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*
Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110;
Department of Microbiology, University of South Dakota School of Medicine, Vermillion, SD 57069
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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1 domain and the
amino acid residue at position 227 in the 3 domain were both found
to be critical for the interaction of class I with calreticulin.
Interestingly, calreticulin displayed sensitivity to class I peptide
binding even in TAP-deficient human or mouse cells. Thus, calreticulin
is clearly more specific than calnexin in the structures and
conformation of the class I molecule with which it can interact. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Calreticulin and calnexin, which are ER chaperones with lectin-like activity, also bind to class I and several other glycoproteins (8, 12, 13, 14). The calcium-binding domains of these two chaperones are also markedly similar in sequence (15). Binding of both calnexin and calreticulin has been shown to be regulated by glucose trimming of nascent N-linked oligosaccharides (14, 16, 17). Calnexin acts initially to stabilize free class I H chains and protect them from degradation (18). Furthermore, calnexin has been reported to promote subunit assembly of class I (18, 19) and retain incompletely assembled class I molecules in the ER (19, 20). In regard to the role of calnexin in ER retention, it is important to note that these studies were done in cells that lack TAP (19) or in cells where class I does not associate with TAP due to a lack of ß2m (20). The importance of this observation is that in wild-type cells class I is associated with the complex of TAP, tapasin, and calreticulin before peptide binding (8). Thus the role of calnexin in ER retention in wild-type cells remains to be determined. In any case, considering the evidence showing calnexin to be a class I chaperone (cf. 18), it was surprising that class I assembly with ß2m, peptide loading, and surface expression were found to be normal in calnexin-deficient cells (21, 22). Thus calnexin appears not to have a unique role in chaperoning class I. Based on this result and the above noted similarities in their binding specificity, it was suggested that calnexin and calreticulin may have redundant functions in class I assembly and expression (22, 23).
Calreticulin has been shown to display steady state association with class I/TAP/tapasin complexes (8, 17, 24). Indeed, the majority of nascent class I molecules associated with calreticulin are simultaneously associated with TAP (17). In contrast, calnexin does not display stoichiometric association with the class I/TAP/calreticulin complex (11, 17, 24) but may bind a small number of assembling TAP complexes (11, 25). Furthermore, at least in human cells, calnexin associates predominantly with free class I H chains (26, 27), whereas calreticulin association requires the H chain be assembled with ß2m (8, 24). Similarly, calreticulin was found to be more restrictive than calnexin in its binding to folding intermediates of influenza hemagglutinin (28). These observations suggest that calnexin and calreticulin have different binding specificities in regard to glycoprotein folding, and that their chaperone functions may be successive and distinct. Furthermore, since calreticulin and not calnexin shows predominant association with the TAP complex, calreticulin could facilitate the retention of ß2m-assembled H chains awaiting peptide.
In this report, we demonstrate that a mutation in the 3 domain of
the H chain or selective removal of the N-linked glycan from
the 1 domain of the H chain ablates association of class I with
calreticulin but not calnexin. Since the binding of calreticulin to
class I is TAP- and tapasin- independent, the simplest explanation of
our findings is that calreticulin, like calnexin, binds directly to the
class I H chain but that the two chaperones bind to different sites. We
also report here that calreticulin preferentially associates with open
forms of both mouse and human class I, and is selective for the class I
open form even in the absence of TAP. These data suggest calreticulin
aids the stabilization and/or ER retention of peptide-free H
chain/ß2m heterodimers. Furthermore, peptide-induced
folding of class I in cell lysates does not alter its association with
calnexin (5, 25), whereas calreticulin and tapasin are associated with
class I only before peptide-induced folding. These cumulative findings
clearly establish that the interactions of calreticulin and calnexin
with class I are quite distinct.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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L-Ld cells were generated by introducing the
Ld gene into murine Ltk- DAP-3
(H-2k) fibroblast cells (29). T1 and T2 cells (30) were
obtained from the American Type Culture Collection (ATCC, Rockville,
MD). T1 (174 x CEM.T1) is a cloned hybrid of 721.174 (an
immunoselected variant of LCL 721) and CEMR.3. T1 has lost
only one copy of CEMR.3-derived chromosome 6; it expresses
TAP1, TAP2, and class II DR Ag. T1 is the parental cell line of T2
(174 x CEM.T2). T2 has lost both copies of
CEMR.3-derived chromosome 6 and so does not express class
II MHC, TAP1, or TAP2. T2-Ld (an Ld
transfectant of T2) was a gift from Peter Cresswell (Yale University,
New Haven, CT). RMA-S (31), a C57BL/6-derived thymoma, was a gift from
Jeffrey Bluestone (University of Chicago, Chicago, IL). RMA-S has a
mutation that causes premature termination of the TAP2 protein, and
therefore it does not have a functional TAP heterodimer.
