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*
Howard Hughes Medical Institute, Department of Medicine, National Jewish Center, and
Departments of Biochemistry, Biophysics and Genetics, Immunology, and Medicine, University of Colorado Health Science Center, Denver, CO 80206;
Department of Pathology, Harvard Medical School, Boston, MA 02115; and
§
Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA 30912
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß
heterodimer (3), and with class II forms a nonameric complex in the
endoplasmic reticulum comprised of an Ii trimer and three ß dimers
of the class II molecule (4, 5). Murine Ii has two forms, the
predominant p31 molecule, and p41, which arises due to alternate
splicing of exon 6b (6, 7, 8). Ii directs newly synthesized class II
molecules to specialized acidic compartments in the endocytic pathway
where Ii is proteolytically destroyed and exogenous peptide is loaded
before the appearance of class II on the cell surface (4, 9). Ii has been shown to bind class II molecules via a conserved sequence, CLIP, that occupies the peptide binding groove of class II. Engagement of CLIP prevents binding of other peptides by class II in the endoplasmic reticulum. However, recent evidence by Zhong et al. indicates that perhaps this function is secondary to stabilization of the class II structure (10). Additionally, Stumptner and Benaroch have shown that it may not even be necessary for CLIP to bind in the peptide groove to prevent peptide binding (11). It is not until the Ii plus class II nonamer reaches the endocytic compartment that CLIP can be displaced and replaced with antigenic peptides. This displacement is probably accomplished by a combination of Ii proteolytic degradation (5, 12) and interaction with another class II-like protein, H2-M (HLA-DM in human systems) (13, 14, 15, 16). In the absence of H2-M, class II molecules bound to peptides derived from the CLIP region of invariant chain accumulate (17).
Some evidence suggests, however, that Ii can bind to class II at sites other than the peptide binding groove. For example, although the superantigen staphylococcal enterotoxin B binds class II at a site that does not primarily involve the peptide binding groove, staphylococcal enterotoxin B engagement can interfere with Ii binding to class II (18). Additionally, Ii engagement inhibits binding to class II of another superantigen, toxic shock syndrome toxin, TSST-1 (19, 20). Also, peptides derived from Ii, not including CLIP, can bind to or interact with class II molecules (18, 21). Stumptner and Benaroch have shown that there are three separate binding sites on Ii for class II, only one of which is CLIP, and that any two of these are sufficient for Ii-class II interaction (11). There is disagreement on this point, however, as attempts to reveal binding sites for class II on Ii distinct from CLIP have sometimes failed (22, 23). To resolve this matter we have taken a different approach to the problem.
Some of us have recently described transgenic mice that lack wild-type
class II ß-chains and are instead transgenic for a gene encoding a
class II ß-chain, Aßb, covalently linked to a peptide,
E52–68 (Ep) (24). The Ep peptide can engage the peptide
binding groove of the class II protein, IAb
(Ab), expressed in these mice. In mice lacking Ii
(AbEpIi-), the peptide binding grooves of all
detectable Ab proteins are occupied by the Ep peptide. In
Ii+ mice, however, some of the Ab proteins no
longer have Ep bound to their grooves, or the Ep bound is no longer
covalently linked, and these Ab proteins are therefore free
to engage other peptides. These results imply that Ii binds to the
AbEp complex and guides it through the endocytic pathway,
resulting in removal of the Ep peptide from the groove. To achieve
this, either the CLIP region of Ii must compete with the covalent
peptide for engagement by the Ab peptide binding groove in
the endoplasmic reticulum, or Ii must interact with the Ab
molecule at a site unaffected by prior engagement of the covalent
peptide. In this paper we show that Ii can indeed interact with a class
II molecule, the peptide binding groove of which is already occupied by
peptide. Hence, Ii can interact with class II via a site distinct from
the peptide binding groove.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mice were bred in the Biologic Resource Center, National Jewish Center (Denver, CO), or were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals transgenic for AbEp were constructed and bred with animals that could not express other class II proteins or Ii as previously described (24, 25, 26).
