species of type I IFN This
trophoblastic IFN-c (TrIFN-c)is an unprecedented
exam-ple of IFN-c being produced spontaneously by an
epithe-lium We therefore studied some of its structural and
biochemical properties, by comparison with pig IFN-c from
other sources, either natural LeIFN-c (from adult
leuco-cytes), or recombinant Biologically active TrIFN-c is a
dimeric molecule, of which monomers are mainly composed
of a truncated polypeptide chain with two glycotypes, unlike
LeIFN-c which is formed of at least two polypeptide chains
and four glycotypes TrIFN-c collected in the uterus lumen
was enzymatically deglycosylated and analysed by mass
spectrometry (MALDI-TOF) The data revealed that the
more abundant polypeptide has a mass of 14.74 kDa,
cor-responding to a C-terminal cleavage of 17 residues from the
expected 143-residue long mature sequence A minor
polypeptide, with a mass of 12.63 kDa, corresponds to a C-terminal truncation of 36 amino acids MALDI-TOF analysis of tryptic peptides from the glycosylated molecule(s) identifies a single branched carbohydrate motif, with six N-acetylgalactosamines, and no sialic acid The only glycan microheterogeneity seems to reside in the number ofL-fucose residues (one to three) The lack of the C-terminal cluster of basic residues, and the presence of nonsialylated glycans, result in a very low net charge of TrIFN-c molecule How-ever, the 17-residue truncation does not affect the antipro-liferative activity of TrIFN-c on different cells, among which
is a porcine uterine epithelial cell line It is suggested that these specific properties might confer on TrIFN-c a partic-ular ability to invade the uterine mucosa and exert biological functions beyond the endometrial epithelium
Keywords: interferon-c; epithelium; mass spectrometry; truncated protein; N-glycosylation
Interferons (IFNs)are proteins or glycoproteins belonging
to an extended family of cytokines IFNs exert a broad
spectrum of biological activities, such as eliciting an
antiviral state in target cells, which provides transient
resistance to infection by numerous viruses [1] Two types of
IFNs have been described, which share no sequence
homology: type I IFNs (a, b, x)include those produced
mainly in response to a variety of viruses, while type II IFN
has only one member, IFN-c, which in mammals is
produced by activated T lymphocytes
(NK)cells, and exerts various modulating effects on the
immune response [2–4]
In pigs, from days 12–20 of development (i.e around the time of implantation), the extra-embryonic trophectoderm secretes huge amounts (up to 250 lg per uterine horn)of IFN-c into the uterine lumen [5,6].This porcine tropho-blastic IFN-c (TrIFN-c)appears to constitute a unique case
of immune IFN being produced by a nonlymphoid cell Moreover, the trophoblast is a polarized epithelium, without any tissue or functional relationship with leuko-cytes In addition, this trophectoderm-derived IFN-c is produced in amounts that are far higher than those found in adult tissues during the immune response The pig tropho-blast, which can reach 1 m in length, is made up of numerous trophectoderm cells, all of which are involved in a polarized (apical)IFN-c secretion through an unusual transcription of the single IFN-c gene, at around days 14–16 [7] To date, the mechanism involved in TrIFN-c secretion has remained unclear, as has whether the epithelial origin of producing cells affects the structure and biological activity
of this embryonic IFN At the same time, the porcine trophoblast secretes another IFN, called IFN-d, which was found to be a novel type I IFN, as yet known only in the pig species, and which plays an unknown role in pregnancy [8]
In all known species, IFN-c is encoded by a single gene, and the protein produced by leukocytes is well characterized [9–11] It consists of a dimer of variant glycotypes derived from the single polypeptide chain, which in man, mouse and pig contains two N-glycosylatable Asn residues [10,12,13]
Correspondence to A Cencicˇ, Faculty of Agriculture, University of
Maribor Vrbanska 30, 2000 Maribor, Slovenia.
Fax: + 386 2 22 96 071, Tel.: + 386 2 25 05 800
Abbreviations: IFN-c, interferon-gamma; TrIFN-c, trophoblastic
interferon-gamma; rGIFN-c, glycosylated recombinant IFN-c;
LeIFN-c, leucocytic IFN-c; rIFN-c, recombinant bacterial IFN-c;
IPTG, isopropyl thio-b- D -galactoside; TMB,
3¢,3¢,5¢,5¢-tetra-methylbenzidine; VSV, vesicular stomatitis virus; MDBK,
Madin-Darby bovine kidney; TBA, trophoblastic cell line; EL,
endometrial glandular cell line; ST, swine testis; DMEM, Dulbecco’s
modified Eagle’s medium; APA, antiproliferative activity.
