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*
Department of Pathology, Section of Immunology, University of Verona, Verona, Italy; and
The Wistar Institute, Philadelphia, PA 19104
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain, formerly defined as p35, disulfide-linked to a heavier
ß-chain, formerly defined as p40. The ß-chain is also produced in
large excess in a free form, and disulfide-linked ß-chain homodimers
with anti-inflammatory effects are produced in the mouse. We
analyzed the biosynthesis and glycosylation of IL-12 in human
monocytes, and in a cell line stably transfected with IL-12 and ß
genes (P5-0.1). The IL-12 heterodimer and free ß-chain were
immunoprecipitated from supernatants and cell lysates of metabolically
labeled cells and resolved in SDS-PAGE. Whereas the ß-chain showed
similar pI pattern whether in the free form or associated in the
heterodimer, either in the secreted or intracellular form, the
-chain in the secreted heterodimer was much more acidic than that
present in the intracellular heterodimer. Deglycosylation experiments
with neuraminidase and Endo-F combined with two-dimensional PAGE of
single bands of the intracellular vs extracellular IL-12 heterodimer
revealed that the -chain was extensively modified with sialic acid
adducts to N-linked oligosaccharides before secretion.
N-glycosylation inhibition by tunicamycin (TM) did not
alter free ß-chain secretion, while preventing the IL-12 heterodimer
assembling and secretion. Pulse-chase experiments indicated that IL-12
persists intracellularly for a long period as an immature heterodimer,
and that glycosylation is the regulatory step that determines its
secretion. ß-chain disulfide-linked homodimers were observed in
TM-treated P5-0.1 cells, but in neither TM-treated nor untreated
monocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. IL-12 is also the major cytokine
responsible for the differentiation of Th1 cells, which are potent
producers of IFN-. IFN-, in turn, has a powerful enhancing effect
on the ability of phagocytes and dendritic cells to produce IL-12
(2), acting therefore as a potent positive feedback
mechanism that leads to a strong, defensive response against
intracellular pathogens and that represents a potentially dangerous
mechanism for uncontrolled cytokine production, shock, or for induction
of autoimmunity.
IL-12 is a heterodimeric molecule (3, 4) composed of an
-chain (formerly the p35 subunit) and a ß-chain (formerly the p40
subunit) linked by a disulfide bridge to form the biologically active
74-kDa heterodimer. Secretion of the isolated -chain has never been
detected; in contrast, the cells that produce the biologically active
IL-12 heterodimer secrete ß-chain in free form in a 10- to 100-fold
excess over the IL-12 heterodimer (5, 6); depending on the
stimulus, consistent amounts of free ß-chain can also be produced in
the absence of the heterodimer (7). A biological function
of free ß-chain has never been observed, and its physiological
significance is still debated. Disulfide-linked homodimers of ß-chain
are produced in the mouse (8); murine ß-chain
homodimers, in contrast to the free ß-chain, have the ability to
block IL-12 functions in vitro and in vivo (9, 10). The
existence of human ß-chain homodimers has been demonstrated up to now
only in ß-chain-transfected cell lines (11). The
physiological relevance of human ß-chain homodimers is still
debated.
The -chain gene is constitutively expressed in most cell types at
low levels, while the expression of ß-chain gene is restricted to
those cells that are able to produce IL-12 heterodimer
(6). Expression of IL-12 - and ß-chains is
transcriptionally and independently regulated (2, 12).
However, for many proteins, posttranslational mechanisms are also
important in the regulation of expression and function of proteins. A
ubiquitous posttranslational modification is glycosylation (13, 14), in which a vast array of oligosaccharide structures can be
assembled on polypeptides and further modified by galactose,
N-acetylgalactosamine, L-fucose,
sialic acid, sulfate, and phosphate groups. Protein glycosylation has a
variety of functions, including involvement in the folding of nascent
proteins in the endoplasmic reticulum, protection of the protein from
the action of proteases, intracellular and extracellular sorting, and
modulation of the biologic activity of the protein; protein
glycosylation is also recognized as a marker in quality control
function of the endoplasmic reticulum (15, 16).
