(Received for publication, March 12, 1997, and in revised form, March 15, 1997)
From Mercator Genetics, Inc., Menlo Park,
California 94025 and the § Edward H. Doisy Department of
Biochemistry and Molecular Biology, Saint Louis University School
of Medicine, St. Louis, Missouri 63104
We recently reported the positional cloning of a
candidate gene for hereditary hemochromatosis (HH), called
HLA-H, which is a novel member of the major
histocompatibility complex class I family. A mutation in this gene,
cysteine 282 tyrosine (C282Y), was found to be present in 83% of
HH patient DNAs, while a second variant, histidine 63
aspartate
(H63D), was enriched in patients heterozygous for C282Y. The functional
relevance of either mutation has not been described.
Co-immunoprecipitation studies of cell lysates from human embryonic
kidney cells transfected with wild-type or mutant HLA-H cDNA
demonstrate that wild-type HLA-H binds
2-microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is
localized exclusively intracellularly. This report describes the first
functional significance of the C282Y mutation by suggesting that an
abnormality in protein trafficking and/or cell-surface expression of
HLA-H leads to HH disease.
Hereditary hemochromatosis (HH)1 is an
autosomal recessive disorder of iron metabolism and represents one of
the most common inherited disorders in individuals of Northern European
descent with an estimated carrier frequency between 1 in 8 and 1 in 10 (1, (2). In patients with HH, excessive iron deposition in a variety of
organs leads to multi-organ dysfunction. Recently, we reported a
mutation in a novel MHC class I-like gene, called HLA-H (3).
Eighty-three percent of HH patient DNAs were found to be homozygous for
this mutation, which consists of a single base transition of G to A and
results in a change of cysteine 282 tyrosine (C282Y). Subsequent
reports have confirmed the high frequency of this founder mutation in
other HH patients (4-6), providing further support that
HLA-H is the primary HH locus. A second missense mutation,
histidine 63
aspartate (H63D), was also reported that was enriched
in heterozygotes with the C282Y mutation (eight of nine cases) (3). The
specific role that either of these mutations in HLA-H play
in the etiology of HH disease has not been elucidated.
The HLA-H protein is similar to MHC class I family molecules including
HLA-A2, nonclassical class I molecules such as HLA-G, and the human
neonatal Fc receptor (FcRn). All four of the invariant cysteine
residues that form disulfide bridges in the 2 and
3 domains of MHC class I family members are present in
the HLA-H protein. One of these conserved cysteine residues is altered
in the C282Y mutation. The integrity of the conserved disulfide
linkages has been suggested to be critical for proper maintenance of
the secondary and tertiary structure of the protein allowing
interactions with accessory molecules such as
2-microglobulin (7). Importantly, the functional
significance of an interaction between
2-microglobulin and an unknown class I-like molecule in HH disease was suggested by
2-microglobulin-deficient mice; these mice display a
progressive hepatic iron overload similar to that observed in human HH
(8-10). Other studies have demonstrated that mutation of cysteine 203 in the
3 domain of the mouse MHC class I family member
H-2Ld prevented intracellular transport of the molecule
from the endoplasmic reticulum to the plasma membrane (11).
As a step toward understanding the role of HLA-H in HH disease, we
examined the effects of the C282Y and H63D mutations on HLA-H cellular
processing. In this report we demonstrate that wild-type HLA-H binds to
2-microglobulin and that the C282Y mutation completely
abrogates this interaction and disrupts intracellular protein
trafficking. The data provide support for the hypothesis that the C282Y
mutation results in intracellular sequestration of the HLA-H protein,
which leads to HH disease.