(Received 11 January 2002, revised 4 April 2002,
accepted 22 April 2002)
Trang 2Full-length IFN-c has a basic net charge, most probably
due to a near C-terminal cluster of Arg and Lys residues
[14–16] However, various forms of C-terminal truncations
have been found to be associated with native IFN-c
(reviewed in [17]) The fact that trophoblastic IFN-c is
translated and secreted by an epithelial cell suggests that
there may be some differences in the molecular structure
and/or biochemical characteristics of TrIFN-c, when
com-pared with leucocyte IFN-c Consequently, the
bioavaila-bility or biological activity of TrIFN-c might be changed
This paper analyses some of the structural, biochemical
and functional properties peculiar to trophoblastic porcine
IFN-c, by comparison with a natural IFN-c produced by
activated porcine leukocytes (LeIFN-c)and a
nonglycosyl-ated, recombinant porcine IFN-c expressed in Escherchia
coli(rIFN-c)
M A T E R I A L S A N D M E T H O D S
Source of porcine IFN-c
Trophoblastic IFN-c (TrIFN-c) Pregnant gilts from the
Chinese Meishan breed were anaesthetized by electric shock
then normally slaughtered on day 15 of pregnancy The
entire reproductive tract was removed, and each uterine
horn was flushed with 50 mL of medium 199 (Life
Technology, Paisley, UK)containing penicillin G
(100 UÆmL)1), Streptomycin (50 lgÆmL)1), and an
antipro-tease cocktail of Trazylol, pepstatin and aprotinin The
flushed fluid was clarified by centrifugation at 2000 g, and
frozen at )20 C Alternatively, for [35S]Met labelling,
TrIFN-c was collected in the supernatant of conceptus
tissue maintained in culture in DMEM for 24 h at 38C
with gentle shaking
Leucocytic IFN-c (LeIFN-c) LeIFN-c was obtained from
pig peripheral blood leukocytes (PBL)stimulated with
4b-phorbol 12-myristate 13-acetate and
phytohaemaggluti-nin according to a previously published protocol [18] The
supernatant containing natural LeIFN-c was collected 48 h
after induction
Recombinant bacterial IFN-c (rIFN-c) The full-length
porcine IFN-c cDNA, encoding the preinterferon sequence
was obtained from a day 15 trophoblastic cDNA library
(unpublished) From this cDNA, a translatable mature
IFN-c sequence was constructed by use of PCR
amplifica-tion, driven by primers designed to insert: (a)an ATG
upstream of the nucleotide sequence encoding the mature
protein (starting with a Gln residue); (b) two restriction sites,
namely EcoRI and HindIII, in 3¢ and 5¢ ends of the coding
sequence, respectively The amplified fragment was digested
with EcoRI and HindIII, and subcloned into pBS+ vector
(Stratagene) The EcoRI–HindIII 456 bp fragment of one
clone with the correct sequence was inserted into the
expression vectors pET14 and pET22 (Novagen) The
resulting plasmids pET14 metPoIFN-c and
pET22met-PoIFN-c were used to transform E.Coli strain BL21 (DE3),
which contains the T7 RNA polymerase under the control
of the lac promoter [19]
Bacteria bearing metPoIFN-c were grown in Luria–
Bertani medium supplemented with 1 mMMgCl2at 37C
until D ¼ 1.0 INF-c expression was induced by the
addition of 1 mM isopropyl thio-b-D-galactoside (IPTG) After incubation for a further 4 h, bacteria were harvested
by centrifugation at 3500 g
extract of rIFN-c was obtained essentially following the protocol developed by Vandenbroeck et al [20]
Glycosylated recombinant IFN-c (rGIFN-c) RGIFN-c was obtained by constructing a tetracyclin-inducible expres-sion system in the RK13 cell line, as previously described [18]
Interferon assays ELISA Coating was carried out with mAb G47 (INRA, Jouy-en-Josas)raised against porcine rIFN-c (CIBA-Geigy)
in NaCl/Pi (1 : 200 dilution) After overnight incubation, samples of IFN-c were diluted in assay buffer (fivefold dilutions in 0.05% Tween/NaCl/Pi) After a 1-h incubation
at 37C, rabbit rIFN-c antiserum was added (1 : 500 dilution in NaCl/Pi/0.05% Tween), and the plate was again incubated at 37C for one hour Finally, 1 : 4000 diluted horseradish peroxidase-conjugated goat anti-(rabbit IgG)Ig (Biosys, France)was added After a further 1-h incubation
at 37C, staining was revealed with 3¢,3¢,5¢,5¢-tetra-methylbenzidine (TMB)at a concentration of 0.4 gÆL)1in
an organic base and 0.02% H2O2 in a citric acid buffer according to the instructions of the supplier (Kirkegaard & Perry Laboratories Inc., or Sigma-Aldrich, USA) As a standard, porcine rIFN-c (CIBA-Geigy)was used at a concentration of 10 lgÆmL)1
Antiviral activity Antiviral activity was assayed by inhibi-tion of the vesicular stomatitis virus (VSV)cytopathic effect
on the Madin–Darby bovine kidney (MDBK)cell line as described previously [21] Titers were expressed in antiviral
IU equivalents by a comparison with a calibrated porcine IFN-a laboratory standard The amount of IFN-c (mg)was determined by ELISA Specific antiviral activity was expressed in IUÆmg)1
Growth inhibition test The antiproliferative effect of purified TrIFN-c was measured by comparison to rGIFN-c and rIFN-c on several porcine epithelial cell lines and bovine MDBK cells The trophoblastic cell line (TBA) was isolated from a 15-day-old pig conceptus and the endometrial glandular cell line (EL)from a cyclic uterus from Large White gilt Both lines were developed at the Unite de Virologie et Immunologie Moleculaires, INRA, France Swine testis (ST)is a previously published cell line [22] In 96-well plates, quadruplicate threefold dilutions of each purified IFN (initial concentration 1 lgÆmL)1)were applied to monolayers of 1· 105 cells (MDBK, ST)or
5· 105 cells (EL, TBA)in Dulbecco’s modified Eagle’s medium (DMEM)/10% fetal bovine serum Incubation was performed at 37C in an humidified incubator for 3 days The plates were stained with Crystal Violet in ethanol, rinsed with water, and destained with 10% (v/v)acetic acid The A590was measured, and the results were expressed, for each dilution, by the mean ratios (%, ± SD)of absor-bances in IFN-treated wells (n ¼ 4)to those in control wells (n ¼ 6) On ST cells, only TrIFN-c was assayed, but the effect of sheep antiserum 166 to type I IFN (a gift of
C Chany
4 , INSERM, Paris), known to neutralize IFN-d,
Trang 3was tested to assess if trace amounts of IFN-d could
partly account for the antiproliferative effect By precaution,
all other tests were performed in the presence of antiserum
166
IFN-c purification
LPC-Hi Trap Heparin purification Crude clarified cell
culture supernatant containing rGIFN-c or bacterial crude
clarified lysate were applied to a 5-mL Hi-Trap heparin