In the present study, we analyzed the biosynthesis and glycosylation of
IL-12 in normal human monocytes and in a IL-12 and ß
gene stably transfected cell line. Our results indicate that the
ß-chain in the heterodimer is indistinguishable from the free
ß-chain with respect to m.w., isoelectric point, and
posttranslational modifications, and that only minor
modifications in the ß-chain occur during biosynthesis. By
contrast, the -chain of the heterodimer is extensively
posttranslationally modified by N-linked adducts and sialic
acid during biosynthesis. These modifications of the -chain
represent the marker that distinguishes the secreted mature heterodimer
from the intracellular immature heterodimer and play a key role in
IL-12 assembling and secretion. Finally, ß-chain homodimers were
observed in the IL-12-transfected cells after N-linked
glycosylation inhibition, but were undetectable in IL-12-producing
normal monocytes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All reagents used in this study were tested for endotoxin
contamination using the Limulus amebocyte assay. PBMC
obtained from healthy human donors were separated by Ficoll-Paque
density-gradient centrifugation. Monocytes were enriched from PBMC by
depletion of T cells using E-rosetting with
2-aminoethylisothiouronium bromide hydrobromide-treated SRBC and
density-gradient separation on Ficoll-Paque. The enriched cell
suspension contained more than 70% monocytes, as evaluated by direct
immunofluorescence with anti-CD14 mAb (Becton Dickinson, San Jose,
CA). Cells were resuspended in RPMI 1640 medium (Biochrom, Berlin,
Germany) supplemented with 10% heat-inactivated FCS (HyClone, Logan,
UT) and L-glutamine. To induce IL-12 protein
synthesis, monocytes (3 x 106 cells/ml)
were cultured for 16 h in the presence of IL-4 (10 ng/ml; Genzyme,
Cambridge, MA) (17) and for an additional 15 h with
IFN- (1000 U/ml; kindly provided by Roussel-Uclaf, Romainville,
France). Cells were stimulated with LPS (1 µg/ml; Sigma, St. Louis,
MO) for 18 h, unless otherwise indicated in Results.
Chinese hamster ovary cells stably transfected with human IL-12 and
ß gene (P5-0.1; kindly provided by Stanley Wolf, Genetics Institute,
Cambridge, MA) were cultured in RPMI 1640 supplemented with 10% FCS
and 0.1 µM methotrexate (Sigma). These cells express constitutively
similar levels of - and ß-chain mRNA, and thus secrete only the
heterodimer at 400 ng/106 cells/24 h, and not the
free ß-chain.
Antibodies
The following mAbs were used: C11.79 and C8.6, which recognize
the IL-12 ß-chain; 12H4, recognizing the IL-12 -chain; and 20.C2,
recognizing the IL-12 heterodimer (6). To increase binding
capacity, mAbs were affinity purified on Sepharose-protein G columns
and covalently immobilized separately or as a mixture of the four mAbs
on CNBr-activated Sepharose 4B beads (Pharmacia, Piscataway, NJ). Each
mAb was coupled at 1-6 mg/g of Sepharose beads. The Sepharose-coupled
mixture of mAbs C11.79, 12H4, 20C2, and C8.6 was used in all
experiments, unless otherwise specified.
[35S]Methionine radiolabeling
For continuous labeling with
[35S]methionine, IL-4-, IFN--primed
monocytes were resuspended at 10 x 106/ml
in methionine-free RPMI 1640 supplemented with 10% FCS and 7% normal
RPMI 1640 as a source of cold methionine, and incubated with
[35S]methionine (165 µCi/ml, 1200 Ci/mmol;
NEN-DuPont, Boston, MA) and LPS. P5-0.1 IL-12 stably transfected cells
were grown in tissue culture flasks at 1 x
106 cells/ml before radiolabeling for 3 h
with 125 µCi/ml [35S]methionine in
methionine-free medium supplemented with 10% FCS and 7% normal RPMI
1640. In some experiments, P5-0.1 cells and IL-4-, IFN--primed
monocytes were preincubated for 1 h with tunicamycin
(TM)4 (4, 18) at 3 µg/ml in methionine-free medium, followed by labeling
for 4 or 7 h, respectively, for P5-0.1 and monocytes, with
[35S]methionine in the presence of TM (3
µg/ml). Viability of TM-treated cells, as determined by trypan blue
exclusion, did not differ from that of control cells and was always
higher than 95%.
For pulse-chase [35S]methionine radiolabeling, P5-0.1 cells were placed in methionine-free medium for 30 min at 37°C, rapidly washed with the same medium, and incubated in methionine-free medium containing 300 µCi/ml of [35S]methionine for 20 min at 37°C (pulse). Radioactive medium was then discarded and cells were rapidly washed with RPMI 1640 medium supplemented with 10% FCS and 4 mM cold methionine. The chase was initiated by the addition of the same medium and continued at 37°C for various times. Cells were rapidly chilled on ice and washed with 10 ml of ice-cold PBS. Radiolabeled Ags were isolated and purified as described below.