RMA-S-Ld is an Ld transfectant of RMA-S (32).
Mutants of Ld that lack 1 domain or 2 domain
glycosylation sites were produced by the PCR in the case of mutants
LdN86K and LdN176Q, or by the Quik Change
method (Stratagene, La Jolla, CA) in the case of mutants
LdN86Q and LdS88W. These Ld mutants
are named as follows: original amino acid residue, position, and
substituted amino acid residue. The mutant cDNAs were cloned into the
expression vector RSV.5neo (33), transfected into L cells with
Lipofectin (Life Technologies, Gaithersburg, MD), and selected in 0.6
mg/ml G418. An Ld mutant that lacks all three glycosyl
groups was donated by Keiko Ozato (National Institute of Child Health
and Human Development (NICHD), National Institutes of Health, Bethesda,
MD) (34). Ld mutant LdD227K has been previously
described (5). Cells were maintained at 37°C, 5% CO2 in
RPMI 1640 (Life Technologies) containing 10% bovine calf serum
(HyClone Laboratories, Logan, UT), glutamine, pyruvate, and
penicillin/streptomycin, or were cultured at 37°C, 7%
CO2 in DMEM (Life Technologies) containing 10% FCS
(HyClone Laboratories), glutamine, pyruvate, and
penicillin/streptomycin.
Antisera and mAb
The mAb 64-3-7 is an IgG2 Ab that recognizes the 1 domain of
open Ld (29, 35, 36, 37, 38), and mAb 30-5-7 is an IgG2 Ab
that recognizes the 2 domain of folded Ld (37, 38, 39).
Ld forms that possess the 64-3-7 serologic epitope lack the
epitopes of several conformation-sensitive mAbs (e.g., 30-5-7) that
surround the peptide-binding groove, which suggests that the
Ld 64-3-7+ form has an open/unfolded cleft
(35). The interaction of 64-3-7+ Ld with
peptide causes a conversion to the folded (30-5-7+) form,
as demonstrated by titration of radioiodinated peptide ligand into cell
lysates and immunoprecipitation of the Ld molecules. With
increasing concentrations of peptide ligand, the amount of
64-3-7+ Ld decreased and the amount of
30-5-7+ Ld increased (37). Furthermore, the
labeled peptide was coprecipitated in dose-dependent fashion with the
30-5-7+ Ld form and could be simultaneously
visualized on the autoradiograph (37). In vivo, a precursor-product
relationship exists between 64-3-7+ Ld and
30-5-7+ Ld, as shown by pulse-chase analysis.
As the chase time increases, 30-5-7+ Ld levels
rise and 64-3-7+ Ld levels drop (37).
