(Received for publication, September 23, 1994; and in revised form, November 7, 1994)
From the
Major histocompatibility complex (MHC) class II molecules are
cell surface heterodimeric glycoproteins consisting of one and
one
polypeptide chain of similar size. These molecules play a
critical role in immune recognition by displaying processed antigens to
CD4-positive T helper cells. Several attempts to express the MHC class
II molecules by recombinant methods in various systems resulted in
either failure or poor recovery of the intact heterodimer. The present
study describes our successful effort to refold and reconstitute HLA
DR2 heterodimer from individually expressed
and
polypeptide
chains lacking the transmembrane hydrophobic regions in Escherichia
coli, in the presence of an immunodominant epitope analog from
human myelin basic protein (b-MBP(83-102)Y
). The
reconstituted DR2 heterodimer complex was selectively purified from
unfolded
and
chains using heterodimer-specific monoclonal
antibody (L243) coupled to a solid support. The detection of two
polypeptide chains in the purified refolded DR2-peptide complex
preparations was accomplished by Western blot analysis and
enzyme-linked immunosorbent assay using heterodimer- and chain-specific
polyclonal antibodies, and the presence of equimolar amounts of both
chain and
chain in the reconstituted complex preparation
was confirmed by a double label experiment. The quantitation of the
bound peptide in complex preparation was measured by incubating two
chains in the presence of
I-labeled peptide. An increase
in the yield of refolded and reconstituted DR2-peptide complexes was
observed with increasing peptide concentration in the reaction mixture.
Finally, the functional activity of the reconstituted DR2 complexes was
measured by their ability to stimulate
-interferon production by
SS8T cloned T cells in an antigen-specific and dose-dependent manner.
These results demonstrate that biologically active complexes of human
DR2
b-MBP(83-102)Y
can be prepared by proper
folding of human leukocyte antigen DR2
and
chains in the
presence of antigenic peptide. The yield of such DR2 heterodimers with
bound peptide is several thousand-fold higher over native DR2 purified
from transformed B cells. Since purified MHC class II-peptide complexes
have been shown to prevent autoimmune diseases in various animal
models, reconstituted heterodimer complexes may have significant
clinical relevance in antigen-specific treatment of various autoimmune
diseases. In addition, such complexes with increased yield will provide
better understanding of the trimolecular interactions between
MHC-peptide and T cell receptor.
The presentation of antigens to CD4-positive T helper cells
involves the binding of processed antigenic peptides to MHC ()class II molecules on the surface of antigen-presenting
cells(1, 2, 3, 4, 5) .
Purified MHC class II molecules isolated from cell surfaces are also
known to bind antigenic peptides in
vitro(6, 7, 8) . The yields of purified
MHC class II molecules from various cell sources are usually very poor
and represent less than 0.5% of the total protein
pool(9, 10, 11) . The low yield of purified
MHC class II molecules has always been an important limiting step in
elucidating structure-function correlation studies of these molecules.
Various attempts to clone and express MHC class II molecules have been
reported recently in prokaryotic (12) and eukaryotic systems (13, 14, 15, 16) . The recombinant
heterodimers prepared by these methods also represent very low yields
due to difficulties in refolding.
Structural information of MHC
class II-peptide interaction has become available recently from x-ray
crystallographic studies(17, 18) . The peptide binding
groove of MHC class II molecules consists of the 1 and
1
domains with eight-stranded
-pleated sheets as the floor of the
peptide binding groove with extended
helices. Peptides play a
related and distinct role in the structure and conformational
maturation of MHC class II molecules(19) . Approximately 2000
different peptides may bind to MHC class II molecules(20) .
Therefore it is difficult to obtain a peptide-free preparation of MHC
heterodimeric molecules because of the slow dissociation rate constant
of the bound peptides(13) . To overcome this problem some of
the MHC class II molecules are expressed in eukaryotic systems which
lack the peptide processing
machinery(13, 14, 15, 16) .
The
folding of MHC class II heterodimer also appears to be an inherently
difficult problem. Unlike MHC class I molecules, where the 1 and
2 domains from the same polypeptide form an intramolecular
dimer(21, 22) , the peptide binding site of class II
molecules consists of two separate domains,
1 and
1, from two
individual polypeptides(17) . The MHC class II protein,
therefore, differs from other heterodimeric proteins of the immune
system that have been successfully folded in vitro, where each
domain is composed of protein segments of a single polypeptide
chain(23, 24, 25) . Reconstitution of
functionally active murine MHC class II peptide complexes from Escherichia coli-expressed, unpurified individual
and
chains have been demonstrated recently(12) . The yield of
such active heterodimer represents 0.5-2% of the starting
protein. In this report, we describe the refolding of E.
coli-expressed recombinant human
and
chains lacking
the transmembrane regions followed by reconstitution of biologically
active HLA DR2. MBP peptide complexes with a yield of over 20% of the
starting protein concentration.
