(Received for publication, March 22, 1995; and in revised form, June 15, 1995)
From the
In this report we demonstrate that the ion pair Arg-80 and
Asp-57
, located in the peptide-binding site of nearly all class II
major histocompatibility complex (MHC) proteins, is important for
surface expression and function of the murine class II heterodimer
I-A
. Charge reversal at either of these two residues by
site-directed mutagenesis generated mutant class II molecules that
failed to appear at the cell surface. This defect in surface expression
was partially reversed when the invariant chain was present or when the
mutants were paired with the corresponding charge-reversed variant of
the opposite chain. Surprisingly, surface expression was restored when
cells expressing the single-site mutants were cultured at reduced
temperature. In addition, the substitution of Asp-57
with residues
found in alleles of class II molecules associated with diabetes
resulted in heterodimers that were inefficiently expressed at the cell
surface and presented foreign peptide poorly. Together, these results
demonstrate that the formation of a salt-bridge between Arg-80
and
Asp-57
is required for efficient surface expression of class II
MHC molecules, therefore representing an important step in the assembly
and transport of functional class II heterodimers to the cell surface.
T lymphocytes respond to foreign antigens by detecting peptide
fragments of those antigens bound to products of the major
histocompatibility complex (MHC) ()and displayed on the
surface of antigen presenting cells (reviewed in (1) ). The
three-dimensional structures of both class I and II MHC molecules
complexed with self- or foreign peptide antigens have recently been
solved, revealing much about the molecular basis of peptide binding to
MHC
molecules(2, 3, 4, 5, 6, 7, 8) .
Structurally, both classes of MHC molecules are similar, as predicted
by sequence comparison(9) ; however, they differ in fine
detail, especially in the peptide-binding site(7, 8) .
These differences partly account for the distinct manner in which each
class of molecule binds peptide. In class I molecules, residues forming
the ``ends'' of the peptide-binding site bury the termini of
bound peptides(3, 4, 5, 6) . In
contrast, in class II molecules, both ends of the peptide-binding site
are ``open,'' allowing the termini of bound peptides to
extend out of the site(7, 8) . Consequently, peptides
bound to class I molecules are predominantly 8-10 residues in
length(10, 11, 12, 13, 14) ,
whereas peptides bound to class II molecules tend to be 13-25
residues in length(15, 16, 17) .
In both classes of MHC molecules, the presence of bound peptides influences their tertiary structure and efficiency of appearance on the cell surface. The binding of self- and foreign peptides to the class I heavy chain promotes the folding, assembly, and surface expression of class I molecules by stabilizing the class I complex(18, 19) . Similarly, peptide binding to class II molecules influences the conformation, stability, and surface appearance of class II molecules (20, 21, 22, 23, 24) . Thus, detailed functional analysis of the peptide-binding sites of both classes of molecules will be necessary to understand the role of peptide binding in determining the tertiary structure, and hence, surface expression of MHC molecules and subsequent display of foreign antigens to the immune system.
We have been interested in the
biochemical basis of recognition of peptide antigens bound to MHC
molecules by T cells. We have characterized the T cell receptor (TCR)
expressed by the murine T cell clone D5(25) , which recognizes
arsonate (Ars)-conjugated peptides presented by the class II molecule
I-A(26) . We have engineered single-site
substitutions in the putative MHC/peptide antigen-binding site of the
D5 TCR and have identified several residues that are important in
recognition of the MHC
peptide complex(27, 28) .
Subsequently, we sought to compensate these single-site TCR mutations
with complementary mutations in the I-A
molecule. In the
course of these studies, we identified an ion pair, Asp-57 of the
-chain and Arg-80 of the
-chain of I-A
, that
plays an essential role in surface expression of I-A
as
well as peptide presentation.