Assay for Ag presentation
Presenting cells were prepared from the spleens of various types of mice as follows. Spleen cell suspensions were prepared in balanced salt solution using nylon screens, and RBC were lysed using ammonium chloride. The spleen cells were then cultured for 24 to 48 h at 106 cells/ml with or without 25 µg/ml LPS (Sigma, St. Louis, MO).
Expression of covalently attached or free Ep in the peptide binding
grooves of Ab on the surface of these spleen cells was
assayed using two T cell hybridomas: BE-16.3, which reacts with Ep
bound to Ab regardless of whether the two are covalently
linked, and BE-20.14, which reacts with free Ep bound to
Ab but not with the covalent combination of Ab
and Ep (L. Ignatowicz, unpublished observations). The spleen cells and
T cell hybridomas were cultured together for 24 h, and stimulation
of T cells by Ab plus Ep on the spleen cells was assessed
by IL-2 production as previously described (27).
The ability of exogenously added peptides to displace Ep from the peptide binding grooves of the Ab proteins of these spleen cells was measured by addition of 75 µg/ml chicken OVA327–339 or OVA323–339 (OVA) to the cells and presentation of OVA to the IAb-OVA-specific T cell hybridoma, 2B10.D2O-22.3. Again, presentation was assessed by the production of IL-2 (27).
Biosynthetic labeling of splenocytes with [35S]methionine/cysteine
Splenocytes were isolated as described above and were cultured at 108 cells/ml in RPMI deficient in cysteine and methionine with 50 µl of EXPRESS/ml (15 mCi/1.2 ml; cysteine, methionine, and protein labeling mix, New England Nuclear, Boston, MA). After 30 min the medium was diluted with a 20-fold excess of complete culture medium, and the cells were spun and washed in complete medium at 37°C. The cells were then resuspended at 1.25 x 107 cells/ml in culture medium, and the pulse was chased by culture at 37°C for various lengths of time. Chases were stopped by spinning the cells out of the culture medium and incubating them on ice.
Immunoprecipitation and SDS-PAGE analyses
35S-labeled cells were lysed at a concentration of 2.5 x 107/ml in lysis buffer (0.5% Nonidet P-40, 50 mM Tris (pH 7.4), and 5 mM Mg2+) on ice for 45 min. Debris was removed by centrifugation, and the supernatant was precleared by two successive incubations with 4 µl of normal rabbit serum and fixed staphylococcus A by a standard method (28), modified by the inclusion of one additional formaldehyde fixation step after the heat inactivation step, for 1 h each, alternated with two successive incubations with 3 µl of normal mouse serum and fixed staphylococcus A for an additional 1 h each. The supernatants were also precleared with 3 µl of the Ab P8 and staphylococcus A.
At this point, TCA precipitations were performed to normalize labeling efficiency. AbEp complexes were immunoprecipitated from the lysates first using anti-AbEp (mAb YAe) (29), and the remaining Ab complexes that lacked the Ep peptide in their grooves were immunoprecipitated by anti-Ab (mAb Y3P) (30), which recognizes mature Ab-peptide complexes regardless of the peptide bound. The resulting immune complexes were isolated using staphylococcus A. Class II molecules were released from staphylococcus A by incubation in sample buffer (0.0156 M Tris (pH 6.8), 1.25% ß-ME, 2.5% glycerol, and 1% SDS) at room temperature for 20 min or by boiling in sample buffer for 5 min. The preparations were run on 12.5% SDS/30- x 25-mm polyacrylamide gels at 80 to 100 V overnight. Gels were processed by soaking in two changes of DMSO, followed by incubation in 27% 2,5-diphenyloxazole/DMSO, then were rehydrated in water. The processed films were dried and exposed to film for 4 wk.
