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*
Department of Surgery and Section of Immunobiology, Yale University School of Medicine, and
Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06510
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible 35-kDa glycoprotein that localizes in MIICs. In this
study, we show that intact IP30 binds to certain HLA-DR alleles via an
N-terminal prosequence. The association takes place in the endocytic
pathway following removal of invariant chain from class II molecules
and before their cell surface expression. We also show that DR-IP30
complexes are SDS stable. The potential precursor-product relationship
between DR-IP30 complexes and the DR-peptide complex is discussed. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and
ß subunits. Immediately after synthesis, the - and ß-chains
associate with trimers of the nonpolymorphic invariant chain forming a
nonameric complex (reviewed in 1 . The invariant chain promotes
the efficient assembly of ß dimers and prevents premature loading
of class II molecules with peptides or intact proteins (2). Following
transit through the Golgi apparatus, the complex is driven into the
endosomal-lysosomal pathway by targeting signals in the cytoplasmic
domain of the invariant chain (3). Following proteolytic degradation of
the invariant chain, its residual fragments (class II-associated
invariant chain peptides,
CLIP3 (4)) in the
peptide-binding groove of class II molecules are replaced with
lysosomally generated peptides by the action of HLA-DM (5, 6, 7). Proteins entering the endocytic pathway encounter an increasingly hydrolytic environment imposed by a progressive decrease in pH and an increase in protease concentrations. Ultimately, most of them are degraded in lysosomes to small peptides and free amino acids. In APCs, peptides of up to 20 or more amino acids with appropriate sequence motifs are rescued from complete degradation by binding to class II molecules (8, 9, 10). CLIP release and peptide binding are believed to occur in specialized lysosome-like compartments containing HLA-DM called MIICs (MHC class II compartments) (11, 12, 13, 14). Characteristics of the compartment, e.g., relatively reduced proteolytic activity or specialized protective mechanisms (15), may be responsible for the generation of relatively long peptides associated with class II molecules.
Davidson et al. (16) showed using Ag-specific B cell lines that class II molecules bind large fragments of radiolabeled tetanus toxoid following Ig-mediated endocytosis. This suggests that binding of such large fragments to class II molecules may be followed by proteolytic trimming in the MIIC, resulting in the generation of class II-peptide complexes that are then transported to the cell surface. An extreme hypothetical variation of such a mechanism is that unfolded, but otherwise intact, proteins may bind to class II molecules before their degradation. A number of in vitro studies have shown that large denatured proteins can bind to class II molecules (reviewed in Refs. 17 and 18). This is not surprising from a structural point of view, because the ends of the class II peptide-binding groove, unlike those of MHC class I molecules, are open (19). Studies from various laboratories suggest that the reduction of disulfide bonds (17, 18, 20, 21, 22) and unfolding (23, 24, 25, 26, 27) are crucial steps in the binding of most proteins to class II. Sercarz and others have shown that class II association can protect a bound segment of a protein from proteolytic degradation (28, 29, 30, 31), leading to the determinant capture model that suggests that class II binding may indeed precede proteolysis.
In this study, we present what we believe is the first example of an
intact endogenous protein that binds in an allele-dependent fashion to
class II molecules in vivo. The protein is an IFN--inducible
glycoprotein, originally defined by Luster et al. (32) and called IP30,
which is expressed constitutively in B-lymphoblastoid cell lines
(B-LCL). IP30 is synthesized as a 224-amino-acid 35-kDa precursor,
derivatized by mannose-6-phosphate addition, and cleaved in the
endosomal/lysosomal pathway, giving rise to a mature 30-kDa form after
removal of both N- and C-terminal peptides (Arunachalam and Cresswell,
manuscript in preparation). Peptides derived from the N-terminal
prosequence of IP30 region were isolated previously from HLA-DR
molecules (33). In this study, we show that intact IP30 binds to
certain DR alleles in vivo and that the association takes place in the
endocytic pathway following removal of invariant chain from the class
II molecules and before their cell surface expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The EBV-transformed cell lines Pala (DR3, DRw52), Swei (DR11, DRw52), Raji (DR3, DR6, DRw52), WT-20 (DR3, DRw52), A2m (DR4), and 8.1.6 have all been previously described (33, 34, 35). T1.DR3 (DR3, DR7) and T2.DR3 (DR3) are previously described T x B hybrid cell lines (36).