Figure 1:
Expression of wild-type I-A heterodimers on the surface of transfected COS cells. COS cells
transfected with WT I-A
- and
-chain DNA or
mock-transfected (no DNA) were analyzed by flow cytometry using control
or I-A
-specific mAb. Shown are histograms of 5000 cells
stained with control mAb 14-4-4S, which recognizes
I-E
, or mAb MK-D6 specific for I-A
, followed by
phycoerythrin-conjugated secondary antibody. Bars indicate the
portions of the histograms used to calculate ``fluorescence
units'' as described under ``Materials and Methods.''
Fluorescence units obtained with MK-D6 staining of COS cells
transfected with WT I-A
were 870,000 compared to 740
obtained with mock-transfected COS.
We individually substituted Lys for several negatively
charged residues in the -helical regions of the
1- and
1-domains of the I-A
heterodimer by site-directed
mutagenesis ( Table 1and Fig. 9). These helices form the
``walls'' of the peptide-binding site of MHC molecules and,
when bound with foreign peptide, are recognized by the TCR. We chose to
express these variant I-A
molecules in COS cells because
COS cells can transiently express murine class II molecules even though
they fail to express the invariant chain(37) . We analyzed
expression of these variant I-A
molecules by flow cytometry
using a variety of mAb recognizing different epitopes on I-A
(Table 2). We then tested the ability of the variant class
II molecules expressed on COS cells to present foreign peptides to
I-A
-restricted, antigen-specific T cell hybridomas.
Figure 9:
Location of tested residues on a predicted
map of I-A. Shown is a ribbon diagram of the top view of
the peptide-binding site of HLA-DR1 bound with an influenza virus
peptide (8) onto which the residues of I-A
tested
in this report were modeled, based on the sequence similarity shared
among class II molecules(9) . Substitution of Asp-57
with
Lys or Arg impaired surface expression of I-A
heterodimers,
whereas substitution of 10 other negatively charged residues with Lys
had little or no effect on surface expression or presentation of
foreign peptide to an I-A
-restricted TCR. The substitution
with Glu or Asp for Arg-80
, predicted to form a salt-bridge with
Asp-57
in I-A
, also impaired surface expression of
I-A
and its ability to present foreign
peptides.
COS cells expressing WT I-A and
the 11 variant Lys-substituted heterodimers listed in Table 1were analyzed by flow cytometry using several mAb reactive
against either the
1- or
1-domains of I-A
(Table 2). The relative efficiency of surface expression of
WT and variant I-A
molecules was estimated by calculating
fluorescence units for each combination of
- and
-chains (Fig. 2). As shown in Fig. 2, of the 11 Lys
substitutions, only one, D57K
, prevented surface expression of
I-A
, since neither the
-chain-reactive mAb
K24-199 nor any of the
-chain-reactive mAb recognized COS
cells transfected with this variant. The remaining 10 Lys substitutions
did not affect surface expression of I-A
since they all
reacted with
-chain mAb K24-199 and at least one
-chain
mAb. Of these, seven (E51K
, E59K
, E70K
, E74K
,
D76K
, E84K
, and E87K
) had no effect on mAb reactivity:
COS cells expressing these variant heterodimers stained with all mAb
tested at intensities within a factor of three compared to COS cells
expressing WT I-A
. In contrast, the remaining three
substitutions (E59K
, E66K
, and E69K
) eliminated
recognition by one or more of the
-chain mAb. The E59K
heterodimer failed to stain with mAb BP107 and N22, the E66K
heterodimer failed to stain with mAb 34-5-3S, and the E69K
heterodimer failed to stain with mAb MK-D6, 34-5-3S, BP107,
28-16-8S, and M5/114. Therefore, Glu-59
, Glu-66
, and
Glu-69
are likely to be solvent accessible in I-A
, in
agreement with the orientation of the corresponding residues in the
human class II molecule HLA-DR1(7, 8) . The loss of
reactivity of mAb with these variant I-A
molecules agrees
with previous reports demonstrating that the N-terminal half of the
-helix of the
1-domain of I-A molecules contributes to the
epitopes of several mAb (for example, see Refs. 36, 52). In addition,
our results agree with previous epitope mapping and provide new or more
detailed information regarding the reactivity of several mAb (see Table 2).