Western analyses
Spleen cells were isolated as described above and lysed at 108 cells/ml in 0.5% Nonidet P-40 or 0.5% CHAPSO, 50 mM Tris (pH 7.4), and 0.1 mM PMSF (Sigma) for 10 min at room temperature. Debris were spun out and the resulting supernatants precleared with normal rabbit serum, normal mouse serum and P8 as described above. The supernatants were then immunoprecipitated with anti-AbEp, anti-Ab, or anti-Ii, and the resulting immune complexes were isolated using protein A beads. Pellets were boiled in sample buffer and run on 12.5% acrylamide SDS minigels for 30 min at 200 V or on regular gels (14 cm x 15 cm x 1 mm) for 2 h at 200 V. The gels were soaked in transfer buffer, and the proteins were transferred to polyvinylidene difluoride membranes using a semidry transfer apparatus (Trans-blot SD, Bio-Rad, Richmond, CA) for 22 min at 15 V for minigels or for 40 min at 20 V for regular gels. Completion of transfer was measured by observing the transfer of Coomassie blue-conjugated m.w. markers, followed by standard fixation and Coomassie blue staining of the gel after transfer of protein to the blot. After blocking, the membranes were exposed to 3.2 µg/ml anti-Ii (mAb In-1) (31) for 1 h, followed by 0.35 µg/ml horseradish peroxidase (HRP)-conjugated goat anti-rat Ab (Boehringer Mannheim, Indianapolis, IN). Bands bound by anti-Ii were revealed using the enhanced chemiluminescence Western blot kit (Amersham, Arlington Heights, IL) and photographic film (Hyperfilm ECL, Amersham) with various exposure times.
In the Western blots shown in Figures 5
and 6, class II and Ii
expression in spleen cells was increased before isolation by incubation
of the cells for 18 h at 106 cells/ml in 1 ng/ml IL-4
(32).
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and 6 used regular sized (14 x 15
cm) SDS gels. In Figure 6, membranes were exposed to 1 µg/ml N22 (33)
followed by 1/2000 biotinylated anti-hamster (1 mg/ml) biotin-HIG
88.2, a mouse anti-Armenian hamster IgG mAb made by one of us
(J.K.), then 0.2 µg/ml streptavidin coupled to HRP. |
Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that the covalent linker between class II
and the peptide in AbEp molecules is cleaved in cells that
express Ii, but not in cells that lack Ii (24). We tested whether this
would also be true in activated cells. Spleen cells from
AbEp mice that did or did not express Ii
(AbEpIi+ or AbEpIi-,
respectively), were cultured with or without LPS for 48 h and then
assayed for their ability to present the Ab binding
peptide, cOVA323–339, to a T cell hybridoma specific for
AbOVA. As shown in Figure 1,
cells from AbEpIi- mice failed to present the
peptide even if they had previously been activated with LPS. In
contrast, cells from AbEpIi+ mice or
from control C57BL/10, Abwt, animals presented the peptide
well, and this ability was somewhat enhanced by preincubation with LPS.
Thus, Ii is required for the displacement of Ep from AbEp
molecules, even in activated cells.
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A peptide bound by a flexible covalent linker to a class II molecule should, due to its high local concentration, compete very efficiently with other peptides for binding to that molecule. Yet the presence of Ii causes this peptide to be displaced with relatively high efficiency. We hypothesized that this was because expression of Ii led to cleavage of the covalent linkage between Ab and Ep, an event that would then allow peptides to compete more effectively for binding to the Ab protein.
We took advantage of a T cell hybridoma, BE-20.14, which
distinguishes between Ab molecules bound to Ep in which the
linker is either present or absent. BE-20.14 recognizes Ep bound to
Ab only if the linker is clipped or missing. Another T cell
hybridoma, BE-16.3, on the other hand, recognizes Ep bound to
Ab regardless of whether the covalent linker remains
intact.