Antisera were raised in rabbits against synthetic peptides derived from
N-terminal (SPLQALDFFGNGPPVNC) (residues 1–16),
internal (CVLDELDMELAFLT) (residues 106–119), and C-terminal
(CPSSTSSLRSVCFK) (residues 211–224) sequences of IP30, and are
referred as R.IP30N, R.IP30i, and R.IP30C, respectively. Note that the
C-terminal sequence is different from that proposed by Luster et al.
(32). The complete correct sequence will be published elsewhere
(Arunachalam and Cresswell, manuscript in preparation). The peptides
were coupled to keyhole limpet hemocyanin (Calbiochem-Behring, La
Jolla, CA) via the sulfhydryl group of the natural or underlined added
cysteine residue (37) for immunization. Mouse mAbs used were DA6.147
(anti-HLA-DR-chain (38)), L243 (anti-HLA-DRß dimer
(39)), XD5.A11 (anti-class II ß-chain (39)), HB10A
(anti-HLA-DRß-chain (40)), and PIN.1 (anti-invariant chain
N-terminal (41)). The rabbit antisera R.DRAB1 (anti-HLA-DRß
dimer (42)), R.DMB-C (anti-HLA-DMß-chain C-terminal peptide
(43)), have been described. A rabbit antiserum to the
mannose-6-phosphate/insulin-like growth factor II receptor (44) was a
kind gift from Dr. Stuart Kornfeld. Rabbit anti-cathepsin D serum
was from Dako (Carpenteria, CA). A rabbit antiserum specific for the
chicken hepatic lectin (CHL; 45) was obtained from Dr. K. Drickamer.
Horseradish peroxidase (HRP)-coupled goat anti-rabbit Ig and
anti-mouse Ig, FITC-coupled goat anti-rabbit Ig, and Texas Red
(TR)-coupled donkey anti-mouse Ig were purchased from Jackson
ImmunoResearch (West Grove, PA).
Generation of mouse mAb specific for IP30
IP30 was partially purified from extracts of different B-LCLs
using Con A affinity chromatography, followed by ion-exchange
chromatography and gel filtration, and used to immunize mice. Cells
were extracted for 1 h on ice in 150 mM NaCl, 10 mM Tris, pH 7.4
(TS), containing 2% C12E9, 0.5 mM PMSF,
0.1 mM tosyl lysyl chloromethyl ketone, and 5 mM iodoacetamide. The
glycoproteins from the postnuclear extract were purified by passing
through a Con A-Sepharose 4B (Sigma, St. Louis, MO) column and eluted
in TS containing 0.1% C12E9, 5% -methyl
D-mannoside (Sigma). The Con A-purified glycoproteins were
applied to a DEAE-Sephacel column, and most of bound IP30 (detected by
Western blot) was eluted with 0.2 M NaCl in 10 mM phosphate buffer, pH
7.4. This was applied to a Sephacryl S-300 column, and fractions
containing IP30 were pooled. At this stage, IP30 was the major protein
based on SDS-PAGE and silver staining. Mice were immunized with the
partially purified material. Spleen cells from an immunized mouse were
fused with Ag.8 myeloma cells, and the culture supernatants were
screened for a vesicular staining pattern by indirect
immunofluorescence. Potential clones were further analyzed by
immunoprecipitation for their specificity. One of the clones (MAP.IP30)
was found to secrete an IP30-specific IgG1 Ab and was used in this
study. MAP.IP30 is a conformation-specific Ab that precipitates the
mature form of IP30 more efficiently than the proform (Arunachalam and
Cresswell, manuscript in preparation).