Immunoprecipitation of radiolabeled IL-12
All procedures (19) were performed at 4°C. Cell culture supernatants were recovered, centrifuged to eliminate residual cells, and immunoprecipitated after addition of 10% of 10 mM Tris-HCl, pH 8.2, 150 mM NaCl, and 0.02% NaN3 containing 1% Nonidet P-40 and 10 µg/ml leupeptin (Sigma), 10 µg/ml antipain (Sigma), 2 mM EDTA, and 2 mM iodoacetamide as protease inhibitors. Radiolabeled cells were washed by centrifugation with PBS, and the cell pellet was resuspended in TBS solution containing 1% Nonidet P-40 and protease inhibitors. After 1-h incubation, lysates were centrifuged at 15,000 x g for an additional 10 min. Supernatants and cell lysates were precleared with Sepharose-protein A and incubated with Sepharose-immobilized mAbs for 2 h with shaking. mAb-coupled Sepharose beads were recovered by centrifugation and washed sequentially with TBS/0.1% Nonidet P-40 (four times) and with 10 mM Tris-HCl, pH 8.2, containing 0.1% Nonidet P-40 (twice). IL-12 chains were eluted from the beads by heating at 100°C for 4 min in SDS-PAGE sample buffer.
SDS-PAGE and isolation of proteins
[35S]Methionine-labeled specific immunoprecipitates were purified by SDS-PAGE conducted essentially as described (20). IL-12 chains were identified by autoradiography (24–48 h), appropriate gel region was cut out, and single polypeptides were eluted for 24–36 h in three steps with 250 µl of PBS containing 0.2% SDS and 50 mM dithiothreitol. Sample glycoproteins were boiled for 4 min, alkylated in the dark with 130 mM iodoacetamide at 37°C for 45 min, precipitated with TCA (final concentration 12% w/v) for 4 h at 4°C, and recovered by centrifugation at 14,000 x g for 5 min on a microfuge. Protein pellets were incubated with cold acetone at -20°C for 2 h, washed three times with cold acetone (14,000 x g for 5 min) on a microfuge, and vacuum-dried and resolubilized in isoelectric focusing (IEF) (21) or 2D-peptide mapping sample buffer (22).
2D-peptide mapping
This was conducted essentially as described (22) with minor modifications. Briefly, Ag eluted from the immunoabsorbent was purified by SDS-PAGE and extracted from the gel, as described above. Vacuum-dried pellets were resolubilized in 100 µl of 1% formic acid/10% acetic acid in water (digestion buffer). Samples were digested with 15 µl of pepsin (Worthington Biochemical, Lakewood, NJ; 150 µg/ml in digestion buffer) for 18 h at 37°C and vacuum dried. Peptide fragments were analyzed by electrophoresis in the first dimension on 200-µm silica gel plates with a Desaphor (Desaga, Heidelberg, Germany) at pH 3.5 for 6 h at 350 V in pyridine/glacial acetic acid/H2O (10:100:890 (v/v)). Ascending chromatography was performed as the second dimension with glacial acetic acid/pyridine/H2O/n-butanol (15:50:40:75 (v/v)). Silica gel plates were dried, exposed to a Kodak phosphor screen for 25–45 days, and developed on a Molecular Dynamics (Sunnyvale, CA) PhosphorImager.
2D-PAGE
This was performed as described (21), with nonequilibrium pH gradient electrophoresis in the first dimension. Immunoprecipitates were purified on SDS-PAGE, and relevant protein bands were extracted as described above. Vacuum-dried pellets were resuspended in 50 µl of a solution containing 9.5 M urea, 2% Nonidet P-40, 5% 2-ME, and 2% ampholines (4:1 Pharmalyte 5/7: Pharmalyte 3/10; Pharmacia). The first-dimension tube gels contained 2% Pharmalyte 3/10 ampholines and were run at 500 V for 4 h. The second dimension was run in 11% SDS-PAGE. For direct comparison of two samples, the top 7 cm of the two tube gels were placed side by side on a slab gel. After electrophoresis, slab gels were dried, exposed to a Kodak phosphor screen for 25–45 days, and developed on a Molecular Dynamics PhosphorImager. Densitometric analysis was performed using ImageQuant software.
Endo-F deglycosylation
Endo-F digestion was conducted essentially as described (23) with minor modifications. Briefly, Ag eluted from the immunoabsorbent was purified by SDS-PAGE and extracted from the gel, as described above. Extracted samples were vacuum dried, and protein pellets were resolubilized in 50 µl of 100 mM Tris-HCl, pH 7.5, containing 1% SDS and 1% 2-ME, and boiled for 5 min. Samples were diluted in 450 µl of 100 mM sodium phosphate, pH 6.1, containing 50 mM EDTA, 1% Nonidet P-40, 1% 2-ME, 10 µg/ml leupeptin, and 10 µg/ml antipain. Endo-F (Boehringer Mannheim, Indianapolis, IN) was added to a final concentration of 6 U/ml, and samples were incubated for 18 h at 37°C. After digestion, samples were precipitated with TCA, washed in cold acetone, vacuum dried, resuspended in 50 µl of IEF sample buffer, and analyzed by 2D-PAGE.