Spleen cells were prepared as described above and then cultured with
each of the T cell hybridomas. BE-16.3 responded well to
AbEp cells regardless of whether they contained Ii (Fig. 2A). BE-20.14 responded to
AbEp cells that expressed Ii but not to those that lacked
Ii. These results confirmed that all detectable Ab
molecules in AbEpIi- cells remain covalently
linked to the Ep peptide. The results also showed that the covalent
linker is clipped on some of the AbEp molecules in
AbEpIi+ cells, even though Ep may still be
engaged to the peptide binding grooves of these proteins. It was not
possible to tell from this assay how much of Ep remained covalently
bound to Ab in AbEpIi+ cells.
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).
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-chain of Ab does not label well with methionine and
cysteine and was therefore less easily visualized than the
Ab ß-chains. After boiling in SDS the AbEp
polypeptide migrated as a glycoprotein of about 34 kDa. This molecular
mass increased slightly during the chase, probably because of
maturation of its attached sugars (Fig. 3). Control
immmunoprecipitations with C57BL/6 spleen cells showed that the
Aßbwt polypeptide migrated at a lower molecular mass
(data not shown). The ß-chains of this molecular mass were observed
in the boiled immunoprecipitates from AbEpIi+
cells (Fig. 3).
The class II material from AbEpIi- cells had
shared characteristics, regardless of whether it was precipitated by
anti-AbEp or by anti-Ab. The ß-chain
always had a molecular weight consistent with that predicted for the
noncleaved AßbEp polypeptide, and the intact
Ab protein precipitated from these cells was always stable
in room temperature SDS (Fig. 3). These results showed that Ep must
occupy the peptide binding groove of the Ab protein to
which it is covalently attached as soon as the protein is assembled.
This conclusion was supported by the fact that
anti-AbEp precipitated ß-chains before their sugars
had completely matured (Fig. 3), when the protein was presumably still
in the endoplasmic reticulum.
Precipitation with anti-AbEp showed that cells from
AbEpIi+ mice also contained Ab
molecules that were still covalently attached to Ep and were stable in
room temperature SDS (Fig. 3). Some of these molecules may also have
been isolated during subsequent precipitation with
anti-Ab, since this Ab precipitated material that was
stable in room temperature SDS immediately after the 30-min pulse.
The T cell hybridoma results described above suggested that
anti-AbEp precipitates from
AbEpIi+ cells should contain some
Aßb chains engaged by Ep but in which the covalent linker
to Ep had been cleaved, and which should therefore run at a lower
molecular mass than that of AßbEp. However, only small
amounts of such material were revealed on the gels, suggesting that the
hybridoma BE-20.14 is sensitive to very low concentrations of
Ab bound to free Ep.
At early time points in the pulse chase of
AbEpIi+ cells, anti-Ab
precipitated some labeled Ab molecules that were not stable
in room temperature SDS (Fig. 3). The mobility of this material on the
gels showed that it was composed of Aßb chains from which
the Ep had been cleaved. As the time of the chase increased,
Ab proteins that were unstable in room temperature SDS
disappeared, and Ab proteins that were stable in SDS at
room temperature increased. These results demonstrated that in the
presence of Ii, the covalent linker between Ep and Ab could
be cleaved within 30 min of synthesis of the polypeptides. Such
cleavage could have occurred either in the endoplasmic reticulum or in
the endosomes. Cleavage in the endoplasmic reticulum would probably
require the cleaved material to be free of the class II peptide binding
groove (34) and thus implies that Ii CLIP successfully competed with
the covalent peptide for binding to a minor proportion of the class II
proteins. Cleavage in the endosomes would require that in the presence
of Ii, some of class II traffick to this compartment of the cell.
Finally, the gels showed that even in the presence of Ii, most of the Ab protein that was precipitated by anti-AbEp was still covalently bound to Ep. These were presumably proteins on which Ep had successfully competed, in the endoplasmic reticulum, with CLIP for binding to their peptide binding grooves. The fact that their covalent linkers were still intact suggested either that the completely folded, Ep-bound Ab protein was resistant to peptide displacement and linker cleavage in the endosomes or that this material had not migrated to the cell surface via the endocytic pathway. In the latter case this must mean that, in the absence of a peptide groove available for CLIP binding, Ii must not be able to bind to all the Ab proteins.