Metabolic radiolabeling and immunoprecipitations
Cells were metabolically labeled, chased, and extracted for immunoprecipitation, as previously described (46). In brief, Pala cells (106–107) were deprived of methionine and cysteine by incubation for 1 h in L-methionine- and L-cysteine-free DMEM (Life Technologies, Grand Island, NY) containing 3% dialyzed FCS, and then pulsed with L-[35S]methionine and L-[35S]cysteine (Amersham Life Science, Cleveland, OH) (0.5–1 mCi) for 1 h at 37°C in the presence of 5 µg/ml brefeldin A (BfA; Epicentre Technology, Madison, WI). For chasing, the cells were washed after labeling and cultured in medium containing 10% FCS and 15-fold excess of nonradioactive L-methionine and L-cysteine for different periods of time. Cells were extracted in 150 mM NaCl, 10 mM Tris, pH 7.4 (TS), containing 1% Triton X-100, 0.5 mM PMSF, 0.1 mM tosyl lysyl chloromethyl ketone, and 5 mM iodoacetamide. Postnuclear supernatants were precleared overnight with normal rabbit serum and either protein A- or G-Sepharose (Pharmacia, Piscataway, NJ), and then precipitated with specific Ab and protein A- or G-Sepharose. Pellets were washed three times with TS/0.1% Triton X-100 and either analyzed by SDS-PAGE or stored at -20°C.
Immunoprecipitation of cell surface vs intracellular DR-IP30 complex and secreted vs intracellular IP30
Cells were labeled and chased for different periods of time, as described above. At each time point, culture supernatant was collected for immunoprecipitation of secreted IP30. To precipitate cell surface DR-IP30 complexes, the cells were washed once with cold, serum-free medium containing 0.1% BSA (SFA) and incubated with the DR-specific mAb L243 in cold SFA for 1 h at 4°C. Cells were then washed twice with cold SFA to remove unbound Abs and detergent extracted as above. Ab-bound complexes were precipitated from the extract using protein A-Sepharose beads. Extracts precleared of cell surface complexes were then incubated with the mAb HB10A and protein A-Sepharose beads for 1 h at 4°C to precipitate intracellular DR-IP30 complexes. The precleared extracts were then used to isolate the residual IP30. R.IP30i recognizes both pro- and mature forms of IP30, but only after reduction and denaturation. Hence, glycoproteins from the cell extracts were isolated using Con A-Sepharose beads (Pharmacia). These were stripped of associated glycoprotein by boiling in 1% SDS under reducing conditions and IP30 isolated following alkylation with iodoacetamide by immunoprecipitation using affinity-purified R.IP30i-Abs and protein A-Sepharose similar to previously described methods (47). Similarly, L243 and HB10A precipitates were SDS stripped, and the released IP30 was reprecipitated with R.IP30N.
Electrophoresis
SDS-PAGE was performed as described (48). 14C-labeled m.w. markers (Amersham, Arlington Heights, IL) were used. Gels were fixed, equilibrated in 150 mM sodium salicylate, dried, and exposed to Kodak Biomax MR film at -70°C. Intensity of specific bands was quantitated with a Bio-Rad (Richmond, CA) GS-250 molecular imager after subtracting a background obtained by integrating a blank area of the appropriate size on each gel.
Western blotting
Samples were separated by SDS-PAGE under reducing or nonreducing conditions. Proteins from the gels were electrophoretically transferred to Immobilon P membranes (Millipore, Bedford, MA). Membranes were incubated in PBS containing 1% BSA and 0.3% Tween-20 for 1 h at room temperature or overnight at 4°C to block nonspecific binding to the membrane. The blots were then incubated for 1 to 3 h at room temperature with specific primary Abs, followed by secondary Abs (HRP-conjugated antispecies Ig) diluted in the blocking solution. The blots were washed extensively after each incubation using PBS containing 0.3% Tween-20. The blots were incubated with SuperSignal CL-HRP substrate working solution (Pierce, Rockford, IL) and exposed to Kodak Biomax MR film to visualize the specific bands. Prestained m.w. markers (Life Technologies, Gaithersburg, MD) were used. For analysis of the SDS stability of DR-IP30 complexes, gels after electrophoresis were incubated in 8 M urea containing 50 mM DTT for 30 min at 80°C. Treated gels were washed three times with water before electrophoretic transfer.