In an attempt to reconstitute human MHC class II
heterodimeric protein in the presence of known antigenic peptide and
without any endogenously bound peptide, individual and
chains lacking the transmembrane region were cloned and expressed in an E. coli system. The recombinant
and
chains
prepared by this method represent approximately 30% of the total cell
protein. The insoluble denatured inclusion body preparations were
solubilized in 8 M urea and purified in large scale quantities
by conventional chromatographic methods as described
previously(28) .
Individual chains were refolded by slow
dialysis at a concentration of 0.5 mg/ml against PBS prior to
reconstitution of the heterodimer. Equal amounts of and
chains were mixed at a concentration of 0.5 mg/ml in the presence of
b-MBP(83-102)Y
peptide. The addition of a biotin tag
at the N terminus of the peptide was created for the detection of bound
MBP peptide in the final complex preparation. In addition, a tyrosine
residue at the N terminus of the MBP peptide allowed radiolabeling with
I. In various quantitative peptide binding studies, the
detection of the biotin moiety of the peptide by enzyme conjugated
streptavidin resulted in either failure or poor detection of the bound
peptide. The reason for an undetected biotin tag in the reconstituted
complex preparation is unclear at present and could be due to steric
hindrance of the large streptavidin molecule to bind biotin molecules
in the peptide binding domain. Based on this unexpected result, the
radiolabeled peptide was used to calculate the percent peptide
occupancy.
The reconstitution of the heterodimeric molecule in the
presence of 10-fold molar excess of b-MBP(83-102)Y peptide was carried out under several different conditions during
optimization of the reconstitution procedure for maximum yield. Among
the various conditions, a reconstitution buffer containing both
dithiothreitol and glutathione gave the best results. The reconstituted
DR2 heterodimers as well as complexes with b-MBP(83-102)Y
peptides were selectively separated from the
and
chains by antibody affinity chromatography using dimer-specific L243
monoclonal antibody. This antibody is known to recognize a monomorphic
region of all human HLA DR molecules and the epitope for this
monoclonal antibody has been mapped on the
chain of the DR
molecule(34) . The lack of reactivity of the L243 antibody to
individual
and
chains by ELISA and Western blot analyses
was also confirmed in our laboratory (data not shown). In various
control experiments, we observed that the
chain alone incubated
in the presence of 10-fold molar excess of the
b-MBP(83-102)Y
peptide showed very weak binding to
the L243 antibody column and the eluted minor peak represents less than
2% of the total eluted heterodimer preparation. In contrast, incubation
of the
chain in the presence of 10-fold molar excess of the
b-MBP(83-102)Y
peptide showed absolutely no binding
to the L243 monoclonal antibody. Since individual chains are known to
form a low level of
-
and
-
homodimers in
solution(35, 36) , the small fraction of
chain
binding to L243 monoclonal antibody affinity column could be due to the
formation of a minor fraction of
-
homodimers in solution.
The reconstitution of functionally active heterodimers carried out
from crude unpurified preparations of and
chains showed
substantially low yield as compared to purified chains. As shown in Fig. 1, reconstitution performed with purified chains in the
presence of equal amount of peptide resulted in 20-fold increase in
yield as compared to unpurified chains. Eluted peak fractions were
analyzed by SDS-PAGE which showed the presence of both heterodimers,
partially dissociated monomers along with various levels of aggregates
(see Fig. 1B, inset). Like the native
heterodimer, various levels of aggregation were also observed for
reconstituted heterodimer under nonreduced gel electrophoresis
conditions. Upon reduction, these bands of high molecular mass merged
into the expected molecular sizes of 21 and 23 kDa for two chains. The
unexpected high molecular aggregates of purified complexes lacking the
transmembrane hydrophobic regions suggest that the aggregation level of
various purified native MHC class II molecules observed consistently is
not dependent upon the hydrophobic transmembrane regions of these
molecules. Using dynamic multi-angle laser light scattering
measurements, we observed that the peptide plays a critical role in
destabilizing such aggregates of MHC class II molecules, (
)suggesting that most of the heterodimer aggregates
observed in our reconstituted samples are the populations containing no
bound peptide.