In summary, these experiments showed that Ii can affect the intracellular trafficking of at least some AbEp proteins, causing them to go to the cell surface via the endosomes. The following experiment was performed to find out how Ii binds to AbEp to have this effect.
Ii can bind class II molecules with peptide binding grooves bearing a non-Ii peptide
The anti-AbEp Ab cannot bind to Ab molecules engaged by peptides other than Ep, including CLIP. The idea that Ii can bind to class II molecules at sites other than the peptide binding cleft could therefore be tested by finding out whether Ii precipitated with Ab during isolation with the anti-AbEp Ab. Since these potential secondary binding sites between Ii and class II might not be of high affinity, various lysis conditions were tested to determine whether any of them would allow detection of Ii bound to peptide-occupied class II. After preliminary experiments two conditions were chosen for additional experiments, lysis with 0.5% Nonidet P-40 and lysis in 0.5% CHAPSO.
Cells from AbEpIi-,
AbEpIi+ and C57BL/6 mice were lysed under the
two conditions described. Lysates were immunoprecipitated with
anti-Ab or anti-AbEp, analyzed on
SDS-PAGE, and Western blotted with an anti-Ii Ab. As shown in
Figure 4, anti-Ab
immunoprecipitates from C57BL/6 and AbEpIi+
cells both contained an Ii-specific band when probed with the
Ii-specific mAb. This was true for both lysates made with 0.5% Nonidet
P-40 and those made under the gentler conditions, with 0.5% CHAPSO.
The band from AbEpIi+ cells was less intense
than that from C57BL/6 cells, because the former cells bore less
Ab (24).
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). These experiments showed that Ii
could bind to Ab even if the peptide binding groove of
Ab were occupied by another peptide. Ii binding under these
circumstances was weak, however, since it was observed only with very
gentle detergents such as CHAPSO. Even relatively mild detergents such
as Nonidet P-40 did not allow coprecipitation of the complex of
AbEp and Ii.
Comparison of the intensities of the various Ii bands in Figure 4
allowed a rough measurement of the amount of Ii bound to
AbEp via sites that included the peptide binding groove or
solely via other sites. The Ii precipitant from
AbEpIi+ cells with anti-Ab was
much more intense if lysates were prepared in CHAPSO than if lysates
were prepared in Nonidet P-40. Since CLIP binding to the groove of
class II should be stable to room temperature Nonidet P-40, this
implied that a respectable percentage of the Ii bound to
AbEp was engaged at sites other than the peptide binding
groove. This conclusion was confirmed by the observation that the Ii
band precipitated from AbEpIi+ Nonidet P-40
lysates by anti-Ab was of roughly the same
intensity as that precipitated from AbEp CHAPSO lysates by
anti-AbEp.
In summary, these experiments showed that Ii could indeed bind to AbEp at sites other than the peptide binding groove.
The low affinity Ii-class II interaction nevertheless resulted in efficient cleavage of the Ep peptide
The T cell hybridoma experiments described above showed that the Ep could be displaced from AbEp cells that were Ii+. However, the experiments did not show how frequently this happened or what percentage of AbEp chains had had their covalent peptide cleaved from them. The following experiment was therefore performed to study this.
Spleen cells were harvested from AbEpIi+ and AbEpIi- mice and incubated in tissue culture for 18 h with up to 10 ng/ml of IL-4 to increase their expression of class II and Ii. A separate experiment used spleen cells from C57BL/10 mice without pretreatment with IL-4. All cells were lysed, precleared, and immunoprecipitated with anti-AbEp or anti-Ab. Protein was eluted from the beads by boiling in SDS and was run on 12.5% polyacrylamide gels. The gels were then analyzed by Western blot using the mAb N22 (26), which reacts in Westerns with the ß-chain of Ab.
The results of this experiment were consistent with those of the
pulse-chase analysis shown in Figure 3. In all immunoprecipitates there
was no sign of ß-chains with unexpected molecular masses on the gels,
indicating that almost all the class II precipitated in these
experiments bore mature sugars (Fig. 6).