The HLA-H
cDNA was fused to the FLAG octapeptide sequence (Eastman Kodak Co.)
to utilize specific available antibodies for detection of the HLA-H
protein. We designed a 3 PCR primer oligonucleotide that altered the
natural stop codon of the cDNA without an amino acid change and
added the FLAG sequence (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) with a
termination codon and a unique NotI site for cloning into pcDNA 3.1 expression vectors (Invitrogen). The 3
primer
sequence was:
5
-ATG-CCG-TAG-CGG-CCG-CTT-ATC-ACT-TGT-CAT-CGT-CGT-CCT-TGT-AGT-CCT-CAC-GTT-CAG-CTA-AGA-CGT-AG-3
. The 5
primer sequence was
5
-GAT-CGG-ATC-CAC-CAT-GGG-CCC-GCG-AGC-CAG-GCC-GGC-GCT-TCT-C-3
, which serves to add a unique BamHI site at the 5
end
and maintains the natural initiation codon.
To isolate the H63D mutant cDNA we started with first strand
cDNA from a patient known to have that mutation and utilized the
primer pair from above to amplify the desired clone. For the C282Y
mutation we utilized a standard PCR mutagenesis approach: two
overlapping fragments with the appropriate G to A base change were
produced in a first-round PCR reaction, and the fragments gel-purified
and combined with the 5 and 3
primers used above to yield the
appropriately mutated cDNA with the FLAG sequence attached to the
3
end.
Three peptides were
synthesized (Multiple Peptide Systems) corresponding to amino acid
sequences of the HLA-H protein. Two were to the predicted extracellular
region of the molecule: peptide EX1 comprising amino acids 164-177 of
the 2 domain (CPRAWPTKLEWERHK) and peptide EX2
comprising amino acids 246-260 of the
3 domain (CKDKQPMDAKEFEPKD). The third, CT1, was from the putative cytoplasmic tail comprising amino acids 326-343 (CRQGSRGAMGHYVLAERE). For each, an
NH2-terminal cysteine residue was incorporated to enable directed coupling to keyhole limpet hemocyanin. Rabbit antisera were
produced and the resulting polyclonal antisera named utilizing the
peptide nomenclature.
Cells were lysed
in 25 mM Tris-HCl, pH 7.5, 150 mM NaCl (TBS)
plus 0.5% Nonidet P-40 and the HLA-H or 2-microglobulin
proteins immunoprecipitated from 1 mg of total cell protein with 50 µg of FLAG M2 monoclonal antibodies (Eastman Kodak Co.) or 6 µg of
2-microglobulin monoclonal antibody B1G6 (Immunotech)
followed by addition of protein G-Sepharose (Pharmacia Biotech Inc.).
After washing three times with TBS containing 0.25% Tween 20, the
antibody-antigen complexes were dissociated by heating at 100 °C for
4 min in standard Laemmli sample buffer and the material separated on a
4-20% Tris-glycine gradient polyacrylamide gel (Novex). Gels were
electroblotted onto PVDF membranes (Novex), incubated with either 2 µg/ml FLAG M2 antibody, 25 µg/ml polyclonal
2-microglobulin antibody (Boehringer Mannheim), or 2 µg/ml CT1 HLA-H antibody and the immune complexes detected by ECL
(Amersham Corp.) utilizing horseradish peroxidase-linked sheep
anti-mouse antibodies or horseradish peroxidase-linked donkey anti-rabbit antibodies as appropriate.
Membrane fractionation was
performed as described previously (12). Three membrane fractions
("light," 20%/35% sucrose interface; "medium," 35%/40%
sucrose interface; "dense", 40%/50% sucrose interface) were
obtained from approximately 1 × 107 cells by sucrose
step-gradient centrifugation of postnuclear membrane fractions.
Membranes from each step-gradient interface representing membranes from
1 × 106 cells were mixed with Laemmli sample buffer
and proteins separated and blotted as before. HLA-H proteins were
detected with a 1:1 mixture of EX1 and EX2 antibodies at 3 µg/ml. For
detection of subcellular markers, protein blots were probed with mouse
monoclonal antibodies to Na+/K+-ATPase (plasma
membrane), calnexin (endoplasmic reticulum, ER), and a rabbit
polyclonal antibody to -COP (Golgi) all obtained from Affinity
Bioreagents. Specific bands were quantitated using a scanning
densitometer (Molecular Dynamics).