column (Pharmacia, Sweden)with a flow rate of 1.5 mLÆ
min)1 After extensive washing (A280 ¼ 0)with a Tris/HCl
buffer, pH 8.0 (0.05 molÆL)1)and NaCl (0.5 mol L)1),
IFN-c was eluted with a linear salt concentration gradient
(0.05–1 molÆL)1NaCl in Tris/HCl, pH 8.0)at a flow rate of
1 mLÆmin)1 Fractions positive for IFN-c were pooled and
processed for further purification
Immunoaffinity chromatography Partially purified
rGIFN-c, rIFN-c or preclarified uterine flushes containing
TrIFN-c were applied to a CNBr-activated Sepharose 4B
(Pharmacia, Sweden)coupled with monoclonal
anti-(por-cine IFN-c)Ig (C5) Unbound impurities were extensively
washed off the column with NaCl/Piat pH 7.4 IFN-c was
eluted with glycine/HCl buffer (0.2 molÆL)1) , pH 3.0, at
which pH porcine IFN-c proved to be stable [19] Eluted
fractions were immediately raised to pH 6.0 by use of 1M
Tris base
Analytical procedures
Gel filtration Crude IFNs were applied to a 1.5· 45 cm
column packed with Sephadex G75 superfine (Pharmacia,
Uppsala, Sweden) The column was equilibrated with
20 mMphosphate buffer, 0.5MNaCl at pH 7.4 The flow
rate was adjusted to 9.5 mLÆh)1 IFN-c was assayed in every
1.5 mL fraction by ELISA and by antiviral assay on
MDBK cells Molecular mass marker proteins were bovine
serum albumin (Mr66 000), ovalbumin (Mr 43 000)and
cytochrome c (Mr12 400) The void volume of the column
was measured by use of Blue Dextran (Mr2000 000)
35S-Labelling of natural IFN-c For LeIFN-c, pig PBL
were washed and suspended in methionine-free medium,
then induced by the sequential addition of 4b-phorbol
12-myristate 13-acetate-phytohaemagglutinin, as described
previously [18] One hundred lCi per mL of a [35S]Met-Cys
mix (Amersham Pharmacia Biotech, Saclay, France)was
added The next day, fresh RPMI containing unlabeled
methionine was added to the culture (1 : 20 dilution)
Metabolically labelled LeIFN-c was harvested after 48 h of
incubation TrIFN-c was produced in the supernatant of
freshly collected day 15 conceptuses as described above,
except that methionine-free MEM and [35S]Met-Cys
(100 lCiÆmL)1)were used
Immunoprecipitation and deglycosylation of IFN-c The
35S-labelled IFN-c were concentrated against poly(ethylene
glycol)(Mr 20 000)to 2 mL and processed for
immuno-precipitation by sheep anti-(mouse IgG)Ig (Biosys,
Compie`gne, France)coupled to Protein A–Sepharose, as
previously described [18] After final washes, the beads were
resuspended in 30 lL of Laemmli buffer (glycosylated
control), or in deglycosylation buffer: 30 lL of 100 mM Tris/HCl, pH 7.4, 1% SDS and 2% 2-mercaptoethanol (deglycosylated sample), and immediately boiled for 5 min
to dissociate IFN-c from the beads Samples of immuno-precipitated rGIFN-c, TrIFN-c and LeIFN-c in deglyco-sylation buffer were diluted 1 : 5 with 50 mMTris/HCl, 1% Nonidet P40; recombinant N-glycosidase F (EC 3.5.1.52, from E Coli, Boehringer, Mannheim, Germany)was added
to a final concentration of 10 UÆmL)1 The enzymatic reaction was carried out overnight at 37C Deglycosylated IFN-c were precipitated with 4 vol acetone Washed pellets were resuspended in Laemmli buffer, then electrophoresed together with the glycosylated controls on a 15% acryla-mide gel [23] The dessicated gel was exposed to autoradi-ography for 48 h at)70 C When necessary, the gels were re-exposed in a radioisotope imager (Phosphorimager, Molecular Dynamics)
N-Terminal microsequence Immunopurified TrIFN-c, obtained from uterine flushes, was subjected to electro-phoresis in SDS/PAGE, then electro-transferred on a ProBlott membrane, which was stained with Coomassie Blue R 250 The two main bands (Mr22 500 and 18 000) were cut out, and analysed for the N-terminal microse-quence Digestion with Pyroglutamate aminopeptidase (EC 3.4.19.3, Sigma–Aldrich)was performed according
to the enzyme supplier’s instructions Automated Edman sequencing was performed using a PE Biosystems Procise
494 HT sequencer, with the reagents and methods des-cribed by the manufacturer
Mass spectrometry of proteins by MALDI-MS Immuno-affinity-purified trophoblastic IFN-c, obtained by flushing pregnant uteri, was subjected to SDS/PAGE after treatment
or mock-treatment with N-glycosydase F After staining the gel with Coomassie blue, bands of interest were cut out and dried Samples were transferred onto a poly(vinylidine fluoride)membrane by passive absorption as described previously [24]; the gel plugs were dried in a Speed Vac concentrator (Savant)for 30 min, then re-swollen in 50 lL 0.2MTris/HCl pH 8.5, 2%SDS, for 30 min After swelling,
200 lL of HPLC water was added and then a 4· 4 mm piece of prewet
added to the solution The procedure required 2 days at room temperature (23C)with gentle vortexing At the end, the gel pieces and the solution were clear, and the membrane was blue The membrane was washed five times with 1 mL 10% methanol with vortexing Protein extraction was carried out by adding 40 lL of trifluoroacetic acid 5% plus
CH3CN 50% and by gentle vortexing for 15 min A second extraction was made, and the two extracts were pooled, then concentrated to 10 lL
One microliter of interferon was mixed on the stainless steel MALDI plate with 1 lL of DHB
(10 mgÆmL)1 in 50% CH3CN, 0.15% v/v trifluoroacetic acid)and dried at room temperature Mass spectra were acquired on a Voyager DE-STR+ time-of-flight mass spectrometer (Applied Biosystems, Framingham, MA, USA)equipped with a nitrogen laser emitting at 337 nm Spectra were recorded in positive linear mode with 25 kV as accelerating voltage, a delayed extraction time of 1200 ns and a 94% grid voltage The spectra were externally calibrated using a mix composed by horse heart
Trang 4cyto-chrome c (M + H)+¼ 12 361.1 Da, horse apomyoglobin
(M + H)+¼ 16 952.6 Da and bovine carbonic anhydrase
(M + H)+¼ 29 024 Da
Tryptic peptide analysis by MALDI-TOF.Tryptic
diges-tions of glycosylated IFNs were achieved directly in the gel
matrix The excised gel plugs were washed in 50% CH3CN
in 50 mMNH4CO3(v/v)and then transferred to Eppendorf
tubes After dessication of the gel in SpeedVac for 30 min,
the digestion was performed in 25 lL of 50 mMammonium
bicarbonate pH 8.0 and 0.5 lg of modified trypsin
(Promega, sequencing grade)for 18 h in a thermomixer
(Eppendorf)at 37C with vortexing at 500 r.p.m
8
A 0.5-lL aliquot of sample was spotted directly onto the
stainless steel MALDI plate The sample was then allowed
to dry at room temperature before addition of a 0.5-lL
aliquot of the matrix solution This dried-droplet sampling