The anti-Aßb Ab detected wild-type Aßb,
with molecular masses of about 30 kDa, precipitated with
anti-Ab from C57BL/10 cells (data not shown). As
expected, no such material was precipitated from the same cells with
anti-AbEp. Also as expected, both
anti-Ab and anti-AbEp precipitated a
single higher molecular mass Aßb-containing band from
AbEpIi- cells. The molecular mass of this
band, 34 kDa, was that expected for Aßb covalently bound
to Ep. The absence of any lower molecular mass Aßb band
precipitated from AbEpIi- cells was consistent
with our previous observation that all the class II ß-chains in
AbEpIi- mice remained covalently linked to the
Ep (see above) (33).
In contrast, anti-Ab and anti-AbEp
precipitated both Aßb-containing bands from
AbEpIi+ cells, albeit in different proportions
for the two Abs. Most of the ß-chains precipitated by
anti-AbEp were of the higher molecular mass, confirming
that most of the class II proteins that were occupied by Ep in these
cells had ß-chains that were still covalently linked to the peptide.
About 7% of these class II proteins were occupied by Ep, however (see
the faint band in lane 1 of Fig. 6), even though the
covalent linker between the ß-chain and Ep had been cleaved. These
must be proteins from which Ep had not been displaced despite linker
cleavage or which had rebound some of the free Ep present, because of
cleavage, in the endosomes.
Anti-Ab immunoprecipitates revealed that in AbEpIi+ cells, the covalent linker had been cleaved on about 70% of all Aßb chains. Only 30% of the total Aßb remained covalently bound to the linker and peptide. The 34-kDa band that was immunoprecipitated by anti-Ab was of nearly the same intensity as that immunoprecipitated by anti-AbEp, demonstrating that the two Abs precipitated material with equal efficiency.
Assuming that the covalent linker would be cleaved from any class II protein that passed through endosomes on its way to the cell surface, these data put a lower limit of at least 70% on the percentage of class II proteins in AbEpIi+ cells that reached the cell membrane by this route. The 30% of uncleaved proteins might all have bypassed the endosomes. However, some of them might still be in the late Golgi before transit to endosomes. Thus, despite the covalently bound Ep, at least 70% of the class II proteins in AbEpIi+ cells must have bound Ii and passaged through the endosomal compartment. Ii must have bound these proteins in two ways, either at sites that did not include their peptide binding grooves or, in addition, at their peptide binding grooves, where it must have successfully competed with the covalent peptide.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alternatively, in the endoplasmic reticulum Ii might be able to bind to Ab molecules with grooves already occupied by Ep. In this case Ii must have bound to Ab at a site(s) other than the peptide binding groove. Ab molecules associated with Ii in this way would then also travel to the cell surface via the endocytic route. In this acidic environment, the Ab molecule would undergo a conformational change to the looser conformation associated with the ability of class II molecules to bind peptides in this acidic environment, increasing both the on and the off rate of the peptide. Also, the covalent linker between Ab and Ep would be cleaved in this protease-rich environment, and the displaced Ep peptide would lose its high local concentration advantage over other peptides for binding to Ab.
It is remotely possible that Ab might bind Ep and CLIP in its groove simultaneously and that this complex could still bind anti-Ab, since class II has been shown to bind two peptides to its groove at the same time (36, 37). If this were so, then Ii might still be bound via the Ab groove to the class II protein. We think this is an unlikely explanation for our results, however, first because class II that is bound at its groove to more than one peptide is in the SDS unstable form, unlike the form found in our experiments. Secondly we think it is very unlikely that anti-AbEp would bind to Ab with such a distorted association with Ep.