Figure 2:
Serological analysis of wild-type and
variant I-A heterodimers expressed on the surface of
transfected COS cells. COS cells transfected with plasmids encoding the
indicated WT or variant
- and
-chains of I-A
were
analyzed for surface expression using control (14-4-4S) or
several I-A
-reactive mAb (Table 2). I-A
mAb K24-199 is
-chain specific, whereas the remaining
I-A
mAb are
-chain specific. Fluorescence units were
calculated for staining of the COS cells and are representative of
several independent experiments.
We determined whether any of the 10 substitutions
that were tolerated with respect to surface expression of I-A affected the presentation of foreign peptides to an
I-A
-restricted T cell hybridoma. Fig. 3A shows representative experiments indicating that COS cells
expressing each of these variant heterodimers were able to present
Ars-Ova(33-50) to D5h cells. Fig. 3B summarizes
the results of several such experiments. When the slight differences in
surface expression between the variant and WT I-A
heterodimers are taken into account (see Fig. 2), each of
these 10 variant heterodimers were within 10-fold as efficient at
peptide presentation as WT I-A
(Fig. 3B).
These results demonstrate that although three of the 10 substitutions,
including E59K
, E66K
, and E69K
, completely eliminated
recognition by at least one mAb, none profoundly affected peptide
presentation.
Figure 3:
Response of D5h T cell hybridoma to
antigenic peptide presented by COS cells expressing wild-type or
variant I-A heterodimers. In A, COS cells
expressing WT or the indicated
- (top) or
-chain (bottom) variant I-A
were tested for the ability
to present the antigenic peptide Ars-Ova(33-50) to the
I-A
-restricted T cell hybridoma D5h. The difference in the
potency of peptide presented by WT I-A
in the two
experiments is due to variation in transfection efficiency and the
response of D5h and IL-2 indicator cells. In B, the relative
ability of variant heterodimers to present antigenic peptide to the D5
TCR in several experiments was calculated as described under
``Materials and Methods'' and is represented by solid
circles (WT = 1.0). Bars represent the geometic
mean of these independent experiments.
Figure 4:
Surface expression of wild-type or variant
I-A containing substitutions at Asp-57
in COS cells.
COS cells transfected with plasmids encoding the indicated WT
-chain and WT or variant
-chains were analyzed by flow
cytometry using control (14-4-4S) or I-A
-reactive
mAb. In B and C, cells were co-transfected with
control (C) 2B4
-
or invariant chain (Ii)
DNA. In C, COS cells co-transfected with I-A
and
2B4
-
DNA were stained with TCR
-chain-specific mAb
H57-597 to detect surface 2B4
-
molecules; in this
experiment, cells co-transfected with WT
-chain and the invariant
chain serve as control cells for staining with mAb H57-597. These
results are representative of several independent
experiments.
Since the invariant
chain has been demonstrated to regulate expression of class II
molecules in vivo(53) and enhance surface expression
of certain class II molecules containing haplotype-mismatched -
and
-chains(40) , we tested whether co-expression of the
invariant chain could enhance expression of WT or variant I-A
molecules on the surface of transfected COS cells. In agreement
with previous reports(40, 54) , co-transfection with
the invariant chain had little effect on surface staining of WT
I-A
by mAb MK-D6 and M5/114 but reproducibly enhanced
staining of WT I-A
by mAb K24-199 by 2.8-fold
(± 0.7-fold, n = 4) (Fig. 4B).