Parental and transfected 293 stable cell lines were seeded on rat-tail collagen (Biomedical Technologies)-coated glass coverslips and grown overnight in standard medium. Cells were fixed in 3.5% paraformaldehyde, stained with 1:1 mixture of affinity-purified antibodies EX1 and EX2 at 50 µg/ml or FLAG-M2 antibodies at 25 µg/ml, and immune complexes detected with fluorescein isothiocyanate-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibodies, respectively (Zymed Laboratories Inc.). To detect intracellular antigen, cells were first fixed and then permeabilized with 0.05% saponin in phosphate-buffered saline for 3 min at room temperature prior to exposure to the primary antibodies. For peptide competition experiments, peptides (EX1, EX2, and M2) were incubated at a 100-fold molar excess with their respective antibodies for 1 h at room temperature prior to application to the cover slips.
We first sought to demonstrate an interaction of the HLA-H protein
with 2-microglobulin and to examine the effects of the C282Y and H63D mutations on that interaction. Human embryonic kidney
cells (293 cells) were transfected with vectors containing the
wild-type HLA-H cDNA or the cDNA with either the C282Y or H63D
mutation. The FLAG octapeptide sequence was fused onto the carboxyl
terminus of each, providing a specific tag for detection of the
expressed proteins (13). We established individual stable cell lines
expressing the three proteins. Immunoprecipitation of cell lysates with
monoclonal antibodies directed to the FLAG sequences (M2 antibodies),
to precipitate the HLA-H/FLAG fusion protein, followed by Western
blotting with
2-microglobulin polyclonal antibodies
demonstrated a clear interaction between the HLA-H protein and
2-microglobulin (Fig. 1A, left
panel, Wild type lane). Significantly,
2-microglobulin was not detected in immune complexes from cell lines expressing the HLA-H protein with the C282Y mutation (Fig. 1A, left panel). This failure to detect
2-microglobulin was not due to lack of HLA-H protein in
the mutant cell lines, since stripping the blots and immunodetection
with rabbit polyclonal antibodies directed to the COOH-terminal 17 amino acids of HLA-H (CT1 antibodies) demonstrated that the amount of
HLA-H protein in the three cell lines was similar (Fig. 1A, right
panel). The results with the H63D mutant were similar to the
wild-type HLA-H protein;
2-microglobulin was
co-immunoprecipitated along with that mutant protein (Fig. 1A,
left panel, compare H63D and Wild type
lanes). It is of interest to note that the wild-type or H63D HLA-H
proteins detected in the right panel appeared to migrate as
a doublet of 49 and 46 kDa in lighter exposures, whereas the C282Y
appeared as only a single band of approximately 46 kDa.
The 2-microglobulin/HLA-H interaction results were
corroborated by performing the inverse experiment in which cell lysates were initially immunoprecipitated with
2-microglobulin
antibodies followed by Western blotting with antibodies directed toward
the COOH-terminal sequence of HLA-H (CT1 antibodies). In this
experiment, the
2-microglobulin antibodies
co-immunoprecipitated HLA-H protein from the wild-type and H63D mutant
expressor cell lines, but failed to do so in the C282Y mutant expressor
cell line (Fig. 1B, left panel). Stripping the blots and
reprobing with
2-microglobulin antibodies demonstrated
that similar amounts of
2-microglobulin protein were
immunoprecipitated from each cell line (Fig. 1B, right
panel). These results further confirm an interaction between wild-type HLA-H protein and
2-microglobulin and
demonstrate that the C282Y, but not the H63D, mutation disrupts this
association.
Previous reports have suggested that association of the MHC class I
heavy chain with 2-microglobulin is critical for
cell-surface expression (14, 15). Because of the failure of the HLA-H
protein containing the C282Y mutation to interact with
2-microglobulin, we next investigated whether this
mutation would also affect cell-surface presentation of the HLA-H
protein. Parental 293 cell lines and those expressing the wild-type
HLA-H protein or the C282Y mutant were examined for cell-surface
protein expression by immunostaining with rabbit polyclonal antibodies
specific to sequences residing in the predicted external domain of the
HLA-H protein (EX1 and EX2 antibodies) followed by detection with
immunofluorescence. Parental 293 cells displayed no detectable surface
labeling by these antibodies (Fig. 2A),
consistent with the undetectable levels of HLA-H protein observed in
the Western blotting experiments (Fig. 1A, right panel).