We believe that both these mechanisms contribute about equally to
the replacement on Ab of Ep by other peptides. Our data
suggest that CLIP could to some extent compete with Ep for binding to
the grooves of newly synthesized Ab proteins. This is
because unstable Ab molecules appeared very quickly after
their synthesis (Fig. 3) and also because some of the conjugates of Ii
and Ab from AbEpIi+ cells
were stable in a relatively harsh detergent, Nonidet P-40 (Fig. 4). On
the other hand, Ii certainly could bind to some Ab proteins
without engaging the peptide grooves of these molecules (Fig. 4), and
the blots suggest that on AbEpIi+ cells, the
amount of Ii associated with class II by this mechanism is about the
same as that which has displaced Ep.
A summary of our current model of class II migration in
AbEpIi+ cells is shown in Figure 7. Assuming that most of the class II in
the cells had reached the cell surface, at least 70% of the protein
must have proceeded to the cell surface via the endosomes, since the
Ep-class II covalent linker was cleaved in 70% of the class II
proteins. All this endosomal migration required Ii binding to class II.
Ii drove this migration by binding in one of two ways, either by
conventional means, including CLIP binding, or by binding at sites that
did not include CLIP binding to the class II peptide binding groove.
These two mechanisms appeared to operate with approximately equal
efficiency in the cells.
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). This ability of Ii to engage class II at sites other than the peptide binding groove may have biologic significance. Different class II alleles vary greatly in their affinity for CLIP, and this was early evidence that CLIP was indeed binding in the peptide binding groove rather than at some other site (12, 38, 39, 40). For class II alleles that have a low affinity for CLIP, secondary binding sites may enhance the possibility that they bind Ii and thus are guided to the endocytic compartment where they can interact with exogenous antigenic peptides.
The experiments described here indicate that in the absence of Ii engagement, properly folded class II proteins can bypass the endocytic compartment on their way to the cell surface. Such an idea was suggested by the experiments of others (41, 42, 43, 44). Class II proteins that follow this route may not avoid the endocytic compartment completely, since it has recently been shown that the dileucine motif in the cytoplasmic tails of class II ß-chains causes the proteins to recycle through a particular type of endocytic compartment (44). Proteolysis is limited in this compartment, however (44), and, as demonstrated by our results in AbEpIi- cells, is clearly not powerful enough to cleave the linker between Ab and Ep.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dr. Philippa Marrack, Department of Medicine, National Jewish Center, Howard Hughes Medical Institute, 1400 Jackson Street, K512, Denver, CO 80206. E-mail address:
3 Abbreviations used in this paper: Ii, invariant chain; CLIP, class II-associated invariant chain peptide; HRP, horseradish peroxidase; CHAPSO, 3-[(3-cholamidopropyl)dimethylammoniol-2-hydroxy-1-propanesulfonate]; COVA, chicken OVA323-339.
Received for publication April 1, 1998. Accepted for publication June 25, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß dimers and facilitates peptide loading. Cell 82:155.[Medline]
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J. B. Ashman and J. Miller A Role for the Transmembrane Domain in the Trimerization of the MHC Class II-Associated Invariant Chain J. Immunol., September 1, 1999; 163(5): 2704 - 2712. [Abstract] [Full Text] [PDF]
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 J. Bender, T. Mitchell, J. Kappler, and P. Marrack CD4+ T Cell Division in Irradiated Mice Requires Peptides Distinct from Those Responsible for Thymic Selection J. Exp. Med., August 2, 1999; 190(3): 367 - 374. [Abstract] [Full Text] [PDF]
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A. Qadri, J. Thatte, C. G. Radu, B. Ober, and E. S. Ward Characterization of the interaction of a TCR {alpha} chain variable domain with MHC II I-A molecules Int. Immunol., June 1, 1999; 11(6): 967 - 977. [Abstract] [Full Text] [PDF]
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J. White, F. Crawford, D. Fremont, P. Marrack, and J. Kappler Soluble Class I MHC with {beta}2-Microglobulin Covalently Linked Peptides: Specific Binding to a T Cell Hybridoma J. Immunol., March 1, 1999; 162(5): 2671 - 2676. [Abstract] [Full Text] [PDF]
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