Co-transfection of the invariant chain reproducibly enhanced surface
staining of variant heterodimers D57S
and D57A
by mAb
K24-199, MK-D6, and M5/114. Enhancement of staining by mAb
K24-199 was 14-fold (± 7-fold, n = 4) for
D57S
and 13-fold (± 5-fold, n = 4) for
D57A
; by MK-D6 was 3.2-fold (± 0.7-fold, n = 4) for D57S
and 2.3-fold (± 0.5-fold, n = 3) for D57A
; and by M5/114 was 3.6-fold (±
1.0-fold, n = 4) for D57S
and 3.8-fold (±
1.1-fold, n = 4) for D57A
. In fact, when the
invariant chain was co-transfected, surface staining of these variant
heterodimers was nearly as intense as observed with WT I-A
(Fig. 4B); for D57S
and D57A
staining
was 89% (± 15%, n = 4) and 78% (± 26%, n = 4), respectively, of wild-type levels using mAb
M5/114. Co-expression of the invariant chain reproducibly enhanced
expression of variants D57K
and D57R
to levels slightly above
background (Fig. 4B); however, this effect was not
observed in experiments in which the efficiency of COS cell
transfection was low. Co-transfection with plasmid DNA encoding the
chimeric cell surface molecule 2B4
-
(38) in place of
invariant chain DNA did not restore surface expression of I-A
variants substituted at Asp-57
(Fig. 4B).
The relative efficiency of transfection of WT or variant DNA in
these experiments was monitored by co-transfection of DNA encoding
2B4-
. Surface expression of the reporter molecule
2B4
-
was approximately equivalent when it was co-transfected
with either WT or variant I-A
DNA (Fig. 4C), demonstrating that the decreased expression
of these variants was not due to nonspecific effects such as toxicity
or inhibition of the expression of cell surface molecules.
We tested
whether the D57S or D57A
substitution had any effect on the
presentation of foreign peptides to I-A
-restricted T
hybridomas in experiments in which surface expression of these variants
was made equivalent to WT I-A
by co-transfecting the
invariant chain. Fig. 5shows that COS cells expressing variant
heterodimers D57S
or D57A
were at least 25-fold less
efficient at presentation of Ars-Ova(33-49) peptide to the D5h T
hybridoma compared to WT I-A
. These substitutions also
diminished presentation of Ova(323-339) peptide to T hybridoma
3DO-54.8 and
cI(12-26) peptide to 9C127 (data not shown),
both of which recognize peptides presented by I-A
. These
results demonstrate that the identity of the side chain at position 57
in the I-A
-chain not only plays a role in surface
expression but also influences peptide presentation.
Figure 5:
Response of D5 T cell hybridoma to
antigenic peptide presented by COS cells expressing wild-type or
variant I-A containing substitutions at Asp-57
. In A, COS cells co-transfected with WT or variant I-A
D57S
and D57A
heterodimers and the invariant chain were
tested for the ability to present Ars-Ova(33-49) to D5h cells.
Relative levels of surface expression of WT and variant heterodimers
determined by flow cytometry are shown in the inset. In B, the relative ability of these variant heterodimers to
present antigenic peptides to D5h in independent experiments is
summarized as in Fig. 3B.
Figure 6:
Flow cytometric analysis of wild-type or
variant I-A substituted at Arg-80
transfected into COS
cells. COS cells transfected with WT
-chain alone or WT or variant
- and
-chains, co-transfected with control (C) or
invariant chain (Ii) DNA, were analyzed by flow cytometry
using control (28-14-8S) or I-A
-reactive mAb. Surface
expression was calculated as fluorescence
units.
We
tested whether charge reversal of Arg-80 could compensate the
defect in surface expression of the charge-reversed variants of
Asp-57
. Fig. 6shows that surface expression of the variant
D57R
was partly restored when it was paired with the variant
R80E
, even in the absence of the invariant chain. Surface
expression of the variant D57R
paired with R80E
was further
enhanced in the presence of the invariant chain. Similar results were
obtained when R80E
was transfected along with D57K
or when
R80D
was transfected with D57K
or D57R
(data not shown).