HC10 is an IgG2a that reacts preferentially with open HLA-B and -C heavy chains (40, 41). W6/32 is an IgG2a that reacts with a monomorphic determinant on all folded HLA/ß2m heterodimers (41, 42). Experiments by Carreno and Hansen showed a reciprocal relationship between HC10+ HLA forms and W6/32+ HLA forms (41). For example, culture of L-B27/hß2m cells with peptides decreases the number of surface HC10-reactive molecules and increases the population of W6/32-reactive molecules. Furthermore, in the presence of peptide ligands, the half-life of W6/32-reactive molecules on L-B27/hß2m is extended by about 3 h and the half-life of HC10-reactive molecules is reduced by the same amount of time.
The 1478 anti-TAP1 antiserum was made against the same TAP1 peptide as the R.RING4C antiserum (43). Anti-calreticulin serum (44) was purchased from Stressgen (Victoria, BC, Canada). MAb 11.4.1 is an IgG2 Ab specific for Kk (45). Affinity-purified rabbit anti-human tapasin Ab (8) was provided by Dr. Peter Cresswell. The anti-calnexin serum specific for a C-terminal segment from mouse and human calnexin (46) was donated by Dr. David McKean (Mayo Clinic, Rochester, MN). Anti-TAP2 hybridoma 435.3 (47) was developed by Dr. Peter van Endert (Institut National de la Santé et de la Recherche Médical (INSERM) Unité, Paris, France) and sent to us by Dr. Peter Cresswell. The 15-5-5 mAb is an IgG2 Ab that recognizes Dk (48).
Immunoprecipitations
Cells were preincubated for 30 to 60 min at 37°C in culture
media that lacked methionine. Then [35S]methionine
(125–250 µCi/ml) was added, and the cells were radiolabeled for 15
to 30 min. The cells were then washed three times in PBS containing 20
mM iodoacetamide (IAA, Sigma, St. Louis, MO) and lysed in buffer that
contained 1%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS,
Boehringer-Mannheim, Indianapolis, IN) in Tris-buffered saline, pH 7.4,
freshly added 0.2 mM PMSF (Sigma), and 20 mM iodoacetamide.
Alternatively, cells were lysed in Tris-buffered saline that contained
1% digitonin (Wako, Richmond, VA), 0.1 mM
7-amino-1-chloro-3-tosylamido-2-heptone (TLCK), and 0.5 mM freshly
added PMSF. The lysis buffer with either CHAPS or digitonin was
supplemented with a saturating volume of mAb or rabbit Ab before its
addition to pelleted cells. After incubation for 30 min on ice, nuclei
were removed by centrifugation, and lysates were incubated with protein
A-Sepharose beads (Pharmacia, Piscataway, NJ). The beads were washed
four times with 0.1% CHAPS or 0.1% digitonin in Tris-buffered saline,
pH 7.4, and the samples were eluted by boiling in 0.125 M Tris pH
6.8/2% SDS/12% glycerol/2% bromophenol blue. All immunoprecipitates
were electrophoresed on 4
20% or 8% acrylamide SDS-PAGE gels
(Novex, San Diego, CA) with the Laemmli buffer system (49). Gels were
treated with Amplify (Amersham, Boston, MA), dried, and exposed to
BioMax MR film (Eastman Kodak Co., Rochester, NY) at -70°C for
varied lengths of time. All immunoprecipitation lanes shown within a
figure are from a single experiment.
In the peptide binding experiments (Figs. 4
and 5), cells were
radiolabeled as indicated above and divided into four aliquots of
2 x 107 cells. The aliquots were solubilized in lysis
buffer supplemented with 0, 0.5, or 50 µM of a synthetic
Ld peptide ligand derived from murine cytomegalovirus
(MCMVpp89, residues 168–176) (50) or with 50 µM of a negative
control Db-binding peptide derived from influenza
nucleoprotein (NP 365–380) (51). Samples were incubated on ice for
1 h and nuclei were removed by centrifugation. Each sample was
divided into two aliquots, one which was precipitated with mAb 64-3-7
and one which was precipitated by mAb 30-5-7 by addition of the
respective Ab and incubation on ice. After 1 h, the lysate was
transferred to protein A-Sepharose, and further steps in the
immunoprecipitation protocol were performed as described above.