Immunofluorescence
Indirect immunofluorescence was performed as previously described (46, 49). In brief, 8.1.6 cells at a concentration of 3 x 104 cells/well were incubated for 30 min at 37°C on Alcian blue (Sigma)-treated coverslips in 24-well tissue culture dishes. Cells were fixed with 3.7% formaldehyde in Iscove’s modified Dulbecco’s medium (IMDM) containing 10 mM HEPES, pH 7.4, for 20 min at room temperature. Fixing was quenched and cells were permeabilized with 0.05% Saponin in IMDM containing 5% calf serum, 10 mM glycine, and 10 mM HEPES, pH 7.4. Cells were incubated with primary Abs (specific to different proteins) as culture supernatant, diluted ascites, or serum, followed by secondary Abs (TR/FITC-conjugated antispecies Ig) on ice in the presence of 0.05% saponin. Coverslips were washed extensively after each incubation and mounted onto slides using mounting solution (10% Mowiol 4-88 (Calbiochem, San Diego, CA), 2.5% 1,4-diasabicyclo[2,2,2]octane (DABCO), 25% glycerol in 0.1 M Tris, pH 8.5). Samples were examined using a fluorescent microscope (Zeiss) under x157.5 magnification.
Flow-cytometric analysis
A total of 0.5 to 1 x 106 cells was washed once with ice-cold PBS containing 1% BSA (PBA) and incubated with primary Abs for 30 min on ice. Cells were washed three times with cold PBA and incubated with secondary Abs (1/50 dilution of goat anti-rabbit IgG conjugated to FITC) on ice. Cells were washed three times with cold PBA and finally once with cold PBS, and suspended in 200 µl of PBS containing 3% formaldehyde. Samples were analyzed on a Becton Dickinson FACScan (Mount View, CA).
SDS-stability analysis
HLA-DR was purified from Pala and A2m cell detergent extracts
using L243 affinity column, as previously described (34). Purified DR
was mixed with reducing SDS-PAGE sample buffer and either boiled or
incubated at room temperature for 5 min. Samples were separated on 12%
SDS-PAGE, followed by Western blotting, as described above. IP30 on the
membranes was detected using either R.IP30N or R.IP30i sera and
DRß-chains using DA6.147 and XD5.A11 Abs.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the original description of IP30, we were struck by the
observation that the molecule was localized to intracellular vesicles,
possibly lysosomes. This, combined with the fact that the molecule was
IFN- inducible, suggested a possible involvement with MHC class II
Ag processing. To more precisely determine the intracellular
distribution of IP30, we examined human B-LCL by indirect
immunofluorescence. The cells were stained for IP30, HLA-DR, HLA-DM,
and the late endosomal and lysosomal markers mannose-6-phosphate
receptor and cathepsin D. The results are shown in Figure 1
. IP30 was clearly localized to
intracellular vesicles containing DR, DM, mannose-6-phosphate receptor,
and cathepsin D. The colocalization with intracellular DR and DM
suggests that the IP30-containing vesicles are MIICs. This has been
confirmed recently by immunoelectron microscopy (H. Geuze, personal
communication).
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The colocalization of IP30 with intracellular HLA-DR and HLA-DM
was consistent with a potential role for IP30 in Ag processing. We
therefore looked for possible physical associations between IP30 and
components of the class II complex. Detergent extracts of a number of
cell lines were immunoprecipitated with mAbs specific for invariant
chain (PIN.1) or DR-chain (DA6.147), and the precipitates were
separated by SDS-PAGE and electrophoretically transferred to membranes.
The membranes were then probed with a rabbit Ab to an internal peptide
of IP30 (R.IP30i). The anti-DR mAb DA6.147 coprecipitated IP30
(Fig. 2). No IP30 was coprecipitated by
the invariant chain-specific Ab, indicating that IP30 is not associated
with DR-invariant chain complexes. In other experiments, Abs specific
for HLA-DM or MHC class I molecules did not coprecipitate IP30 (data
not shown). Interestingly, IP30 association with HLA-DR molecules was
detected only in cells expressing the DRw52 supertypic allele, namely
Pala (DR3), Swei (DR11), Raji (DR3, DR6), and WT-20 (DR3). No
association was seen in T1 cells (DR7) transfected with a genomic clone
encoding the DR3ß-chain or T2 cells transfected with genomic clones
encoding DR- and DR3ß-chains. Additional cell lines tested,
including DR1 and DR2 homozygous lines, showed no association. Thus,
among the alleles analyzed, IP30 only appears to associate detectably
with DRw52. In separate experiments, extracts from metabolically
labeled DRw52-negative cells were incubated with unlabeled extracts
from DRw52-positive cells. Anti-class II Abs did not coprecipitate
labeled IP30 from the mixture, which argues against potential postlysis
association (data not shown). All of the cell lines shown in Figure 2
expressed similar amounts of IP30, and reprobing of immunoblots with
DA6.147 indicated that for each cell line, comparable amounts of class
II molecules were precipitated (data not shown). These data suggested
that the class II-IP30 association was class II restricted and might be
mediated by the peptide-binding groove.