Wild-type HLA-H-expressing cells demonstrated a distinct pattern of
surface labeling as evidenced by a punctate pattern of labeling that
was much more intense at the edges of the cells (Fig. 2B).
By contrast, cells expressing the C282Y mutation displayed no surface
labeling and were indistinguishable from the parental controls (Fig. 2,
compare C and A). The specificity of the antibody
labeling was demonstrated by preincubating the EX1 and EX2 antibodies
with their respective peptides; in these experiments the punctate
surface labeling observed in the wild-type HLA-H expressor cells was
completely abolished (data not shown).
We examined the possibility that the C282Y mutant protein was expressed in the transfected cells but remained intracellularly localized. Immunostaining was performed following treatment of the cells with saponin to permeablize them. Staining of these cells for the FLAG-tagged C282Y mutant HLA-H protein demonstrated strong perinuclear fluorescence, which was absent in the parental control cells (Fig. 2, compare D and F). Permeablized wild-type HLA-H protein expressor cells showed similar intracellular staining with the FLAG-M2 antibody, suggesting that not all of the wild-type protein in these transfected cells reaches the cell surface (E). Experiments utilizing the EX1, EX2, or CT1 antibodies yielded the same results (data not shown). These results clearly demonstrate that the C282Y mutation specifically disrupts cell-surface presentation of the HLA-H protein.
To examine the distribution of wild-type and mutant HLA-H proteins
within the cell in more detail, we performed subcellular fractionations
on stepwise sucrose gradients to separate the various membrane
components. Three separate postnuclear membrane fractions were
obtained; the 20/35% interface contained the lightest density membranes (L); dense membranes (D) partitioned at the 40/50%
interface, whereas the 35/40% medium-density (M) interface contained a
mixture of light and dense membrane-derived components. We initially
characterized the efficacy of our subcellular membrane fractionation
scheme by assaying these fractions for marker proteins. Antibodies to Na+/K+-ATPase were utilized as markers for
plasma membrane, -coatomer protein (
-COP) for Golgi, and calnexin
for ER membrane identification. Samples representing membranes from
equal numbers of cells from each interface were analyzed by Western
blotting and quantitated on a Molecular Dynamics scanning densitometer.
Plasma membranes were found primarily in the light-density interface
and to a lesser extent in the medium-density layer: L, 90%; M, 10%;
D, 0%. Golgi membranes were distributed nearly equally throughout the
three interfaces: L, 30%; M, 40%; D, 30%. ER membranes were found
mostly in the dense membrane interface: L, 0%; M, 20%; D, 80%. The
fractionations from each of the three cell lines gave equivalent
results.
We determined the specific distribution of HLA-H proteins in the
sucrose gradient interfaces by Western blotting and probing with HLA-H
antibodies. As with the co-immunoprecipitation results (Fig. 1), the
immunostaining suggested that the wild-type HLA-H protein migrated as a
doublet of the 49 and 46 kDa forms (Fig. 3, left
panel). The slower migrating 49-kDa form was found principally in
those fractions containing plasma membranes, whereas the lower molecular mass 46-kDa form was distributed in a pattern similar to that
of the Golgi marker, -COP. By contrast, the C282Y mutant HLA-H
protein consisted only of the faster migrating 46-kDa species. Like the
wild-type 46-kDa protein, the mutant 46-kDa protein was distributed in
a pattern that most closely resembled that of the Golgi marker protein,
suggesting the possibility of incomplete posttranslational processing
or modification (Fig. 3, middle panel). The H63D mutant
proteins migrated in a pattern resembling that of the wild-type
protein, implying that this mutation had little or no effect on
intracellular HLA-H protein trafficking (Fig. 3, right
panel). In other studies, no HLA-H protein was detected in the
cytosolic fractions of any of the wild-type or mutant cell lines,
suggesting that neither the C282Y nor the H63D mutation cause a
redistribution of the protein to the cytoplasm (data not shown).