Neuraminidase (NANAse) treatment
For samples to be treated with NANAse (24), Ag eluted from the immunoabsorbent was purified by SDS-PAGE and extracted from the gel, as described above. Vacuum-dried pellets were resuspended in 70 µl of PBS, pH 7.2, containing 1 mM CaCl2 and 5 U/ml of type V Cl. perfringens NANAse (Sigma) for 2 h at 37°C. Control precipitates were incubated in the same buffer without enzyme. Desialized glycoproteins were precipitated overnight with 950 µl acetone at -20°C and washed twice in cold acetone. Vacuum-dried samples were resuspended in 50 µl of IEF sample buffer and resolved in 2D-PAGE.
Data analysis
Radiolabeled material in each band or spot was quantitated by PhosphorImager scanning (Molecular Dynamics) and evaluated as Volume Analysis (ImageQuant software). Molar ratios of secreted free ß-chain (sum of the p39 and p36 bands) to IL-12 heterodimer were calculated as: (volume data of free ß-chain/2)/(volume data of IL-12 heterodimer/11), where 2 and 11 indicate methionine composition in the free ß-chain and in the IL-12 heterodimer, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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[35S]Methionine biosynthetically labeled
culture supernatants and cell lysates were obtained from 1) monocytes
primed with IL-4 and IFN- and stimulated with LPS, and 2) P5-0.1
cells stably transfected with human IL-12 and ß genes that
produce constitutively the IL-12 heterodimer, but not the free
ß-chain. Samples were immunoprecipitated with several anti-IL-12
mAbs and analyzed by SDS-PAGE under nonreducing conditions (Fig. 1
). The mixture of the four mAbs
(lane a) or the anti-ß-chain mAb C11.79 alone
(lane c) immunoprecipitated three distinct species of
74, 39, and 36 kDa from supernatants of stimulated monocytes. The
anti--chain mAb 12H4 (lane b) and the
anti-IL-12 heterodimer mAb 20C2 (lane d)
immunoprecipitated a single 74-kDa species from the same supernatants.
Thus, we can identify the p74 band as the IL-12 heterodimer, and the
p36 and p39 bands as the free ß-chain. The mixture of the four mAbs
immunoprecipitated a single 74-kDa band, but not the 39- or 36-kDa
species from supernatant of P5-0.1 cells (lane e),
confirming that this cell line produces the IL-12 heterodimer, but not
free ß-chains.
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2D-peptide mapping
To characterize and confirm the common derivation of the
intracellular and the mature secreted forms of IL-12 heterodimer,
2D-peptide mapping was performed on
[35S]methionine-labeled P5-0.1 intracellular
CLp67 and secreted SNp74 bands (Fig. 2,
inset). Fingerprints obtained from the CLp67 (Fig. 2a) and the SNp74 (Fig. 2b) IL-12 heterodimers
were virtually superimposable, indicating a common derivative
polypeptide. Differences, marked by arrows, probably reflect
posttranslational modifications during maturation of the chains.
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d) was virtually superimposable to that of SNp74 from
P5-0.1 cells. In the monocyte SNp40 free ß-chain map (Fig. 2c), all of the major spots were clearly shared with those
of the monocyte SNp74 map. Thus, the SNp40 band represents the actual
free IL-12 ß-chain. Minor differences (asterisks in Fig. 2, c and d) probably reflect slight differences
between the free and the heterodimerized ß-chain in protease
sensitivity or in posttranslational modifications. Comparison of intracellular and extracellular IL-12 heterodimers by 2D-PAGE
To characterize the biosynthetic steps of IL-12 heterodimer, cell
lysates and supernatants were obtained from primed, stimulated
[35S]methionine-radiolabeled monocytes, and
from IL-12-transfected P5-0.1 cells and immunoprecipitated with
anti-IL-12 mAbs. Relevant bands in P5-0.1- and monocyte-derived
cell lysates and supernatants (far left panels in
Fig. 3) were separately eluted from
SDS-PAGE, reduced, alkylated, and resolved in 2D-PAGE. SNp74 chain
(Fig. 3, c and i, from P5-0.1 and monocytes,
respectively) migrated as two different series of spots: the first
series, representing the ß-chain, appeared as three to four acidic
spots at 44 kDa, and three to four intense, slightly more basic spots
at 41 kDa; the second series, representing the -chain, migrated in a
larger pI range, as two parallel series at 33 and, with greater
intensity, at 35 kDa. No 74-kDa spots were detectable (not shown),
confirming that the 74-kDa band represents the IL-12 heterodimer. As
compared with monocytes, P5-0.1 cells were characterized by a markedly
more acidic pI of the -chain in the mature IL-12 heterodimer,
despite the superimposable migrational pattern of the ß-chain.