Thus, the defect in surface expression of I-A
chains
containing individual charge reversals at Asp-57
or Arg-80
can be partially reversed by expression of the partner charge-reversed
chain, suggesting direct interaction between Arg-80
and
Asp-57
.
We tested the consequences of charge reversal at
Arg-80 or Asp-57
on the presentation of foreign peptide. Fig. 7shows that the singly substituted variants R80E
or
R80D
paired with the WT
-chain were 50-100-fold less
efficient at presentation of Ars-Ova(33-49) peptide to D5h cells
than WT I-A
when expressed on the cell surface in COS cells
co-transfected with the invariant chain. In addition, when matched for
surface expression with WT I-A
, the doubly substituted
variant R80E
/D57R
was also 50-100-fold less efficient
at peptide presentation than WT I-A
(Fig. 7). These
single- and double-site substitutions also decreased presentation of
Ova(323-339) peptide to 3DO-54.8 cells (data not shown).
Therefore, as do substitutions at Asp-57
, substitutions at
Arg-80
affect surface expression of I-A
and its
ability to present foreign peptides.
Figure 7:
Response of D5h T cell hybridoma to
antigenic peptide presented by COS cells expressing variant I-A heterodimers substituted at Arg-80
and Asp-57
. In A, COS cells co-transfected with the invariant chain and WT or
variant I-A
as indicated were tested for the ability to
present Ars-Ova(33-49) peptide to T cell hybridoma D5h. In the insets, relative levels of surface expression of I-A
heterodimers were determined by flow cytometry and calculated as
fluorescence units. In B, the relative ability of these
variant heterodimers to present antigenic peptide to D5h cells in
independent experiments is summarized as in Fig. 3B.
Fig. 8shows that singly substituted variant class II
heterodimers containing charge reversal at Asp-57 or Arg-80
,
which were expressed poorly at 37 °C in the absence of the
invariant chain, appeared on the surface of COS cells cultured at room
temperature. After room temperature incubation, the level of surface
expression of singly substituted charge-reversed variants R80E
or
D57R
was identical to expression of the doubly substituted
charge-swapped variant R80E
/D57R
and WT I-A
heterodimers, as determined by staining with mAb MK-D6 (Fig. 8A) and
- and
-chain reactive mAb (Fig. 8B). The increase in fluorescence of singly
substituted charge-reversed variants at room temperature was due to an
increase in both the number of cells expressing I-A
as well
as the intensity of those cells (Fig. 8A). On the other
hand, the decrease in fluorescence of R80E
/D57R
and WT
heterodimers (Fig. 8B) was primarily due to the
reduction of cells expressing I-A
at room temperature,
rather than a decrease in the intensity of staining (Fig. 8A). It is important to note that the pattern of
staining of heterodimers containing WT or variant
- and
-chains expressed in COS cells at room temperature and at 37
°C was similar when tested with the
- and
-chain-specific
mAb (Fig. 8B), demonstrating that the differences in
temperature did not cause selective changes in the epitopes or
conformations of I-A
molecules detectable by these mAb.
These results demonstrate that surface expression of mutant class II
molecules expressed poorly at 37 °C in the absence of the invariant
chain can be enhanced at reduced temperature.
Figure 8:
Surface expression of mutant I-A heterodimers in COS cells cultured at reduced temperature. COS
cells transfected with WT or variant
- and
-chain DNA of
I-A
were cultured for 2 days at 37 °C (top
row) or 1 day at 37 °C followed by 1 day at room temperature (RT, bottom row), and analyzed by flow cytometry
using I-A
-reactive mAb. In A, results are
expressed as histograms of 5000 living cells analyzed by staining with
mAb MK-D6. Bars indicate regions used to calculate
fluorescence units. In B, fluorescence units were calculated
for staining with control (28-14-8S) or I-A
-reactive
mAb. These results are representative of two separate
experiments.