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The possibility that the IP30 molecule might associate with a
subset of DR molecules by the peptide-binding groove is consistent with
the finding of Chicz et al. (33) that two peptides derived from the
N-terminal region of IP30 could be isolated from DR3/DRw52 molecules
purified from the B-LCL WT-20. Many class II-peptide complexes are
stable in SDS unless heated, suggesting that the DR-IP30 complex might
also be stable in SDS if the association was via the binding groove. To
assess this, DR molecules were affinity purified from detergent
extracts of unlabeled Pala (DR3, DRw52) and A2m (DR4) using an L243
column. After incubation in SDS-containing sample buffer under reducing
conditions and with or without boiling, proteins were separated by
SDS-PAGE. Before transfer to membranes, the gels were incubated in 8 M
urea containing 50 mM DTT for 30 min at 80°C. This treatment was
found to dramatically improve the subsequent detection of unheated
samples of IP30 by R.IP30i. Blots were probed with either a mixture of
DA6.147 and XD5.A11 (Fig. 3A), R.IP30N (Fig. 3B), or R.IP30i (Fig. 3C).
SDS-stable DRß dimers were detected both in A2m and Pala under
nonboiling conditions, and upon heating to 100°C they separated into
free - and ß-chains, as expected (panel
A). IP30 was detected only in purified DR from Pala cells
(panels B and C). More free IP30
was detected both with R.IP30N and R.IP30i when the samples were first
heated to 100°C (last lane in panels B
and C). In the nonboiled samples, both R.IP30N and
R.IP30i detected a band with an approximate m.w. of 90 to 100 kDa only
in the Pala-derived DR preparation. This is the expected size for an
ß.IP30 complex, and the result suggests that the complex is SDS
stable. There are R.IP30N- and R.IP30i-reactive bands below the
dominant high m.w. band in the nonboiled sample and below the IP30 band
in the boiled sample (panels B and
C). This may correspond to C-terminally proteolyzed
IP30. We have been unable to confirm this by probing with R.IP30C,
which is unfortunately a less sensitive reagent in Western blots.
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The two IP30-derived peptides isolated from DR3/DRw52 by Chicz et
al. (33) correspond to residues 1–20 and 1–22 of the protein
following signal sequence cleavage. The R.IP30N antiserum was raised to
residues 1–16. Thus, it seemed possible that this antiserum might
react with the peptide, presumably DRw52 associated (Fig. 2
), at the
cell surface. To test this, Pala (DR3, DRw52), Raji (DR3, DR6,
putatively DRw52), and A2m cells (DR4) were stained with R.IP30N and
analyzed by flow cytometry (Fig. 4). Pala
and Raji cells were both positive, and the reactivity of the Ab was
inhibited by the immunizing peptide. Swei cells (DR11, DRw52) were also
positive with R.IP30N (data not shown). The A2m cell line was negative,
as were a number of other DRw52-negative B cell lines (data not
shown).
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shows that Pala and Raji cells are also positive with this Ab,
and again the reactivity is inhibited by the immunizing peptide
(residues 211–224; Arunachalam and Cresswell, manuscript in
preparation). The staining intensity with the C-terminal-specific Ab is
lower than that of the N-terminal-specific Ab. This may be because the
N-terminal-specific Ab is also recognizing DRw52-peptide complexes,
although inherent differences in efficiency of the two antisera may be
responsible.