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Immunoprecipitates electrophoresed on SDS-PAGE as described above were transferred to Immobilon P membranes (Millipore, Bedford, MA). After overnight blocking, membranes were incubated in a dilution of Ab for 2 h, washed three times with PBS/0.05% Tween-20, and incubated for 1 h with biotin-conjugated goat anti-mouse or anti-rabbit IgG (Caltag Laboratories, San Francisco, CA). Following three washes with PBS/0.05% Tween-20, membranes were incubated for 1 h with streptavidin-conjugated horseradish peroxidase (Zymed, San Francisco, CA), washed 3 times with PBS/0.3% Tween 20, and incubated with Western blot developing reagents (Amersham). Membranes were exposed to BioMax MR film for varied lengths of time. Western blot lanes shown within a figure are from a single experiment, beginning from the same immunoprecipitation samples displayed in the "Immunoprecipitation" 35S-methionine autoradiographs above the Western blot lanes.
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Calreticulin can associate with the open H chain even in the absence of TAP
To determine whether calreticulin was associated with open vs
folded class I molecules, we used mAb that distinguish these
alternative class I conformations. For the mouse Ld
molecule, open forms were specifically detected with mAb 64-3-7, and
folded forms were detected with 30-5-7 (29, 35, 36, 37). As shown in Figure 1, calreticulin was found to be
specifically associated with open forms of Ld expressed by
L-Ld cells. For human class I molecules, open forms were
specifically detected with mAb HC10, and folded forms were detected
with W6/32 (40, 41, 42). As shown in Figure 2, calreticulin is uniquely associated
with open human class I molecules expressed by T1 cells. It should be
noted that TAP, like calreticulin, is primarily associated with open
class I molecules, whereas calnexin has been detected in association
with both open and folded forms of Ld and HLA class I (5).
These findings thus support the model that calreticulin and TAP are
uniquely and simultaneously associated with open class I forms (cf.
23). To determine whether the association of class I with calreticulin
is TAP dependent, we studied molecular associations in TAP-deficient
human (T2) and mouse (RMA-S) cell lines. As shown in Figure 2, the
association of calreticulin with open human class I was found to be
comparable in the presence (T1) or absence (T2) of TAP. Furthermore,
open forms of Ld were detected in association with
calreticulin in TAP-deficient mouse (RMA-S-Ld) or human
(T2-Ld) cells (Fig. 3). Thus
the association of calreticulin with class I is clearly not TAP
dependent. Previous studies demonstrated that calreticulin is also
associated with class I in the absence of tapasin (8, 24). The simplest
explanation of these combined data is that there is a direct
interaction site between the class I H chain and calreticulin.
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It has been previously reported that addition of peptide can cause
the dissociation of class I from TAP (3, 4, 5). To determine whether this
dissociation was dependent upon TAP itself, or the TAP-associated
proteins (tapasin and calreticulin), we monitored peptide-sensitive
molecular associations in TAP-deficient cells. As shown in Figures 4
and 5,
peptide-induced folding of class I sharply diminishes the population of
open class I molecules and causes a parallel reduction in calreticulin
association in mouse RMA-S cells and human T2 cells transfected with
the Ld gene. Using an Ab specific for human
tapasin (8), we also monitored the tapasin association in
T2-Ld cells and found its binding paralleled that of
calreticulin (Fig. 5). Thus, even in the absence of TAP, the
association of calreticulin and tapasin with class I is specific for
open forms before peptide-induced folding. In contrast to these
findings, the association of calnexin with class I is not affected by
peptide-induced folding (5, 25).