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a, and CLp70 of monocytes, Fig. 3g) revealed much more basic spots for -chains than those
resolved from the secreted mature form of the heterodimer (SNp74 of
P5-0.1, Fig. 3c, and SNp74 of monocytes, Fig. 3i). By contrast, the higher m.w. form of the intracellular
heterodimer (CLp74 in P5-0.1, Fig. 3b, and CLp74
in monocytes, Fig. 3h) showed a migrational pattern similar
to that of the mature, secreted molecule, most likely representing the
mature IL-12 before secretion. The basic spots of immature
intracellular -chain were never found in the -chain derived from
extracellular mature IL-12 heterodimer in any donor tested.
The ß-chain resolved from the lower m.w. forms of intracellular
heterodimer (CLp67 + 70 of P5-0.1, Fig. 3a, and CLp70 of
monocytes, Fig. 3g) was indistinguishable from that in the
mature secreted SNp74 for the major p41 component, while the minor p44
component derived from P5-0.1 cells was slightly more acidic in the
CLp74 and in the mature secreted form (Fig. 3c).
In conclusion, in monocytes and in P5-0.1 cells, the -chain, unlike
the ß-chain, was heavily acidified during maturation of the IL-12
heterodimer.
Deglycosylation of IL-12 heterodimers with NANAse
To determine the nature of the acidic adducts of the IL-12
heterodimer during maturation, P5-0.1-derived CLp67 + 70-,
CLp74-, and SNp74-eluted bands and monocyte-derived SNp74-eluted
band were digested with NANAse and resolved in 2D-PAGE under
reducing conditions (Fig. 3, d, e, f,
and j). NANAse treatment did not affect the major p41
component of the ß-chain derived from the mature SNp74 (compare
c with f, and i with j in
Fig. 3), from the mature CLp74 (compare b with
e), and from the immature CLp67 + 70 (compare a
with d), indicating the absence of sialic acid adducts on
this protein. The minor p44 component of the mature heterodimer shifted
slightly toward the basic end of the gel, becoming superimposable on
the p44 component of the immature heterodimer. On the other hand, the
-chain derived from monocyte or P5-0.1 SNp74 showed a marked shift
toward the basic end of the gel after NANAse treatment, indicating
extensive sialic acid modification to this glycoprotein chain; the
-chain of the immature CLp67 + 70 band was unaffected by NANAse
treatment. Note that after NANAse treatment, the extracellular mature
form of IL-12 showed a 2D-PAGE identical to that of the immature
intracellular form. These results indicate that the mature secreted
IL-12 heterodimer derives from an -chain that is heavily modified
with sialic acid adducts and a ß-chain that is slightly modified by
sialic acid adducts only on the minor p44 component.
Characterization of intracellular and secreted free ß-chain
Comparison of the free ß-chain p39 and p36 bands in
immunoprecipitates obtained from cell lysates and supernatants of
primed, radiolabeled, stimulated monocytes revealed that the two CLp39
and CLp36 bands were very similar to the extracellular SNp39 and SNp36
bands (Fig. 4, far left
panel). In 15 different donors, levels of SNp36 were consistently
6-fold greater than those of SNp39. The free ß-chain derived from
cell lysates (CLp39 and CLp36 bands) and supernatants (SNp39 and SNp36
bands) were cut from the gel, eluted, reduced, alkylated, and resolved
under reducing conditions in 2D-PAGE. 2D-migration patterns of
intracellular (Fig. 4a) and secreted (Fig. 4b)
free ß-chains were superimposable on the pattern of ß-chain derived
from intracellular and secreted IL-12 heterodimers (see Fig. 3). After
reduction, the p39 and p36 ß-chain components migrated with apparent
m.w. of 41 and 44 kDa, respectively, most likely due to the opening of
the four intrachain disulfide bridges of the IL-12 ß-chain. Like the
ß-chain in the heterodimer, the major p41 component of the ß-chain
derived from the secreted free form was unaffected by NANAse treatment
(inset in Fig. 4b), while the minor p44 component
of the secreted free ß-chain shifted slightly toward the basic end of
the gel. Thus, the intracellular and the secreted free ß-chain
appears indistinguishable from the ß-chain linked in the
heterodimer.
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3 kDa)
reduction in size and a slight shift toward the acidic end of the gel
(Fig. 4d), as compared with the control (Fig. 4d'), while the SNp36 more representative component remained
unchanged (Fig. 4, c and c'). These modifications
indicate the presence of Endo-F-sensitive uncharged sugar(s), with a
slightly acidic shift following a partial asparagine to aspartic acid
conversion due to the pH of the enzyme treatment (25).
Note that after Endo-F treatment, SNp39 showed the same molecular and a
slightly more acidic pI as compared with SNp36, indicating that p39
chain differs from the p36 chain only by the addition of a neutral
sugar. Role of glycosylation in IL-12 assembling and secretion
To define the role of glycosylation in the biosynthesis of IL-12, P5-0.1 cells and monocytes were metabolically labeled in the presence or absence of TM (3 µg/ml). This treatment did not alter cell viability as evaluated by trypan blue exclusion.