Alternative explanations for the reactivity of the IP30
C-terminal-specific Ab with the cell surface could be that the
C-terminal peptide is independently expressed or that the intact
molecule is present, but not class II associated. To approach this
question, we isolated surface HLA-DR molecules by prebinding the
anti-DR mAb L243 to intact Pala cells that had been radiolabeled
and chased for 6 h to ensure cell surface expression of labeled
class II molecules. The cells were washed to remove free Ab, and
detergent solubilized. The DR-Ab complexes were isolated using protein
A-Sepharose, and dissociated by boiling in SDS under reducing
conditions. After alkylation with iodoacetamide, the supernatants were
diluted with Triton X-100 and reprecipitated with HB10A, R.IP30N,
R.IP30i, or R.IP30C. The results are shown in Figure 5. Lanes 1 to 4show that while DA6.147 (lane 1) (which
reacts with the cytoplasmic domain of DR and therefore fails to bind
at the cell surface) and an irrelevant rabbit antiserum (anti-CHL,
lane 2) failed to bind surface DR molecules, L243
(lane 4) effectively captured them, as
assessed by reprecipitation of DRß subunits with HB10A. While the
irrelevant rabbit anti-CHL Ab (lane 3)
failed to react with any protein released from the surface class II
molecules, all three IP30-specific Abs specifically immunoprecipitated
released IP30 (lanes 5–7). Thus, complexes of
DR with intact IP30 protein are clearly present at the cell
surface.
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The observation that IP30 is detectable in MIICs suggested that
binding to DR molecules might occur in this compartment before
expression of the complex on the cell surface. If this were true, the
level of association could potentially be affected by reagents that
affect peptide loading. Alternatively, binding could occur in the ER,
before invariant chain association. To test these ideas, we examined
the effects on the class II-IP30 interaction of various drugs that
perturb intracellular trafficking and processing. Pala cells were
incubated with BfA, monensin, chloroquine, or leupeptin for 13 h
at 37°C, and extracted with detergent, and HLA-DR molecules were
immunoprecipitated. Preliminary experiments showed that all of these
agents induced the accumulation of the proform of IP30 (data not
shown), similar to results for many lysosomal enzymes (reviewed in 50 . Associated IP30 was detected by SDS-PAGE, followed by Western
blotting. The only agent that substantially affected the amount of
DR-associated IP30 was chloroquine, which induced a significant
increase (Fig. 6). BfA caused a
reproducible, but slight, decrease. The most straightforward
explanation of these results is that the class II-IP30 association
occurs in an acidic compartment, putatively the MIIC. Neither BfA nor
monensin has a significant effect because both prevent newly
synthesized ß-invariant chain complexes, IP30, and other lysosomal
proteins from getting to the MIIC, arresting transport in the ER and
medial Golgi, respectively. Chloroquine, by neutralizing the MIIC,
presumably causes the accumulation of intact IP30 by inhibiting the
proteolysis of the N- and C-terminal prosequences, favoring the
association of intact IP30 with liberated class II molecules. Leupeptin
might have been expected to have a similar effect, except that it
profoundly affects invariant chain degradation, causing the
accumulation in MIICs of class II complexes with the LIP
(leupeptin-induced protein) fragment (51, 52). LIP consists of
approximately the N-terminal two-thirds of the invariant chain, and
includes the CLIP region (53). Thus, class II-LIP complexes are not
capable of binding peptides. Chloroquine also can inhibit invariant
chain degradation (54), but this effect is incomplete at the
concentration used (data not shown). In fact, the processing and
presentation of certain epitopes are unaffected by chloroquine under
similar conditions (55, 56).
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The enhanced association of IP30 with DR molecules induced by
chloroquine treatment (Fig. 6) is consistent with it occurring in an
acidic compartment. In addition, the SDS-stable nature of DR-IP30
complexes suggests that they have probably been exposed to endosomal
environment (27). Together with the observation that IP30 is localized
in MIICs, the data suggest that IP30 association is likely to occur
intracellularly, before cell surface expression of the complex. To test
this, we wished to determine the kinetics of DR-IP30 association, and
to compare it with that of cell surface expression of the complex. This
experiment was combined with an analysis of IP30 processing to the
mature form, as well as secretion of the molecule.
Pala cells were labeled for 1 h with [35S]methionine and [35S]cysteine in the presence of BfA to accumulate sufficient labeled IP30 in the ER for analysis. BfA was removed by washing, and the cells were resuspended in chase medium at 37°C containing unlabeled methionine and cysteine. At various times, supernatants were collected and secreted IP30 immunoprecipitated with R.IP30N. Surface DR molecules were isolated by prebinding the L243 mAb before solubilization, as described above. After isolation of the surface complexes with protein A-Sepharose, intracellular DR molecules were immunoprecipitated using the DRß-specific mAb HB10A and protein A-Sepharose. Finally, free intracellular IP30 together with the mature 30-kDa form was isolated from the extract by binding the total cellular glycoproteins to Con A-Sepharose, stripping by heating to 100°C in SDS under reducing conditions, and reprecipitating with R.IP30i after alkylating with iodoacetamide and diluting the samples with Triton X-100. IP30 was similarly released from L243 and HB10A precipitates and reprecipitated with R.IP30N.