method was employed using a freshly prepared solution at
3 mgÆmL)1 of a-cyano-4-hydroxycinnamic acid matrix in
50% (v/v)acetonitrile and 0.1% (v/v)trifluororacetic acid
For acquisition, the accelerating voltage used was 20 kV
Peptide spectra were recorded in positive reflector mode and
with a delayed extraction of 130 ns and a 62% grid voltage
To analyse some peptides, spectra were recorded by the
positive linear method with a delayed extraction of 160 ns
and a 62% grid voltage
The spectra were calibrated using an external calibration
which was composed of: Des-Arg 1 Bradykinin (M + H)+¼
904.468 Da, human angiotensin I (M + H)+¼
1296.685 Da, neurotensin (M + H)+¼ 1672.917 Da,
melittin from bee venom (M + H)+¼ 2845.762 Da and
bovine insulin B chain disulfonate (M + H)+¼
3494.651 Da Samples digest with trypsin were calibrated
using internal calibration with autolysis of trypsin:
(M + H)+¼ 2211.104 and 842.509 Da
R E S U L T S
Active trophoblastic IFN-c is a dimer
In order to determine the form in which TrIFN-c is present
in the uterine lumen and therefore available to the
endometrium, the Mrof native TrIFN-c was measured by
gel-filtration, in comparison with those of crude LeIFN-c
and unglycosylated rIFN-c Each column fraction was
tested by antiviral assay and by IFN-c specific ELISA
Elution profiles (Fig 1)show that the antiviral activity
eluted mostly as a single peak, around an Mrof 43 000 for
TrIFN-c (Fig 1A), 50 000 for LeIFN-c (1B), and 34 000
for nonglycosylated rIFN-c The scheme with TrIFN-c
(Fig 1A)was however, more complex; in ELISA, a single
peak eluted at around 43 000, while the antiviral assay
detected two peaks, one at 43 000 and slightly above, and
one around 17–19 000 This second peak was most
prob-ably due to the presence of IFN-d in the crude uterine flush,
which had previously been shown to be monomeric, with an
Mr around 19 000 [25] This peak was not detected by
ELISA
Unexpectedly, for leucocytic IFN-c, and to a lesser extent
for rIFN-c, the maximum ELISA score was delayed by one
and two fractions with regard to antiviral activity One
possibility is that our ELISA is more specific for shortened
IFN-c molecules (see below)
TrIFN-c therefore appears to be essentially, if not entirely, dimeric, similar to natural LeIFN-c and unglycos-ylated recombinant IFN-c
Monomers of TrIFN-c are glycosylated and have shorter polypeptide chains
Both TrIFN-c and LeIFN-c were metabolically radio-labelled with [35S]Met, then immunoprecipitated with rabbit antiserum as described in Materials and methods The
Fig 1 Sephadex G-75 gel filtration profiles of three preparations of crude porcine IFN-c (A)TrIFN-c derived from uterine flushes of a day-15 pregnant gilt (B)crude natural LeIFN-c (C) recombinant IFN-c (E.coli) In each fraction IFN-c concentration (d)was meas-ured by ELISA, and antiviral activity (h)was determined by antiviral assay on MDBK cells Molecular weight standards and void volume (V 0 )are indicated by arrows, and the black rectangle designates the elution area of expected IFN-c monomers.
Trang 5monomers were analysed by denaturing SDS/PAGE
(Fig 2) The results were clearly contrasted: in the
immu-noprecipitate from leukocytes, LeIFN-c consisted of four
major bands (lane 1: Mr24 800; 22 000; 19 800; 17 500), the
Mr24 800 band being slightly more pronounced These four
bands resolved into two bands on deglycosylation (lane 2:
16 000 and 14 000) As for TrIFN-c, only two main bands
were visible at 22 500 and 18 000 (lane 3), which yielded one
main band with an Mr of 14 400 after N-glycosidase F
treatment (lane 4), suggesting a single major polypeptide
chain, but macroheterogeneity at the two potential
glyco-sylation sites present on the IFN-c polypeptide core They
could differ in the rate and site of glycosylation, considering
the 22 500-Da band as bi-glycosylated and the 18 000-Da
band as monoglycosylated (Fig 2) The deglycosylated
14 400-Da band may correspond to the truncation of about
20 amino acids in the embryonic IFN-c molecule, as the
expected mass of full-length porcine IFN-c polypeptide is
around 16 780 Da
In order to check if a full-length TrIFN-c form could be
found in the trophoblast cells, which would be indicative of
extracellular degradation, the same immunoprecipitation
was performed on the conceptus cell lysate in parallel with
the supernatant (Fig 3) SDS/PAGE revealed only one
major band in the cell lysate, at an apparent Mrof 23 000–
24 000 (lane 2), which was slightly higher than the largest
of the major monomers found in the supernatant (lane 1)
Because of the scarcity of intracellular material, it was not
possible to analyse the effect of N-Glycosydase F on this
band, which casts a doubt on its glycosylation status
However, no equivalent of the largest LeIFN-c species
(24 800)was found Furthermore, the larger amount of
TrIFN-c found, when compared to the previous
experi-ment (Fig 2), revealed that the 22 500 and 18 000 bands
were the major but not the only components of TrIFN-c;
two minor bands at Mr 24 000 and 20 500 were also
visible These band most probably correspond to the
diglycosylated and monoglycosylated forms of the minor
polypeptide of Mr16 000 obtained after N-glycosidase F
treatment (lane 3)
TrIFN-c has an intact N-terminus, and a truncated
C-terminus
Immunoaffinity-purified trophoblastic IFN-c, obtained
from pregnant uterine flushes, was electrophoresed in
SDS/PAGE, after treatment (or mock treatment)with N-glycosydase F (Fig 4A) Again, two major bands were found at Mr 22 500 and 18 000 (lane 1), and upon deglycosylation, one major band at Mr 14 400 was seen But unlike TrIFN-c collected in the supernatant of cultured conceptuses, a minor deglycosylated band was obtained at
Mr12 000 (lane 2) The two major TrIFN-c polypeptides yielded no residue by Edman microsequencing, a result compatible with a blocked pyroglutamate N-terminus (the expected mature sequence is Q-A-P-F-F-K-E-I-T-I-L-K-) Immunopurified TrIFN-c was then treated with
pyroglu-Fig 2 SDS/PAGE profiles of [35S]Met metabolically labelled native
TrIFN-c and LeTrIFN-c Lane 1, control LeTrIFN-c Lane 2,
N-glycosidase F treated LeIFN-c Lane 3, control TrIFN-c Lane 4,
N-glycosidase F-treated TrIFN-c. Fig 3 SDS/PAGE profiles of [35
S]Met-labelled TrIFN-c after immu-noprecipitation by rabbit anti-(porcine IFN-c) Ig Lane 1, glycosylated conceptus IFN secreted in the supernatant Lane 2, intracellular TrIFN-c Lane 3, conceptus secreted IFN-c treated with N-Glycosi-dase F.