Taken together these results demonstrate that the C282Y mutation
prevents the HLA-H molecule from interacting with
2-microglobulin and eliminates cell-surface
presentation. Cysteine 282 is one of four cysteine residues that are
invariant in both classical and nonclassical MHC class I molecules and
forms a critical disulfide bridge in the
3-immunoglobulin-like domain (7). Thus, the integrity of
this structure is critical to the formation of the heterodimer of
2-microglobulin and HLA-H and also for proper intracellular processing of the protein. Class I MHC molecules are
noncovalently linked heterodimers between an
heavy chain and
2-microglobulin (light chain) (7). The role of the
2-microglobulin/heavy chain interaction is to facilitate
and stabilize the folding of the heavy chain during biosynthesis
through interactions with the
1-
2
platform and the
3 domain (16) (7). Previous work demonstrated that mutating the reciprocal cysteine residue (cysteine 203) in mouse H-2Ld protein abolished cell-surface
presentation (11). Interestingly, the mutant H-2Ld molecule
retained the ability to associate with
2-microglobulin. Mouse H-2Ld belongs to the family of classical
antigen-presenting molecules, whereas HLA-H is nonpolymorphic and,
therefore, resembles nonclassical molecules such as HLA-G or the human
Fc receptor (3). It is conceivable that the integrity of the
3 domain may be less stringent for
2-microglobulin association with classical
antigen-presenting proteins (such as H-2Ld) than for
nonclassical molecules (such as HLA-H).
The biogenesis of MHC class I molecules is well documented. The heavy
chain of a MHC class I molecule is synthesized on membrane-bound polysomes, and N-linked glycosylation occurs
co-translationally in the ER (17). Subsequently, the modified heavy
chain associates with chaperone proteins and
2-microglobulin in the ER and is then transported
through the cis-Golgi network, to the middle and trans-Golgi cisternae
where the glycosyl side chain is modified to a more complex form en
route to the plasma membrane (18-20). Class I molecules that fail to
assemble properly are recycled between the ER and Golgi, rather than
being retained exclusively in the ER (21). In our studies the C282Y
mutant of HLA-H is retained on intracellular membranes in a pattern
that would be consistent with these earlier observations. The mutant
protein migrates similar to the Golgi marker protein
-COP in
subcellular fractionations, but because of the limited resolution of
the step-gradient, we cannot rule out that some protein may also be in
the ER. The perinuclear pattern of staining noted in the
immunofluorescence studies does not definitively resolve this. More
detailed studies will be necessary to ascertain the specific point at
which intracellular transport of the C282Y mutant is disrupted.
In contrast to our results with the C282Y mutation, we found no
detectable changes in the 2-microglobulin interaction or intracellular processing of the H63D mutant form of HLA-H, which is
enriched in C282Y heterozygous patients (3). Other studies have
demonstrated alterations in intracellular transport of class I
molecules by mutations in the peptide-binding groove of HLA-A (22). The
H63D mutation is localized in the
1 domain between the
third and fourth
strands of the external peptide-binding region. It
is possible that the effect of this mutation is to alter the affinity
of the HLA-H protein for an as yet unidentified ligand or to alter the
manner that the mutant protein interacts with other proteins in the
cell membrane. Alternatively, this mutation may represent a common
polymorphism with little or no effect on the biological functioning of
the protein. The definitive answer to this question will await further
investigation as we elucidate how the HLA-H molecule regulates iron
metabolism in the body.
We acknowledge M. Ellis, A. Gnirke, and E. Sigal for critical reading of the manuscript; D. A. Ruddy for DNA sequencing; D. J. McCarley for assistance with the scanning of subcellular protein distribution films; and colleagues at Mercator Genetics for continued support.