Figure 1:
Isolation of
reconstituted DR2 heterodimer by L243 antibody affinity chromatography.
Elution profiles of reconstituted DR2 heterodimer from unpurified
chain (-TM) and
chain (-TM) inclusion body
preparation in the presence of 10-fold molar excess of
b-MBP(83-102)Y
peptide is shown in Panel A.
Panel B represents the reconstitution elution profiles of DR2
heterodimers from purified
and
chains in the presence of
10-fold molar excess peptide. The inset figure in Panel B represents peak fractions analyzed by
SDS-PAGE.
The presence of both and
polypeptide
chains in the eluted, reconstituted peak was demonstrated by Western
blot analysis and ELISA. Western blot analysis was performed using
heterodimer and chain-specific polyclonal antibodies. As shown in Fig. 2A, the reconstituted heterodimer was recognized
by all three polyclonal antibodies under both reduced and nonreduced
conditions. Similarly, the presence of both
and
chains was
characterized by antibody capture ELISA (Fig. 2B). In
this assay, the reconstituted and native DR2 were captured by
heterodimer-specific polyclonal antibody and detected by
HRP-conjugated, L243-purified monoclonal antibody. The
chain and
the
chain alone, incubated with equivalent amounts of
b-MBP(83-102)Y
peptide, did not show any reactivity
in this assay.
Figure 2:
Characterization of reconstituted DR2 by
Western blot analysis and ELISA. Panel A represents the
Western blot analysis of reconstituted DR2 by anti-DR2, anti- and
anti-
polyclonal antibodies, under reduced and nonreduced
conditions. Panel B represents antibody capture ELISA of
heterodimer using dimer specific antibodies. Closed circles,
native DR2; open circles, reconstituted DR2; closed
squares,
alone; and open squares,
alone.
To demonstrate the presence of two chains in
equimolar amounts, the and
chains were separately
radiolabeled with
S and
I, respectively. The
selection of the labeling tag for each chain was based on their
sequences. The
chain contains 9 lysine residues whereas the
chain contains 9 tyrosine residues. Equimolar amounts of the two
differently labeled chains were refolded and mixed with 10-fold molar
excess amount of b-MBP(83-102)Y
peptide. The
reconstituted heterodimers were eluted from the L243 monoclonal
antibody affinity column and analyzed for the presence of two chains. Fig. 3shows the absorbance of eluted fractions along with
I and
S counts of each fraction measured in
and
counters, respectively. The quantitation of each chain
in the final eluted pool was carried out using the specific activities
of the two chains. Results presented in Table 1show that both
chains are present in almost equal amounts. A small increase in the
total
chain correlates well with our observation that a small
fraction of
-
homodimers are capable of binding L243
antibody.
Figure 3:
Elution of double-labeled reconstituted
DR2 heterodimer from L243 column. and
chains were
radiolabeled with
S and
I, respectively, as
described under ``Materials and Methods.'' Purified labeled
chains were allowed to refold in the presence of 10-fold molar excess
of unlabeled b-MBP(83-102)Y
peptide. Panel A represents the elution profile of reconstituted DR2 by absorbency
at 280 nm. Panel B represents the profile of radiolabeled
I of
chain, as measured by the
radiation
count. Panel C represents the profile of radiolabeled
S of
chain as measured by the
radiation
count.
The effect of antigenic peptide concentration on the yield
of reconstituted heterodimers was next examined by incubating equimolar
amounts of the two chains in the presence of increasing peptide
concentrations. As shown in Fig. 4, increasing the peptide
concentration has a significant effect on the yield of reconstituted
DR2 heterodimers as seen by the elution profile and SDS-PAGE analysis
of peak fractions. At a peptide concentration of 50-fold molar excess,
the yield of reconstituted DR2 was more than 20% (Fig. 4). We
observed a further increase in yield up to 29% with 100-fold molar
excess of the MBP peptide. Thus, the presence of peptide is critical in
increasing the yield of reconstituted complexes. A low level of
reconstituted DR2 heterodimer representing 1-3% of the starting
protein was also observed in various experiments in the absence of
b-MBP(83-102)Y peptide. The peptide-dependent
refolding and assembly of complexes correlates well with various
earlier reports with MHC class I refolding (37, 38) in vitro. In vivo, the presence of
peptide is also a prerequisite for the correct folding and transport of
MHC class II-peptide complexes to the surface of the antigen-presenting
cells(39, 40, 41, 42, 43, 44) .