Fig 4 Mass determination of deglycosylated TrIFN-c species (A) SDS/PAGE profiles of native TrIFN-c obtained in flushings of Day-15 pregnant uterus, control (lane 1)and N-glycosidase F treated (lane 2) (B)Mass spectrum obtained by MALDI-TOF of the M r 14 400 polypeptide (C)Mass spectrum of the M 12 000 polypeptide.
Trang 6tamate aminopeptidase, and re-submitted to the
microseq-uencing process In the largest band, the A-P-F-F-K
sequence appeared with a moderate yield, thus confirming
that the native N-terminus was intact
In a second step, the mass analysis of the two
deglycos-ylated bands was performed by MALDI-TOF The 14
400-Da species yielded a main peak at (M + H)+14 742.0 Da
(Fig 4B) This measured mass is compatible with a
nonglycosylated polypeptide starting with an N-terminal
pyroglutamate and ending at C-terminal L126 Indeed such
a peptide has a theoretical sequence mass (average)of
14 712.0 Da, to which 17 Da must be substracted for
N-terminal pyroglutamate, and 2 Da must be added for two
N-glycosidase F-induced Nfi D transitions, and 48 Da
added for oxidation of three residues (probably the three
M), respectively The calculated (M + H)+ obtained is
then 14 746.0 Da, a value which differs by 4 Da from the
observed mass
The MALDI-MS analysis of the minor peak with an Mr
of 12 000 yielded an observed (M + H)+of 12 635.0 Da
(Fig 4C) This is compatible with a deglycosylated
poly-peptide with R107 as C-terminus Indeed such a 1–107
polypeptide with N-terminal pyroglutamate, three oxidized
residues and two Asn/Asp transitions gives a calculated
(M + H)+of 12 635.5, that is a 0.5-Da difference with the
measured value The second peak of the MALDI spectrum
was measured at 12 762.4 Da (Dmass ¼ 127.4 Da), a mass
compatible with a peptide cleaved behind R107
Therefore, it is most probable that TrIFN-c is mostly
composed of a polypeptide in which the C-terminus is
cleaved after L126 (a lack of 17 residues), and of a minor
polypeptide which is further cleaved, that is after R107 (a lack of 36 residues)
TrIFN-c N-glycans contain no sialic acid, and have limited heterogeneity
The tryptic peptide analysis of the four main bands obtained
in PAGE were performed We chose to point to data obtained for the Mr 22 500 species Figure 5 shows the complete sequence of pig IFN-c [26] with its theoretical trypsin cleavage sites, the peptides found upon MALDI analysis (underlined), and the deduced C-termini of each 14.74 and 12.63 kDa species (arrows) The coverage of the molecule was rather high as peptide analysis amounted to 87.3% of the sequence Q1-L126 Table 1 shows the comparison between theoretical and measured masses of tryptic peptides as provided by MALDI Three main conclusions could be drawn: (a)on the peptide 1–6, the
Fig 5 Complete amino-acid sequence of mature porcine IFN-c [26] Being 143 residues long, it includes two glycosylatable Asn at positions
16 and 83 (in grey) Expected trypsin cleavages are marked by slashes, and peptides analysed by MALDI-TOF are underlined The two arrows point to the inferred C-termini of each 14.74 and 12.63 kDa species.
Table 1 MALDI-TOF tryptic peptide analysis of the M r 22 500 TrIFN-c species.