Intracellular IL-12 was immunoprecipitated from TM-treated P5-0.1 cells
in similar amount than from control untreated cells, but, instead of
migrating at 74–70-67 kDa, it appeared as a 60-kDa band in SDS-PAGE,
with a considerable amount of material aggregated at the origin of the
gel (>200 kDa) and a small amount migrating at 34 kDa (Fig. 5, left panel). In 2D-PAGE
under reducing conditions, the 60-kDa band (Fig. 5c)
appeared as a heterodimer with charge characteristics similar to those
of the 67–70-kDa immature form of intracellular IL-12 in untreated
cells (shown in Fig. 3a). The >200-kDa band (Fig. 5a) migrated in 2D-PAGE as - and ß-chains identical to
those of the 60-kDa heterodimer, with the majority of the material
represented by -chain, indicating that the >200-kDa material
represents the - and ß-chains of IL-12 in differently aggregated,
poorly soluble forms. 2D-peptide mapping confirmed the common peptide
origin of the 60- and >200-kDa bands (not shown). The region around
the 34-kDa band, which upon longer exposure showed heterogeneity (not
shown), resolved in 2D-PAGE as free ß-chains and a small amount of
free -chains (Fig. 5e).
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, left panel). The 60-kDa band
(Fig. 5d) resolved in 2D-PAGE in a pattern comparable with
that of the corresponding intracellular IL-12 (Fig. 3a), but
characterized by a more acidic pI of the -chain. The 74-kDa band
consisted of disulfide-linked ß-chain homodimers (Fig. 5b). The region around the 34-kDa band consisted of free
ß-chain and a small amount of free -chain (Fig. 5f).
Similarly to TM-treated P5-0.1 cells, TM-treated monocytes (Fig. 6A) secreted less than 5% of
the IL-12 heterodimer than untreated cells, while secretion of free
ß-chain remained almost unaffected (85% of the control). IL-12
heterodimers and free ß-chains secreted from TM-treated monocytes
migrated at 64, 35, and 33 kDa under nonreducing conditions in
SDS-PAGE, instead of 74-, 39-, and 36-kDa migration with IL-12 secreted
from untreated monocytes, and no band corresponding to the 74-kDa
disulfide-linked ß-chain homodimers secreted by TM-treated P5-0.1
cells was detected.
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- and ß-chains in
TM-treated P5-0.1 cells, together with the results of the NANAse
experiments indicate that posttranslational oligosaccharide
modifications of the IL-12 heterodimer by sialic acid adducts on the
-chain of the IL-12 heterodimer are required for IL-12 heterodimer
secretion and correct assembling. In contrast to the heterodimer, the
amount of free ß-chain secreted from stimulated monocytes was largely
unaffected by TM treatment, indicating that N-linked
oligosaccharides are crucial elements for the secretion of the
heterodimer, but not of the free ß-chain. Lack of production of disulfide-linked ß-chain homodimers in stimulated monocytes
The finding of disulfide-linked ß-chain homodimers in TM-treated
P5-0.1 cells, but not in the TM-treated monocytes strongly suggested
that the formation of these structures was peculiar to the
IL-12-transfected cell line. To exclude the production of such
covalently linked ß-chain homodimers in monocytes,
[35S]methionine-labeled supernatants of primed,
stimulated monocytes were immunoprecipitated with the
anti--chain 12H4 mAb (Fig. 6B, line 1) and
further depleted of IL-12 heterodimers by repeated preclearing with the
12H4 mAb. Depleted supernatants were subsequently immunoprecipitated
with the pool of anti-IL-12 mAbs (line 2). After
depletion of the 74-kDa IL-12 heterodimer, no material was left
corresponding to ß-chain homodimers analogous to those observed in
TM-treated P5-0.1 cells. Thus, ß-chain homodimers are not secreted by
IL-12-producing human monocytes.
Processing of IL-12 heterodimers
The relevance of glycosylation of immature IL-12 heterodimer for
its secretion was further confirmed by pulse-chase experiments in
P5-0.1 cells. No shift of the intracellular immature CLp67 toward the
mature CLp74 band was observed during the time course (Fig. 7); the parallel decrease of the
intracellular heterodimer forms indicates that polypeptide
glycosylation is the regulatory step ultimately determining IL-12
heterodimer secretion. Similar results were obtained in monocytes (not
shown).