We have conducted extensive mutational analysis of negatively
charged residues in the peptide/TCR-binding domain of the class II
molecule I-A, which has been modeled onto the structure of
HLA-DR1 (Fig. 9). We have demonstrated the importance of the ion
pair Arg-80
and Asp-57
in both surface expression of and
peptide presentation by class II heterodimers. Our results suggest that
interactions in the peptide-binding site, including the formation of
the salt-bridge between Arg-80
and Asp-57
and possibly
peptide binding, are important for folding and surface expression of
class II molecules at 37 °C. These results agree with previous
studies demonstrating that residues pointing into the peptide-binding
site within the
1- and
1-domains influence surface expression
of class II
molecules(32, 36, 57, 58) . The
mutant class II molecules described here resemble haplotype-mismatched
class II
- and
-chains, which are partially blocked in egress
to the medial Golgi compartment(40) , and mutant heterodimers
that are blocked in a relatively late stage of transport to the cell
surface(57) . We hypothesize that an important step in the
transport of class II molecules to the cell surface can be blocked by
disrupting the salt-bridge between Arg-80
and Asp-57
or the
hydrogen bonds between these residues and bound peptides.
The ion
pair Asp-57 and Arg-76
, which corresponds to Arg-80
in
murine class II
-chains, is present in nearly all class II
molecules (9) and is likely to represent an important
universal structural feature of class II molecules(8) . Based
on the structure of HLA-DR1 (8) , the formation of this
salt-bridge replaces the hydrogen-bonding network found at the end of
the peptide-binding site of class I molecules in which the C termini of
bound peptides are buried. Asp-57
, present in the
-chain of
most class II molecules, occupies a position analogous to Thr-143
conserved in all class I
2-domains; similarly, Arg-80
,
conserved in all
-chains of class II molecules, occupies a
position analogous to Tyr-84, conserved throughout all class I
1-domains(9) . Together Tyr-84 and Thr-143 in class I
heavy chains form hydrogen bonds with C termini of foreign and
self-peptides(3, 4, 5, 6) . The side
chains of Asp-57
and Arg-80
in HLA-DR1 occupy roughly the
same position as the side chains of Tyr-84 and Thr-143 in class I
molecules, yet are located about 2 Å lower in the class II
molecule due to slight changes in the
-helices(7) . This
lowering ``opens'' this end of the peptide-binding site of
class II molecules, allowing longer peptides to bind and extend out of
the site(7, 8) . In addition, Asp-57
and
Arg-80
form hydrogen bonds with amide hydrogen and carbonyl oxygen
atoms of bound peptides.
The interchain salt-bridge formed between
Asp-57 and Arg-80
and the hydrogen bonds they form with the
backbone of peptides bound to class II heterodimers might contribute a
great deal to the stability of the class II heterodimer and its ability
to bind peptides. The reversal of charge in singly substituted mutants
D57R
, D57K
, R80E
, and R80D
would bring together in
close proximity similarly charged residues in the peptide-binding
domains of assembling class II heterodimers, potentially destabilizing
a portion of the interface between the
- and
-chains and
preventing these mutant class II heterodimers from adopting a
conformation such that they can bind peptides efficiently. Similarly,
the substitution of Ser or Ala for Asp-57
disrupts the
salt-bridge, but unlike charge reversal at Asp-57
, this
substitution might be less disruptive because only one charged residue,
Arg-80
, remains unpaired. However, since the remaining unmutated
charged residue is still available to hydrogen bond to the main chain
of a bound peptide, the singly substituted mutant class II molecules
might be stabilized to some degree when provided with peptide. The
pairing of charge-swapped
-chains R80E
or R80D
with
-chains D57R
or D57K
restores a salt-bridge between
- and
-chains; however, these variants might not bind
peptides efficiently because hydrogen bonding between residues at these
positions and carbonyl oxygen and amide hydrogen atoms of bound
peptides is disrupted. Alternatively, the new charge-swapped
salt-bridge might not be as strong as the original WT salt-bridge
because the I-A
microenvironment that stabilizes the WT
salt-bridge might destabilize the charge-swapped salt-bridge, as
predicted on theoretical grounds(59) .