Peptide
start
Peptide
Theoretical (M + H)+
Measured (M + H)+
Dmass (meas – theor)Remarks
4394.224 4250.802
2143.062 1997.026 1853.604
Glycosylation Glycosylation Glycosylation
708.351
)0.016 15.987 Oxidized Met
840.379
0.023 16.007 Oxidized Met
2951.970
1996.455 2142.554
Glycosylation Glycosylation
947.480
0.036 16.014 Oxidized Met
Trang 7presence of N-terminal pyroglutamate is confirmed (b)On
peptides 69–74, 75–80 and 116–123, the three Met residues
are oxidized (c)On peptides 13–34 and 81–88, a mass excess
of 2143 Da is the most probable signature of the same
glycan conjugate This mass is compatible with a sugar
moiety made of three fucoses plus six N-acetylglucosamines
plus three hexoses (monoisotopic mass ¼ 2142.80 Da)
Table 1 and Fig 6 show that other peaks differed from the
major one by 146 Da, corresponding to the mass of fucose
Therefore, a microheterogeneity exists with at least three
glycoform variants at each site, depending on the fucose
content (one, two or three) Whether this is the real situation
on the native molecule, or a consequence of laser-induced
cleavage of fucose in the course of MALDI analysis is not
known
The same analysis performed on the band at Mr18 500
(data not shown)indicated that the same glycan motif was
present on the tryptic peptide 13–34, but absent on the
peptide 81–88, for which the measured value was 809.42 Da
[theoretical (M + H)+value is 809.41 Da] We can
there-fore conclude that if the main deglycosylated peptide is
indeed 14.74 kDa, then the two main species of natural
TrIFN-c found in uterine flushings have molecular masses
of 19.03 kDa and 16.88 kDa, corresponding to
diglycosyl-ated and monoglycosyldiglycosyl-ated isoforms, respectively, the latter isoform being glycosylated on N16 As expected, the correspondance between the measured masses and observed
Mr in SDS/PAGE is quite good for nonglycosylated proteins, but not for glycosylated ones, as the latter have lowered electrophoretic mobility
Specific antiVSV activity of TrIFN-c is reduced Table 2 shows results concerning the antiviral activity of TrIFN-c, in comparison with LeIFN-c and two species of recombinant IFN-c, including the glycosylated rGIFN-c produced in transfected RK13 cells [18] The specific activity
of TrIFN-c on MDBK cells challenged with VSV was 1–5· 105UÆmg)1of IFN-c (ELISA reactive), i.e approxi-mately 10 times lower than that of its adult equivalent (LeIFN-c), and 20–50 times less than the two recombinant forms
TrIFN-c has an antiproliferative activity (APA) Immunoaffinity-purified IFN-c from uterine flushes did exert an APA on different cells We first checked on pig swine testis cells that possible residual IFN-d was not a
Fig 7 Compared antiproliferative effect of TrIFN-c and other porcine IFN-c on several cell lines Dilutions 1–6 represent serial threefold dilutions of purified IFNs, all of them being adjusted before assay to
1 lgÆmL)1(A)ST cells treated with TrIFN-c in the absence (hatched bars)or presence (black bars)of rabbit antiserum 652 to type I IFN (B,C,D): EL cells, TBA cells and MDBK cells, respectively, treated with serial dilutions of TrIFN-c (black bars), rGIFN-c produced in RK13 cells (grey bars)and rIFN-c expressed in E coli (white bars) Values are means of four replicate assays per dilution, and the errors bars give the positive value of the SEM.
Fig 6 MALDI-TOF analysis of tryptic peptide 13–34 from the 22 500
IFN-c species The area shown is an enlargement of the total mass
spectrum The main peak at an (M + H)+of 4540.26 is compatible
with a N-glycosylation on N16 having the proposed structure drawn
above the peak, including three fucose residues Two other peaks on
the left, with Dmass of 146.03 and 146.42 Da with each other, are
compatible with masses of the peptide 13–34 with 2 and 1 fucose,
respectively (monoisotopic mass of fucose: 146.04) The two peaks
marked with an asterisk represent the Na adducts of the two main
peaks The unmarked peak could not be identified Schematic structure
of the glycan conjugate was inferred by analogy with data obtained for
human IFN-c [27] N-acetylglucosamine (j) ; hexose (d); fucose ( fi ).
Table 2 Specific antiviral activity of TrIFN-c by comparison with other natural and recombinant IFN-c.
Cell line
IFN-c origin
1–5 · 10 6
2–3.5 · 10 6
5–10 · 10 6
(UÆmg)1IFN-c)
Trang 8significant effector of any APA by comparing the effect of
dilutions from 300 ngÆmL)1to 1.2 ngÆmL)1in the absence
or presence of antiserum to porcine type IFN (Fig 7A),
known to neutralize IFN-d [8] Other cells were tested for
their proliferation in the presence of TrIFN-c, and two
purified recombinant proteins, one glycosylated (rGIFN-c
produced in eucaryotic cells), the other free of carbohydrate
chains (rIFN-c produced in E coli) Figure 7B–D shows
that, with cell-related differences, trophoblastic IFN-c
exerted the same (in MDBK cells)or even more pronounced
APA (in endometrial cells and trophoblast cell line TBA)
than its recombinant counterparts On pig EL and TBA
cells, TrIFN-c was the most active on cell growth inhibition,
especially in the first four dilutions, that is in the range of
300–11 ngÆmL)1 On these same cell lines, recombinant
E coli-derived IFN-c was found the least active, which
suggests that the glycosylation status is important for cell
growth inhibition
D I S C U S S I O N
Embryonic TrIFN-c is the only IFN-c secreted by a
nonlymphoid tissue It is also a unique case among all IFN
species, because it is intensely induced under physiological
conditions (at the time of trophoblast implantation)
TrIFN-c is secreted in substantial amount, simultaneously
with IFN-d, in a polarized manner, by the trophectoderm
The precise structure and function of this embryonic,
epithelial IFN-c has not been clarified to date In this work,
we have demonstrated that structural, biochemical and
biological differences exist between TrIFN-c and LeIFN-c
As shown by gel filtration chromatography, TrIFN-c is
accessible to the uterine lumen in the form of relatively
heterogeneous glycosylated dimer with an apparent Mrof
43 000 A shift towards a lower Mrwas noted for TrIFN-c,
when compared to LeIFN-c, which eluted as a major
heterogeneous peak at a Mrbetween 50 000 and 60 000 On
the other hand, rIFN-c exhibits no macroheterogeneity, as
it elutes as one homogeneous peak at around 34 000, a size
compatible with the correct predicted size of a biologically
active dimeric protein We can therefore conclude that
functional embryonic IFN-c (TrIFN-c), like LeIFN-c, is a
dimer The weak antiviral activity found in fractions
corresponding to monomers is certainly that of IFN-d,
with an Mraround 19 000, which is also present in uterine
flushes [25]
As revealed by the electrophoretic profiles of35S-labelled
TrIFN-c and LeIFN-c immunoprecipitates, TrIFN-c
monomers differ from the LeIFN-c in terms of their