Figure 7 shows the kinetics of IP30
secretion and intracellular processing to the mature 30-kDa form. IP30
secretion begins at 1.5 h and reaches a maximum at 6 h. The
accumulation of the intracellular mature form begins at approximately
the same time as secretion, but does not increase significantly after
3 h. The 30-kDa form does not react with the N-terminal- and
C-terminal-specific antisera (data not shown). The fraction that is
secreted is difficult to assess from this experiment, because of the
many manipulations involved in generating the data describing the
intracellular material. However, other experiments involving more
direct comparisons suggest that approximately 25% is secreted, which
is consistent with the fraction of starting material that remains
intracellular in the mature form in the lower right-hand panel of the
figure.
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.
Intracellular complexes are close to maximal at 1.5 h of chase,
and after 3 h they begin to decline until a plateau level is
reached at 8 h. Cell surface DR-IP30 complexes reach a peak at
3 h, when they also decrease, with the rate of decline also
decreasing at 8 h. Thus, at the time when IP30 degradation begins
to be seen (1.5 h, Fig. 7), DR-IP30 association is close to maximal.
This suggests that the IP30 interaction with DR molecules may occur
before it is exposed to the proteolytic machinery normally involved in
its maturation. Accumulation of DR-IP30 complexes on the cell surface
lags behind their intracellular detection, consistent with the proposed
model that binding occurs in an acidic intracellular vesicle and is
followed by transport of at least some of these complexes to the cell
surface. The remaining fraction presumably undergoes intracellular
proteolytic degradation, perhaps generating DR-IP30 peptide complexes
that are also expressed on the cell surface. The accumulation of steady
state levels of DR-IP30 on the cell surface is clear from Figures 4 and 5. This presumably reflects a balance between the decline visible in
Figure 8 and new synthesis. Similar kinetics of IP30 secretion and
intracellular processing and DR-IP30 complex generation was observed
when cells were chased after pulse labeling in the absence of BfA (data
not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The presence of an
IFN--inducible protein in MIICs has potential implications for Ag
processing. While searching for a function for IP30, we determined that
it associates with DR molecules (Fig. 2) and that the complex is
expressed on the cell surface (Figs. 4, 5, and 8). Based on analysis of
a number of cell lines, all of which express IP30 at similar levels, we
believe that it associates with the product of the DRw52 allele (Fig. 2), and that the binding is therefore likely to occur via the
peptide-binding groove. Evidence in favor of this is that the
association is SDS stable (Fig. 3), as are many class II-peptide
interactions (57). Further evidence is that N-terminally derived
peptides from IP30 have previously been isolated from DR molecules
purified from the DR3/DRw52-positive cell line, WT20 (33), one of the
cell lines in which we have shown the DR-IP30 interaction (Fig. 2).
Although exogenous intact proteins have been shown to bind to class II
molecules (17, 18, 58, 59), this is the first example of which we are
aware of class II association with a defined intact endogenous protein
other than the invariant chain. The fact that DR-IP30 peptide and
DR-IP30 protein complexes are present in the same cell line raises a
number of questions concerning regulation of the class II-binding
process.
Following synthesis in the ER, IP30 is transported through the Golgi
apparatus and a fraction is secreted. The majority, however, remains
intracellular, presumably being transported to MIICs, where proteolytic
processing takes place (Fig. 7). The near simultaneous appearance of
intracellular processed IP30 and unprocessed IP30 in the supernatant
argues for an intracellular segregation of the two pools. We have
evidence that IP30 is modified by addition of mannose-6-phosphate
residues to one or more of its N-linked glycans (Arunachalam
and Cresswell, manuscript in preparation), which argues that this
segregation probably occurs at the Trans Golgi Network (TGN), in which
lysosomal enzymes so derivatized are captured by the
mannose-6-phosphate receptor for delivery to late endosomes or
prelysosomes (reviewed in 60 . Missorted lysosomal enzymes are
also often secreted. Processing to remove the DR-binding N-terminal
peptide, as well as the C-terminal peptide, of IP30 would be expected
to begin when the molecule enters proteolytically active lysosomal
compartments, or MIICs. Similarly, invariant chain proteolysis and
release of class II ß dimers are thought to occur in MIICs
following transport of ß-invariant chain complexes from the TGN
(reviewed in Refs. 1, 61, and 62). Current models suggest that this
results in the generation of ßCLIP complexes, and that CLIP
release induced by HLA-DM molecules reveals the peptide-binding groove
(reviewed in 63 . Thus, a plausible scenario is that peptide-free
DRß dimers are being generated in proteolytically active,
DM-positive organelles in which IP30 processing is simultaneously
occurring.