The N-linked glycan in the 1 H chain domain is
uniquely required for the interaction of H chain with calreticulin and
not calnexin
Earlier reports demonstrated that calreticulin and calnexin,
consistent with their lectin-like activity, interact with a glycosyl
group on the class I H chain (8, 17). The experiments presented in
these reports involved drugs that affect processing of all glycosyl
moieties of all proteins. To study the effects of class I glycosylation
without altering other cellular proteins and to determine whether
certain class I H chain oligosaccharides are of predominant importance,
we created a panel of mutants with selected omissions of
N-linked glycans from the 1, 2, and/or 3 domains of
Ld. We have found that the removal of the Ld
1 domain glycosylation site by site-directed mutagenesis
(LdN86K) prevents association of calreticulin and TAP with
the class I molecule (Fig. 6). Calnexin,
however, still binds strongly to LdN86K (Fig. 6).
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1 domain
carbohydrate group and not the amino acid substitution per se that
prevented interaction of the H chain with calreticulin and TAP, two
other mutants were tested that had different amino acid substitutions
but lacked the same glycosyl group (LdN86Q and
LdS88W). As shown in Figure 6, these two mutants also were
not associated with calreticulin or TAP, but were associated with
calnexin. As a further confirmation of this result, an additional
Ld mutant that lacks all three carbohydrate groups
(LdCHO-) was tested and found not to associate
with calreticulin and TAP (Fig. 7).
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1 domain glycosyl group alone was
necessary for calreticulin and TAP association with class I, or whether
absence of a different carbohydrate moiety would have the same effect.
An Ld mutant with an amino acid substitution that prevented
glycosylation in the 2 domain (LdN176Q) was made, and it
was found to have the same level of calreticulin, TAP, and calnexin
interaction as wild-type Ld. This indicates that the 1
domain carbohydrate is uniquely required for calreticulin association
with class I, whereas calnexin’s association with class I is clearly
not dependent on the 1 domain carbohydrate. Indeed, calnexin may
also contact the H chain polypeptide chain (52, 53, 54), even though the
specificity of complex formation with non-MHC glycoproteins appears to
be provided by the carbohydrate moieties (55). In any case the findings
reported here provide clear evidence that the two lectins, calnexin and
calreticulin, bind glycan moieties at different locations on the H
chain. This difference may reflect differential accessibility of glycan
moieties for the membrane-associated calnexin vs the lumenally located
calreticulin. Our finding that the 1 glycan is necessary for
calreticulin association with class I is interesting in light of the
fact that all mouse and human H chains have an 1 glycan at this
position, even though mouse H chains have a second, and sometimes a
third, glycan in the 2 and 3 domains (56, 57, 58, 59, 60).
Mutation in the 3 H chain domain affects class I association
with calreticulin and not calnexin
Studies of both Ld (5) and Dd (25)
have shown that mutations in the 3 domain can prevent class I
association with TAP. For example, an aspartic acidlysine mutation
at position 227 of the 3 domain of Ld
(LdD227K) ablates TAP association (Ref. 5, and Fig. 7).
Given previous results, which demonstrated that the binding of
calreticulin to class I is TAP and tapasin independent (8, 24),
calreticulin, like calnexin, seems to bind directly to class I. Thus it
was of considerable interest whether the D227K 3 mutation ablated
the interaction of class I with calnexin and calreticulin. As shown in
Figure 7, this mutation disrupts calreticulin interaction with class I
and does not interfere with the association between calnexin and class
I (data not shown). Therefore, the 1 domain glycan and the 3
position 227 are both uniquely required for the association of the
class I H chain with calreticulin, but not calnexin.
Overall, our data point to discrete roles for calreticulin and calnexin
in class I assembly and expression. Our data extend earlier models (8, 17) of calnexin and calreticulin function by better distinguishing
structural features necessary for class I binding. We have observed
that calreticulin and calnexin bind to different sites on class I. More
specifically, the 1 domain glycan and the 3 residue at position
227 both affect class I association with calreticulin and not calnexin.
Importantly, the experiments showing the role of the 1 domain
carbohydrate moiety were done by site-directed mutagenesis and not by
chemical treatment, which would broadly affect protein glycosyl groups.