polypeptide length and glycosylation pattern
Electropho-retic profile of TrIFN-c exhibits two major bands that are
equimolar, with an apparent Mr values of 22 500 and
18 000, thus suggesting that dimers are composed of equal
proportions of mono glycosylated and biglycosylated
monomers The two glycoforms resolve into a major
Mr 14 000 band upon enzymatic deglycosylation with
N-glycosidase F The fact that TrIFN-c secreted in the
supernatant of conceptus in culture presents with the same
truncation as TrIFN-c collected in uterine fluids suggests
that the cleavage of natural TrIFN-c is not due to
endometrial peptidases The pig trophectoderm has been
shown to express various proteases, among which
plasmi-nogen activator and different matrix metalloproteinases,
which could, directly or by activation of protease cascades, lead to the observed cleavage of TrIFN-c [27,28] In addition, in the flushed fluids, where TrIFN-c is supposed to
be present in its bioavailable form, a minor polypeptide variant was observed after treatment with N-glycosidase F, with an apparent Mrof 12,500, corresponding to a still more cleaved polypeptide
The MALDI-TOF resolution of deglycosylated TrIFN-c monomers, obtained from the uterine flush, confirmed results obtained by SDS/PAGE electrophoresis The major form of TrIFN-c molecule is a polypeptide with a mass of
14 741 Da and a minor one with a mass of 12 634 Da As confirmed by MALDI analysis, the two polypeptides found
in TrIFN-c are truncated at the C-terminus The major polypeptide lacks 17 C-terminal amino acids, as compared
to the full length sequence, and a minor one is further truncated by 36 residues Porcine LeIFN-c and TrIFN-c monomers are glycosylated, unlike human IFN-c, where nonglycosylated forms have also been found in crude preparations [29] In the TrIFN-c molecule, little variability
in the glycan structure are observed Only three variants in glycan composition were found at both N-glycosylation sites, which differ only by the number of boundL-fucose molecules Surprisingly, TrIFN-c glycans terminate with N-acetylglucosamine and not with sialic acid like for human IFN-c Indeed, post-translational modifications, including glycosylation, are strongly dependant upon the type and physiological status of producing cells, and may signifi-cantly influence the characteristics of a glycoprotein [16,17,29] From this point of view, no direct comparison has been possible with the glycan structure and heterogen-eity of porcine LeIFN-c, for which low amounts obtained in phytohaemagglutinin-activated pig PBL did not allow the same mass spectroscopy analysis
As a consequence of the C-terminal truncation, the native TrIFN-c lacks seven basic residues, in particular the R-K-R-K-R cluster (residues 127–131) It is therefore expected to
be less positively charged than LeIFN-c or rIFN-c, which comprise full-length molecules Indeed, unlike the two other IFNs, TrIFN-c, when analyzed by chromatofocusing, did not yield a readable pI, as it did not bind to a Mono-P column In addition, attempts at binding TrIFN-c onto CM-cellulose columns at neutral pH were unsuccessful (data not shown) Although the calculated pI is 10.66 for the full length IFN-c molecule and 9.66 for the 1–126 polypep-tide, TrIFN-c behaves as a molecule without measurable net charge
Concerning biological activities, we found divergent results for antiviral and APA The data shown in Table 2 suggest that TrIFN-c is much less antiviral than LeIFN-c and rIFN-c, as far as VSV challenge is concerned It is possible however, that the relative values for TrIFN-c specific activity are underestimated, if it happened that the specificity of our ELISA test was slightly or significantly better for truncated molecules In any case, the C-terminal truncation of TrIFN-c is most probably not the only reason for the reduced antiviral activity of TrIFN-c on MDBK cells (Table 2), such as that previously described for HuIFN-c [14] The specific glycan composition that we found for TrIFN-c might also contribute to this reduced antiviral activity
On the other hand, we observed that TrIFN-c exhibits an APA on homologous (ST, TBA, EL)and heterologous
Trang 9(MDBK)cells, that does not significantly differ from intact
nonglycosylated rIFN-c and intact glycosylated porcine
rGIFN-c (Fig 7) Moreover, the APA of TrIFN-c on EL
and TBA cells was even higher as compared to the intact
porcine IFN-c It seems that, especially in homologous cell
lines, an intact IFN-c C-terminus is not essential for its
biological function, as was shown for human IFN-c [30]
Our results also confirm previous data, namely that IFN-c
antiviral and APAs can be dissociated [31–33]
Our results shed some light on the specific structure and
properties of this atypical porcine trophoblastic IFN-c,
produced by a polarized epithelium It is probable that the
structural and chemical characteristics of TrIFN-c affects its
bioavailability and biological effect(s)on the maternal
uterus In particular, this shortened version of IFN-c,
lacking a well known ECM-binding sequence and with very
weak net charge, could be more prone than full-length
IFN-c to IFN-cross the endometrial epithelium, and to reaIFN-ch IFN-cellular
targets located in the uterine mucosa (e.g lymphoid or
endothelial cells) It is possible that the very particular
context
9 in which this embryonic IFN-c is produced, namely
between two opposite epithelia, has favoured the selection
of a functionally adapted molecule, which differs from adult
lymphoid IFN-c more by its bioavailability in this particular
context than by its biological activity
A C K N O W L E D G E M E N T S
We would like to thank Christiane De Vaureix for her technical help.
This work was supported by grants from the Slovenian Scientific
Foundation and from the French Ministry of Foreign Affairs.
R E F E R E N C E S
1 De Maeyer, E & De Maeyer-Guignard, J (1988) Interferons and
Other Regulatory Cytokines John Wiley & sons, New York.
2 Ijzermans, J.N.M & Marquet, R.L (1989)Interferon-gamma: a
Review Immunobiol 179, 456–473.
3 Baron, S., Tyring, S.K., Fleischmann Jr, R.W., Coppenhaver,
D.H., Niesel, D.W., Klimpel, G.R., Stanton, J & Hughes, T.K.
(1991)The interferons, Mechanisms of action and clinical
appli-cations J Am Med Assoc 266, 1375–1383.
4 Degre, M (1996)Interferons and other cytokines in bacterial
infections J Interferon Cytokine Res 16, 417–426.
5 Cross, J.C & Roberts, R.M (1989)Porcine conceptuses secrete an
interferon during the pre-attachment period of early pregnancy.
Biol Reprod 40, 1109–1118.
6 La Bonnardie`re, C., Martinat-Botte´, F., Terqui, M., Lefe`vre,
F., Zouari, K., Martal, J & Bazer, F.W (1991)Production of
two species of interferon by Large White and Meishan pig
con-ceptuses during the peri-attachment period J Reprod Fertil 91,
469–478.