Simultaneous processing of IP30 and DR-invariant chain complexes
in MIICs would appear to present newly generated DRw52 ß dimers
with a choice of binding N-terminal IP30-derived peptides, among other
peptides, or intact IP30 protein. The DR-IP30 interaction is initiated
intracellularly, consistent with its occurring in MIICs (Fig. 8).
Chloroquine treatment enhances the association of IP30 with DR
molecules (Fig. 6), which could be explained if it reduces N-terminal
processing of IP30 by raising the pH of MIICs. Chloroquine
would appear to have a greater inhibitory effect on IP30 processing
than on invariant chain degradation, because invariant chain-associated
class II molecules do not bind IP30 (Fig. 2), and yet IP30-class II
association is increased. Following their association, DR-IP30
complexes are transported from MIICs to the plasma membrane with a lag
time of 1 to 2 h (Fig. 8). Anderson et al. (64) observed that a
small fraction of invariant chain-free I-Ak molecules is
also associated with a protein on the cell surface. This protein had an
apparent Mr of 55 kDa, but remains
uncharacterized.
An alternative idea to the "choice" hypothesis presented above is
that DR-IP30 protein complexes, once formed, are processed to DR-IP30
peptide complexes by proteolysis at the C-terminal end of the
N-terminal propeptide of IP30. This would be a natural example of
determinant capture (31). It is clear that the number of intracellular
and cell surface DR-IP30 complexes declines between 4 and 8 h
(Fig. 8), which could reflect such processing. It is also clear that
the number of DR-IP30 complexes is almost maximal at a time (1.5 h)
when processed IP30 is considerably submaximal (Fig. 8). This could
reflect initial DR-IP30 complex formation and subsequent rapid cleavage
to generate DR-IP30 peptide complexes. The maintenance of an
approximately steady state level of intracellular DR-IP30 protein
complexes between 1.5 and 4 h (Fig. 8) may reflect a balance
between new complex formation and degradation of preformed complexes
into DR molecules associated with IP30 peptide. However, we have been
unable to date to show a clear precursor-product relationship between
DR-IP30 protein complexes and DR-IP30 peptide complexes. The decline
between 4 and 8 h may in fact represent loss of the
protein-protein complex independent of the generation of the DR-peptide
complex. Binding of IP30 by its N-terminal prosequence could
conceivably inhibit the cleavage at position 31, which normally occurs
during maturation of IP30 (Arunachalam and Cresswell, manuscript in
preparation).
The function of IP30 is unknown, although the combination of
MIIC/lysosomal localization and IFN- inducibility is intriguing and
suggests a possible role in Ag processing. It is unclear whether the
formation and surface expression of a DRw52-IP30 complex have any
functional significance, or whether they represent an opportunistic
interaction of a class II molecule with a region of a protein extended
and available because its normal function is to be cleaved and removed.
If the DR-IP30 complex represents a processing intermediate, then it
may provide an avenue to the identification of additional molecules
involved in Ag processing. These questions await further analysis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter Cresswell, Howard Hughes Medical Institute, Section of Immunobiology, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06510. E-mail address:
3 Abbreviations used in this paper: CLIP, class II-associated invariant chain peptide; B-LCL, B-lymphoblastoid cell line; BfA, brefeldin A; CHL, chicken hepatic lectin; ER, endoplasmic reticulum; HRP, horseradish peroxidase; LIP, leupeptin-induced protein; MIIC, major histocompatibility complex class II compartment; TR, Texas Red.
Received for publication August 28, 1997. Accepted for publication February 13, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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