The porcine trophoblastic interferon-c, secreted by a polarized
epithelium, has specific structural and biochemical properties
Avrelija Cencic
ˇ
1,2
,Ce
´
line Henry
3
, Franc¸ois Lefe
`
vre
1
, Jean-Claude Huet
3
, Srecko Koren
4
and Claude La
Bonnardie
`
re
1
1
Unite
´
de Virologie et d’Immunologie Mole
´
culaires, INRA, Jouy en Josas, France;
2
Faculty of Agriculture, University of Maribor,
Slovenia;

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 of
L
-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 apartic-
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
Ôantiviral stateÕ 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
1
and natural killer
(NK) cells, and exerts various modulating effects on the

, 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)
Eur. J. Biochem. 269, 2772–2781 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02950.x
Full-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-

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 m
M
MgCl
2
at 37 °C
until D
600
¼ 1.0. INF-c expression was induced by the
addition of 1 m
M
isopropyl thio-b-
D

)1
in
an organic base and 0.02% H
2
O
2
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
expressedinIUÆmg
)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

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 (A
280
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
)1
NaCl in Tris/HCl, pH 8.0) at a flow rate of
1mLÆmin
)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/P

12 400). The void volume of the column
was measured by use of Blue Dextran (M
r
2000 000).
35
S-Labelling of natural IFN-c. For LeIFN-c,pigPBL
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 [
35
S]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 [
35
S]Met-Cys
(100 lCiÆmL
)1
) were used.
Immunoprecipitation and deglycosylation of IFN-c. The
35
S-labelled IFN-c were concentrated against poly(ethylene
glycol) (M
r
20 000) to 2 mL and processed for immuno-

phoresis in SDS/PAGE, then electro-transferred on a
ProBlott membrane, which was stained with Coomassie
Blue R 250. The two main bands (M
r
22 500 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
0.2
M
Tris/HCl pH 8.5, 2%SDS, for 30 min. After swelling,
200 lL of HPLC water was added and then a 4 · 4mm
piece of prewet
5
(methanol) PVDF membrane (Problott) was
added to the solution. The procedure required 2 days at
room temperature (23 °C) with gentle vortexing. At the end,

2774 A. Cencic
ˇ
et al. (Eur. J. Biochem. 269) Ó FEBS 2002
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% CH
3
CN
in 50 m
M
NH
4
CO
3
(v/v) and then transferred to Eppendorf
tubes. After dessication of the gel in SpeedVac for 30 min,
the digestion was performed in 25 lLof50 m
M
ammonium
bicarbonate pH 8.0 and 0.5 lgofmodifiedtrypsin
(Promega, sequencing grade) for 18 h in a thermomixer

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.
RESULTS
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 M
r
of native TrIFN-c was measured by
gel-filtration, in comparison with those of crude LeIFN-c
and unglycosylated rIFN-c.Eachcolumnfractionwas
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 M
r
of 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

monomers 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: M
r
24 800; 22 000; 19 800; 17 500), the
M
r
24 800 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 M
r
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

22 500 and 18 000 (lane 1), and upon
deglycosylation, one major band at M
r
14 400 was seen.
But unlike TrIFN-c collected in the supernatant of cultured
conceptuses, a minor deglycosylated band was obtained at
M
r
12 000 (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 [
35
S]Met metabolically labelled native
TrIFN-c and LeTrIFN-c. Lane 1, control LeTrIFN-c.Lane2,
N-glycosidase F treated LeIFN-c.Lane3,controlTrIFN-c.Lane4,
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.Lane3,conceptussecretedIFN-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

The MALDI-MS analysis of the minor peak with an M
r
of 12 000 yielded an observed (M + H)
+
of 12 635.0 Da
(Fig. 4C). This is compatible with a deglycosylated poly-
peptide with R
107
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 (D
mass
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 M
r

7 12 EITILK 716.455 716.452 )0.003
13 34 DYF…ILK 2397.198 4540.260
4394.224
4250.802
2143.062
1997.026
1853.604
Glycosylation
Glycosylation
Glycosylation
44 55 IIQSQIVSFYFK 1472.814 1472.847 0.033
56 61 FFEIFK 830.444 830.459 0.015
62 68 DNQAIQR 844.427 844.452 0.025
69 74 SMDVIK 692.364 692.348
708.351
)0.016
15.987 Oxidized Met
75 80 QDMFQR 824.372 824.395
840.379
0.023
16.007 Oxidized Met
81 88 FLNGSSGK 809.416 2805.871
2951.970
1996.455
2142.554
Glycosylation
Glycosylation
98 107 IPVDNLQIQR 1195.679 1195.762 0.083
89 94 LNDFEK 765.377 765.383 0.006
109 115 AISELIK 773.476 773.480 0.004

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 monoglycosylated isoforms, respectively, the latter
isoform being glycosylated on N16. As expected, the
correspondance between the measured masses and observed
M
r
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 · 10
5
UÆmg
)1
of IFN-c (ELISA reactive), i.e. approxi-
mately 10 times lower than that of its ÔadultÕ 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

5
1–5 · 10
6
2–3.5 · 10
6
5–10 · 10
6
(UÆmg
)1
IFN-c)
2778 A. Cencic
ˇ
et al. (Eur. J. Biochem. 269) Ó FEBS 2002
significant effector of any APA by comparing the effect of
dilutions from 300 ngÆmL
)1
to 1.2 ngÆmL
)1
in 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,andtwo
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,

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,isa
dimer. The weak antiviral activity found in fractions
corresponding to monomers is certainly that of IFN-d,
with an M
r
around 19 000, which is also present in uterine
flushes [25].
As revealed by the electrophoretic profiles of
35
S-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 M
r
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
M
r
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

-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

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.
ACKNOWLEDGEMENTS
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.
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