7 Lefe`vre, F., Martinat-Botte´, F., Guillomot, M., Zouari, K.,
Charley, B & La Bonnardie`re, C (1990)Interferon-gamma gene
and protein are spontaneously expressed by the porcine
tropho-ectoderm early in gestation Eur J Immunol 20, 2485–2490.
8 Lefe`vre, F & Boulay, V (1993)A novel and atypical type one
interferon gene expressed by trophoblast during early pregnancy.
J Biol Chem 268, 19760–19768.
9 Arakawa, T., Hsu, Y.R., Chang, D., Stebbing, N & Altrock, B.
(1986)Structure and activity of glycosylated human interferon-c.
J Interferon Res 6, 687–695.
10 Ealick, S.E., Cook, W.J., Kumar, S.V., Carson, M., Nagabhushan,
T.L., Trotta, P.P & Bugg, C.E (1991)Three-dimensional structure
of recombinant human interferon-c Science 252, 689–701.
11 Denesyuk, A.I., Zavyalov, V.P & Korpela, T (1994)Common structural patterns of cytokine outer surfaces Bioch Biophy Res Comm 201, 1396–1400.
12 Dorner, F., Scriba, M & Weil, R (1973)Interferon: evidence for its glycoprotein nature Proc Natl Acad Sci USA 70, 1981–1985.
13 Sareneva, T., Pirhonen, J., Cantell, K., Kalkkinen, N & Julkunen,
I (1994)Role of N-glycosylation in the synthesis, dimerization and secretion of human interferon-c Biochem J 303, 1–10.
14 Arakawa, T., Narachi, M.A., Hsu, Y.R., Everett, R.R., Lai, P.H.
& Fish, E.N (1989)The effect of C-terminal processing on the activity of human interferon-c Drug Des Del 4, 217–225.
15 Honda, S., Asano, T., Kajio, T., Nakagawa, S., Ikeyama, S & Ichimori, Y (1987)Differential purification by immunoaffinity chromatography of two carboxy-terminal portion-deleted deriv-atives of recombinant human interferon-c from Escherichia coli.
J Interferon Res 7, 145–154.
16 James, D.C., Goldman, M.H., Hoare, M., Jenkins, N., Oliver, R.W.A., Green, B.N & Freedman, R.B (1996)Posttranslational processing of recombinant human IFN-c in animal expression systems Protein Sci 5, 331–340.
17 Hooker, A & James, D (1998)The glycosylation heterogeneity of recombinant human IFN-c J Interferon Cytokine Res 18, 287– 295.
18 Cencic, A., Lefe`vre, F., Koren, S & La Bonnardie`re, C (1999) Tetracycline-controlled expression of glycosylated porcine inter-feron-c in mammalian cells Anim Biotechnol 10, 63–79.
19 Cencic, A (1995)Porcine immune interferon M.Phil Thesis Biotechnical faculty, University of Ljubljana.
20 Vandenbroeck, K., Willems, L., Billiau, A., Opdenakker, G & Huybrechts, R (1994)Glycoform heterogeneity of porcine inter-feron-c expressed in Sf9 cells Lymphok Cytok Res 13, 253–258.
21 La Bonnardie`re, C & Laude, H (1981)High interferon titer in newborn pig intestine during experimentally induced viral enteritis Infect Immun 32, 28–31.
22 McClurkin, A.W & Norman, J.O (1966)Studies on Transmis-sible gastroenteritis of swine II selected characteristics of a cyto-pathogenic virus common to five isolates from transmissible gastroenteritis Can J Comp Med Vet Sci 30, 190–198.
23 Laemmli, U.K (1970)Cleavage of structural proteins during the assembly of the head of the bacteriophage T4 Nature 227, 680–685.
24 Messer, M., Griffiths, M., Rismiller, P.D & Shaw, D.C (1997) Lactose synthesis in a monotreme, the echidna (Tachyglossus aculeatus): isolation and amino acid sequence of echidna alpha-lactalbumin Comp Biochem Physiol 188B, 403–410.
25 Niu, P.D., Lefe`vre, F., Me`ge, D & La Bonnardie`re, C (1995) Atypical porcine type I interferon Biochemical and biological characterization of the recombinant protein expressed in insect cells Eur J Biochem 230, 200–206.
26 Dijkmans, R., Vandenbroeck, K., Beuken, E & Billiau, A (1990) Sequence of the porcine interferon-gamma (IFN-gamma)gene Nucleic Acids Res 25, 4259.
27 Geisert, R.D & Yelich, J.V (1997)Regulation of conceptus development and attachment in pigs J Reprod Fertil 52 (supplement), 133–149.
28 Menino, A.R., Hogan, A., Schultz, G.A., Novak, S., Dixon, W & Foxcroft, G.H (1997)Expression of proteinases and proteinase inhibitors during embryo-uterine contact in the pig Dev Genet.
21, 68–74.
29 Mortz, E., Sareneva, T., Julkunen, I & Roepstorff, P (1996)Does matrix-assisted laser desorption/ionization mass spectrometry allow analysis of carbohydrate heterogeneity in glycoproteins? A study of natural human interferon-c J Mass Spectr 31, 1109– 1118.
30 Luk, S.K., Jay, E & Jay, F.T (1990)Structure-function analysis
of the human interferon-gamma The COOH terminus is not essential for functional activity J Biol Chem 265, 13314–13319.
Trang 1031 Carter, W.A., Swartz, H & Gillespie, D.H (1985)Independent
evolution of antiviral and growth-modulating activities of
inter-feron J Biol Resp Mod 4, 447–459.
32 Goldberg, M., Belkowski, L.S & Bloom, B.R (1989)Regulation
of macrophage growth and antiviral activity by interferon-gamma.
J Cell Biol 109, 1331–1340.
33 Caruso, A., Tiberio, L., De Rango, C., Bonfanti, C., Flamminio, G., Gribaudo, G., Monti, E., Viani, E., Manca, N., Garotta, G., Landolfo, S & Turano, A (1993)A monoclonal antibody to the NH2-terminal segment of human IFN-c selectively interferes with the antiproliferative activity of the lymphokine J Immunol 150, 1029–1035.