Because the
substitutions for Arg-80 and Asp-57
affect both suface
expression of I-A
and presentation of peptides to the TCR,
we propose that the defect in efficient surface expression of the
variant heterodimers results from intracellular editing of the
heterodimers as incorrectly folded proteins. Class II
- and
-chains are synthesized initially in the endoplasmic reticulum as
heterodimers complexed with the invariant chain and devoid of tightly
bound peptides(21) . The invariant chain forms multimers with
class II heterodimers(60) , precludes the binding of endogenous
peptides (61, 62) and may, like low affinity peptide
binding, prevent the aggregation and denaturation of peptide-free class
II heterodimers at physiological temperature(24) . The
invariant chain directs the intracellular transport of class II
heterodimers(63) , which includes passage through the Golgi
complex and subsequent entry into the endocytic pathway. In specialized
endocytic compartments(64, 65, 66) , the
invariant chain is proteolytically processed and dissociates from class
II heterodimers, resulting in the stable binding of peptides to class
II molecules and their transport to the cell surface. Class II
heterodimers that fail to acquire peptide undergo aggregation and
denaturation, are presumably recognized as mis-folded proteins, and are
blocked from transport to the cell surface(23) . The class II
mutants described in this report that are defective in surface
expression might be inactivated by aggregation, denaturation, or
degradation more rapidly than WT I-A
heterodimers. In the
presence of the invariant chain, surface expression of these variants
is partly restored, perhaps because the invariant chain stabilizes the
association of the nascent polypeptides, provides a peptide to the
peptide-binding site, or enhances the transport of the variant
heterodimers to a subcellular compartment where peptides can be
supplied.
In addition, the mutant class II molecules described here resemble class I molecules that fail to appear on the surface of RMA-S cells at physiological temperature but reappear at the cell surface at reduced temperature(56) . It is likely that at reduced temperature, both class I and II MHC molecules do not require bound peptide for surface expression because, when expressed in insect cells cultured at reduced temperature, they appear on the cell surface apparently devoid of bound peptide(22, 55) . Perhaps at reduced temperature, rates of inactivation of peptide-deficient MHC molecules and the mutant class II molecules described here are decreased. Our results support the hypothesis that both class I and II molecules might be best suited for surface expression at physiological temperature only when bound with peptide.
Finally, the importance of
Arg-80 and Asp-57
in surface expression and the function of
I-A
might explain why these residues are conserved
throughout nearly all class II molecules. Several naturally occurring
class II molecules have been identified that contain substitutions at
position
-57, which are analogous to the variants D57S
and
D57A
described in this report. It is noteworthy that these alleles
have been implicated in autoimmune disease. Deviations from Asp-57 in
human class II molecule
-chains have been observed in alleles
associated with insulin-dependent diabetes mellitus, including DQw3.2,
DQw2, DQw1.1, and DQw1.AZH, in which Ser, Val, or Ala has been
substituted for Asp-57
(67) . In the I-A
-chain of
non-obese diabetic mice, Ser is substituted for
Asp-57
(68) . Although such alleles have other
substitutions in addition to the change for Asp-57
that might
account for the association of these alleles with diabetes, the
identity of the side chain at
-chain position 57 appears to
determine susceptibility and resistance to diabetes: the presence of
Asp appears to be protective against diabetes(67) . Expression
of these variant forms of class II molecules might result in the
presentation of an abnormal array of self- or foreign peptides or an
altered conformation of class II molecule on the cell surface. Indeed,
self-peptides eluted from I-A
molecules of non-obese
diabetic mice are reportedly distinct from other peptides that bind
class II molecules in that they possess acidic C-terminal
residues(69) . Thus, antagonists directed at the
peptide-binding site of the variant class II molecule might be designed
that selectively inhibit presentation of these abnormal polypeptides.