In addition, we demonstrate that calreticulin is responsive to class I
folding since it, unlike calnexin, preferentially binds to open
conformations of class I. Furthermore, we show here that
calreticulin’s sensitivity to peptide-induced folding is TAP
independent.
Our data and those from other laboratories (17, 23) suggest sequential interaction of calnexin and calreticulin with class I. In this model, the open H chain initially associates with calnexin. Before interaction with the TAP/tapasin complex, the class I H chain exchanges calnexin for calreticulin. It should be noted that this switch from calnexin to calreticulin is not strictly governed by ß2m assembly. Although ß2m assembly is a requisite for H chain association with calreticulin, mouse and human H chains with or without ß2m are detectable in association with calnexin (5, 61). It is likely that calnexin functions in the ER retention of free H chains. Indeed, this conclusion is strongly supported by the observation that calnexin retards class I transport in ß2m-deficient cells (20). However, the data presented here, that calreticulin and tapasin are sensitive to peptide loading of class I, suggest that these proteins could be involved in ER retention of peptide-empty class I/ß2m heterodimers. In total, these data show that the interactions of calreticulin with class I are structurally and functionally distinct from those of calnexin.
Note added in proof.
Consistent with findings reported here showing that calreticulin and calnexin interact with different glycan determinants, Q. Zhang and R. D. Salter (1998. J. Immunol. 160:831) added a second N-glycan at position 176 of the HLA-A*0201 molecule and found that this mutant interacted better with calnexin and less well with calreticulin.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Ted H. Hansen, Department of Genetics (Box 8232), Washington University School of Medicine, 4566 Scott Avenue, St. Louis, MO 63110. E-mail address:
3 Abbreviations used in this paper: H, heavy; ER, endoplasmic reticulum.
Received for publication September 18, 1997. Accepted for publication February 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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2 domain of HLA-A2.1 define a functionally relevant interaction with TAP. Curr. Biol. 6:873.[Medline]
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H. R. Turnquist, S. E. Vargas, A. J. Reber, M. M. McIlhaney, S. Li, P. Wang, S. D. Sanderson, B. Gubler, P. van Endert, and J. C. Solheim A Region of Tapasin That Affects Ld Binding and Assembly J. Immunol., October 15, 2001; 167(8): 4443 - 4449. [Abstract] [Full Text] [PDF]
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 M. R. Harris, L. Lybarger, N. B. Myers, C. Hilbert, J. C. Solheim, T. H. Hansen, and Y. Y. L. Yu Interactions of HLA-B27 with the peptide loading complex as revealed by heavy chain mutations Int. Immunol., October 1, 2001; 13(10): 1275 - 1282. [Abstract] [Full Text] [PDF]
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K. M. Paulsson, P. Wang, P. O. Anderson, S. Chen, R. F. Pettersson, and S. Li Distinct differences in association of MHC class I with endoplasmic reticulum proteins in wild-type, and {beta}2-microglobulin- and TAP-deficient cell lines Int. Immunol., August 1, 2001; 13(8): 1063 - 1073. [Abstract] [Full Text] [PDF]
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T. Chun, A. G. Grandea III, L. Lybarger, J. Forman, L. Van Kaer, and C.-R. Wang Functional Roles of TAP and Tapasin in the Assembly of M3-N-Formylated Peptide Complexes J. Immunol., August 1, 2001; 167(3): 1507 - 1514. [Abstract] [Full Text] [PDF]
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B. E. Gewurz, R. Gaudet, D. Tortorella, E. W. Wang, H. L. Ploegh, and D. C. Wiley From the Cover: Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2 PNAS, June 5, 2001; 98(12): 6794 - 6799. [Abstract] [Full Text] [PDF]