In this regard, peptides are also known to stabilize the quaternary
structure of MHC class II molecules(45) .
Figure 4:
Effect of the peptide concentration on
reconstitution of heterodimers. Equivalent amounts of and
chains were incubated with no peptide (Panel A), 1-fold molar
excess (Panel B), 10-fold molar excess (Panel C), and
50-fold molar excess (Panel D) b-MBP(83-102)Y
peptide. Reconstituted DR2 complexes were purified over L243
antibody column. Fractions of each elution profile (10 µl) were
analyzed by 13.5% SDS-PAGE followed by silver
staining.
The quantitation
of bound peptide in the refolded and reconstituted DR2 heterodimer
preparation was carried out by increasing concentrations of
radiolabeled peptide. The b-MBP(83-102)Y peptide was
radiolabeled by
I using the chloramine-T method and was
incubated at various concentrations with equivalent amount of purified
and
chains. Although increasing the peptide concentration
had significant effect on the yield of reconstituted DR2 heterodimer,
the percent of DR2 occupied with bound peptide remained constant (Table 2).
Finally, the functional activity of reconstituted
heterodimeric complexes was analyzed by measuring the increased level
of -IFN produced by H. saimiri-transformed SS8T cloned T
cells in a dose-dependent manner. An increase in
-IFN production
has been correlated with the occupancy of TCR on the surface of T
cells(33) . The specificity and restriction of the SS8T
transformed T cell clone for the HLA DR2 and MBP(84-102) peptide
has been demonstrated recently (33) . As shown in Fig. 5, both native and reconstituted
DR2
b-MBP(83-102)Y
complexes were able to
recognize TCR on the surface of transformed T cells. The reconstituted
DR2
b-MBP(83-102)Y
complexes appeared to be
more effective in producing
-IFN than native
DR2
b-MBP(83-102)Y
complexes in several
experiments (data not shown). In various controls, DR2 alone, DR2 with
irrelevant peptide b-MBP(124-143) and b-MBP(83-102)Y
peptide complexed with irrelevant MHC class II (HLA DR3), showed
no significant increase in the level of
-IFN production. Human T
cells are known to express low levels of MHC class II molecules on
their surfaces and can be stimulated in the presence of antigenic
peptide(46, 47) . In order to demonstrate that the
observed level of increased
-IFN is not due to the release of
bound peptide in the culture medium, the b-MBP(83-102)Y
peptide was complexed with HLA DR3 as a control which showed no
increase in
-IFN level. Similarly in mock experiment equivalent
amount of b-MBP(83-102)Y
peptide incubated under
identical refolding conditions in the absence of chains did not show
any increase in
-IFN level.
Figure 5:
Level of -IFN production of SS8T T
cells in the presence of reconstituted
DR2
b-MBP(83-102)Y
complex. Occupancy of TCR by
complexes of reconstituted DR2. b-MBP(83-102)Y
was
shown by increased level of
IFN using SS8T transformed T cells,
restricted to DR2 and MBP(84-102) complexes. Purified native DR2
and DR3 proteins at a concentration of 0.5 mg/ml were incubated with
50-fold molar excess of respective peptides in citrate buffer pH 6.0 at
37 °C for 96 h. The complexes of
DR2
b-MBP(83-102)Y
,
DR2
b-MBP(124-143), and DR3
b-MBP(83-102)Y
were prepared and the unbound free peptide was removed by passing
through Sephadex G-75 gel filtration column. The level of
-IFN was
measured by ELISA as described under ``Material and
Methods.'' Closed circles, native
DR2
b-MBP(83-102)Y
complex; open
circles, DR2 alone; open squares, DR2 with irrelevant
b-MBP(124-143) peptide; closed squares, DR3 with
b-MBP(83-102)Y
; and closed triangles,
reconstituted DR2
b-MBP(83-102)Y
complex.
In conclusion, results presented
here demonstrate the formation of functionally active HLA DR2
heterodimeric complexes containing antigenic epitopes. The yield of
such complexes is approximately 8000-fold higher than the native DR2
molecules. Present results from our laboratory showed that purified MHC
class II-peptide complexes can be used for the prevention and treatment
of experimental autoimmune disease in various animal
models(48) . The reconstituted heterodimeric MHC
class II containing known antigenic epitope may have significant
clinical relevance in developing antigen-specific therapeutics for
various autoimmune diseases. In addition, such complexes in sufficient
quantities, will facilitate several structure-function studies
including x-ray crystallographic studies for better understanding of
the trimolecular interactions among MHC class II, peptide, and TCR.