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In supernatants from primed monocytes stimulated for 18 h
with LPS, the ratio of the free ß-chain to the IL-12 heterodimer
ranged from 6 to 30 times (18 ± 7) in 15 different donors. To
determine whether the ratio of free ß-chain to IL-12 heterodimer
secretion might depend on the duration of stimulation, anti-IL-12
mAb immunoprecipitates from supernatants of primed, labeled monocytes,
stimulated with LPS for different times, were analyzed by SDS-PAGE
under nonreducing conditions (Fig. 8A). Ratios of secreted free
ß-chain to IL-12 heterodimer increased over time (Fig. 8B), indicating that their secretion is independently
regulated and that ß-chain accumulates in the supernatant at longer
times after stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß genes, and we determined the importance of
N-linked oligosaccharide adducts and sialylation for the
correct assembling and secretion of the molecule.
2D-PAGE and deglycosylation analysis of single bands purified by
SDS-PAGE after immunoprecipitation with anti-IL-12 mAbs allowed us
to analyze in detail the intracellular and extracellular IL-12
heterodimers and the free ß-chain. Both the intracellular and the
secreted IL-12 heterodimers are formed by a ß- and an -chain,
linked by a disulfide bridge. The intracellular IL-12 heterodimer
appears as a doublet band of 70 and 74 kDa in monocytes and as a
triplet of 67, 70, and 74 kDa in P5-0.1 cells, while a single 74-kDa
band represents the secreted mature heterodimer in both monocytes and
P5-0.1 cells, indicating that structural modifications of the molecule
take place before its secretion. In monocytes, a large amount of free
ß-chains was present intracellularly and secreted as a doublet of 36
and 39 kDa.
Comparison of the 2D-PAGE migration pattern of the - and ß-chains
in the secreted IL-12 heterodimer and in the different forms of
intracellular heterodimers identified the 70-kDa band in monocytes and
the 67- and 70-kDa bands in P5-0.1 cells as immature molecules, while
the intracellular 74-kDa band represents the mature molecule before
secretion in both cell types. The ß-chain of the intracellular
heterodimer was essentially unmodified as compared with that of the
secreted heterodimer, and with the intracellular and secreted free
ß-chain. The invariability of these ß-chain forms was further
suggested by the equal molar ratios between p36 and p39 derived from
IL-12 heterodimers and from free ß-chains in all donors tested. The
p39 component of the ß-chain represents the same polypeptide chain as
the p36 component, posttranslationally modified by addition of
N-linked, uncharged sugars. In contrast to the invariability
of the ß-chain, at least in the predominant p36 form, the -chain
appeared in different posttranslationally modified forms. The -chain
derived from the intracellular immature IL-12 heterodimer has a much
more basic charge than that derived from the mature intracellular or
secreted IL-12 heterodimer. The secreted, mature IL-12 and the
intracellular p74 mature form derive from discrete sialylation steps on
the -chain of intracellular immature heterodimer, as demonstrated by
NANAse experiments. The basically charged immature forms of -chain
that were observed in the intracellular heterodimers and in
NANAse-treated, secreted mature heterodimer were never found in the
-chain derived from secreted mature heterodimers in 10 different
donors analyzed. Thus, sialylation of the -chain appears to be a key
requirement for the secretion of the IL-12.
Inhibition of N-linked sugar adduction by TM at
concentrations that did not affect the IL-12 biosynthesis, as
demonstrated by unaltered amounts of intracellular immunoprecipitated
IL-12 in P5-0.1 cells, and by comparable amounts of secreted free
ß-chain in TM-treated and control monocytes, drastically inhibited
IL-12 heterodimer secretion in both cell types. Moreover, assembly of
deglycosylated - and ß-chains to form the heterodimer was
profoundly altered, as demonstrated by the presence of intracellular
high m.w. aggregates composed prevalently by -chains, and by the
presence of intracellular and secreted free - and ß-chains, never
observed in untreated P5-0.1 cells. High m.w. aggregates were not found
in secreted products, suggesting that incompletely
glycosylated/sialylated IL-12 is intracellularly degraded.
Moreover, the -chain of the intracellular IL-12 heterodimer in
TM-treated cells, the -chain of the immature intracellular IL-12 in
untreated cells, and the NANAse-treated -chain of the mature and
secreted IL-12 heterodimer had a similar pI pattern, supporting the
conclusion that terminal addition of sialic acid to N-linked
oligosaccharides of -chain represents the key regulatory element in
the correct production and secretion of IL-12 heterodimer. Noteworthy,
the small amount of IL-12 heterodimer secreted from TM-treated cells
presents an -chain more acidic than the -chain from the
intracellular heterodimer, suggesting that acidification of moieties
other than N-linked sugars might mediate alternative
secretory pathways. Polypeptide acidification by sulfate groups
represents a frequent posttranslational modification in secreted
proteins (26). Indeed, we found that IL-12 is a sulfated
molecule (not shown). The presence of acidic moieties other than
N-linked sialic acid may also explain the heterogeneous pI
observed in -chain after treatment with NANAse or tunicamicyn.