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M. R. Harris, L. Lybarger, Y. Y. L. Yu, N. B. Myers, and T. H. Hansen Association of ERp57 with Mouse MHC Class I Molecules Is Tapasin Dependent and Mimics That of Calreticulin and not Calnexin J. Immunol., June 1, 2001; 166(11): 6686 - 6692. [Abstract] [Full Text] [PDF]
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N. B. Myers, M. R. Harris, J. M. Connolly, L. Lybarger, Y. Y. L. Yu, and T. H. Hansen Kb, Kd, and Ld Molecules Share Common Tapasin Dependencies as Determined Using a Novel Epitope Tag J. Immunol., November 15, 2000; 165(10): 5656 - 5663. [Abstract] [Full Text] [PDF]
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U. G. Danilczyk, M. F. Cohen-Doyle, and D. B. Williams Functional Relationship between Calreticulin, Calnexin, and the Endoplasmic Reticulum Luminal Domain of Calnexin J. Biol. Chem., April 21, 2000; 275(17): 13089 - 13097. [Abstract] [Full Text] [PDF]
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 A. Vambutas, V. R. Bonagura, and B. M. Steinberg Altered Expression of TAP-1 and Major Histocompatibility Complex Class I in Laryngeal Papillomatosis: Correlation of TAP-1 with Disease Clin. Vaccine Immunol., January 1, 2000; 7(1): 79 - 85. [Abstract] [Full Text] [PDF]
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Y. Y. L. Yu, N. B. Myers, C. M. Hilbert, M. R. Harris, G. K. Balendiran, and T. H. Hansen Definition and transfer of a serological epitope specific for peptide-empty forms of MHC class I Int. Immunol., December 1, 1999; 11(12): 1897 - 1906. [Abstract] [Full Text] [PDF]
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G. Lauvau, B. Gubler, H. Cohen, S. Daniel, S. Caillat-Zucman, and P. M. van Endert Tapasin Enhances Assembly of Transporters Associated with Antigen Processing-dependent and -independent Peptides with HLA-A2 and HLA-B27 Expressed in Insect Cells J. Biol. Chem., October 29, 1999; 274(44): 31349 - 31358. [Abstract] [Full Text] [PDF]
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Y. Y. L. Yu, H. R. Turnquist, N. B. Myers, G. K. Balendiran, T. H. Hansen, and J. C. Solheim An Extensive Region of an MHC Class I {alpha}2 Domain Loop Influences Interaction with the Assembly Complex J. Immunol., October 15, 1999; 163(8): 4427 - 4433. [Abstract] [Full Text] [PDF]
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J. D. Hansen, P. Strassburger, G. H. Thorgaard, W. P. Young, and L. Du Pasquier Expression, Linkage, and Polymorphism of MHC-Related Genes in Rainbow Trout, Oncorhynchus mykiss J. Immunol., July 15, 1999; 163(2): 774 - 786. [Abstract] [Full Text] [PDF]
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F. A. Arosa, O. de Jesus, G. Porto, A. M. Carmo, and M. de Sousa Calreticulin Is Expressed on the Cell Surface of Activated Human Peripheral Blood T Lymphocytes in Association with Major Histocompatibility Complex Class I Molecules J. Biol. Chem., June 11, 1999; 274(24): 16917 - 16922. [Abstract] [Full Text] [PDF]
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W.-K. Suh, M. A. Derby, M. F. Cohen-Doyle, G. J. Schoenhals, K. Fruh, J. A. Berzofsky, and D. B. Williams Interaction of Murine MHC Class I Molecules with Tapasin and TAP Enhances Peptide Loading and Involves the Heavy Chain {alpha}3 Domain J. Immunol., February 1, 1999; 162(3): 1530 - 1540. [Abstract] [Full Text] [PDF]
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A. R. Patil, C. J. Thomas, and A. Surolia Kinetics and the Mechanism of Interaction of the Endoplasmic Reticulum Chaperone, Calreticulin, with Monoglucosylated (Glc1Man9GlcNAc2) Substrate J. Biol. Chem., August 4, 2000; 275(32): 24348 - 24356. [Abstract] [Full Text] [PDF]
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