In monocytes, TM treatment determined a reduction in m.w. of the free ß-chain, while it left its secretion unaltered, implying that N-linked glycosylation is not involved in free ß-chain secretion. The finding that TM treatment results in a reduced m.w. of the ß-chain, while the same molecule was unaffected by NANAse and Endo-F deglycosylation, at least in the prevalent p36 form, suggests the presence of unsialylated, N-linked, Endo-F-insensitive sugars, most likely of bi-, tri-, and/or tetraantennary nature (25).
The heterodimeric structure of IL-12 is unusual among cytokines. Based
on sequence homology of IL-12 ß-chain with receptors for growth
hormone (27) and for IL-6 (28), it has been
suggested that IL-12 represents an ancestral soluble receptor (the
ß-chain) in a complex with a cytokine (the -chain). The
invariability of ß-chain in different cellular systems and in
different biosynthetic steps, as opposed to the heavy posttranslational
modifications of the -chain that occurs after binding to the
ß-chain, is consistent with the hypothesis of a ligand-receptor
complex. The binding of -chain to ß-chain might sterically modify
the -chain to favoring addition of N-linked sialylation.
The persistence of the immature intracellular IL-12 heterodimer, with
no shift of the immature form toward the mature high m.w. secreted
heterodimer during the time course of our pulse-chase experiments,
indicates that sialic acid addictions to the -chain are very late
events in the biosynthesis of IL-12. Mechanisms that depend on
recognition of sugar moieties might determine the secretion of mature
heterodimers. In contrast, secretion of free ß-chain, unaltered after
inhibition of N-linked sugar addiction, may follow different
secretory pathways.
Treatment of P5-0.1 cells with TM allowed the detection of secreted,
covalently bound homodimers of the ß-chain, consistent with findings
in murine systems (8, 10) and in human IL-12
ß-chain-transfected cells (11); however, similar
homodimers have not been detected in TM-treated or untreated
IL-12-producing human monocytes. Covalently linked ß-chain homodimers
were also not detectable in supernatants from human monocyte-derived
dendritic cells, stimulated to produce considerable amounts of free
ß-chain in the complete absence of IL-12 heterodimer (F. Gerosa and
G. Carra, unpublished results), indicating that synthesis of ß-chain
alone in the absence of -chain is not a condition favoring ß-chain
homodimers secretion. Murine ß-chain homodimers antagonize IL-12
biological functions without mediating biological activity, and in vivo
significantly inhibit IL-12-mediated inflammatory responses (8, 29). The finding that ß-chain homodimers are not detectable in
supernatants of IL-12 heterodimer-producing normal human monocytes, or
in TM-treated monocytes is important in consideration of the potential
functional relevance of these molecules. Although we did not analyze
whether ß-chain homodimer secretion is a unique property of this cell
line also in the absence of TM treatment, it is tempting to hypothesize
that the altered mechanisms of association of deglycosylated ß-chains
are responsible for homodimer assembly.
The large excess of free ß-chain secretion with respect to the
biologically active IL-12 heterodimer, which we observed in IL-4-,
IFN--primed monocytes stimulated overnight with LPS, is well
documented (5, 6). We found that the ratio of IL-12
heterodimer secretion over free ß-chain is higher at earlier times
after stimulation, while a great excess of free ß-chain accumulates
at longer times after stimulation. This is consistent with the notion
that - and ß-chains are independently regulated (12).
It has been reported that -chain is the limiting factor in
controlling IL-12 heterodimer production in monocytes
(30). The -chain expression is, in turn, regulated by
transcription of -chain mRNA variants deriving from different
initiation sites (31, 32). Now we demonstrate that IL-12
heterodimer secretion is controlled by posttranslational addiction of
acidic sugar moieties to the -chain, further pointing out the
importance of -chain in the control of the biologically active IL-12
heterodimer production.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Giuseppe Carra, Department of Pathology, Section of Immunology, University of Verona, Policlinico di Borgo Roma, 37100, Verona, Italy.
3 Current address: Schering-Plough Laboratory of Immunological Research, 27, Chemin des Peupliers, 69571 Dardilly Cedex, France.
4 Abbreviations used in this paper: TM, tunicamycin; 2D, two-dimensional; IEF, isoelectric focusing; NANAse, neuraminidase.
Received for publication November 5, 1999. Accepted for publication February 23, 2000.
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-dependent inducible expression of the human interleukin-12 p35 gene in monocytes initiates from a TATA-containing promoter distinct from the CpG-rich promoter active in Epstein-Barr virus-transformed lymphoblastoid cells. Blood 91:4645.This article has been cited by other articles:
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) labeled with [35S]methionine and stimulated with
LPS for 18 h (lanes a–d) or for 6 h
(lane f), or derived from
[35S]methionine-labeled P5-0.1 cells (lanes
e and g). Samples were immunoprecipitated with
Sepharose-linked anti-
