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J C Solheim

Publications and source records attributed to J C Solheim.

At least 19 recordsLinked to original sources
PubMed2002

Calreticulin binds to the alpha1 domain of MHC class I independently of tapasin.

Prior to binding to antigenic peptide, the major histocompatibility complex (MHC) heavy chain associates with an assembly complex of proteins that includes calreticulin, tapasin, and the transporter associated with antigen processing (TAP). Our data show that calreticulin can bind weakly to Ld without tapasin's assistance, and that deglycosylation of the alpha1 domain results in a primary loss of binding to calreticulin rather than tapasin. We have also shown that high amounts of wild-type tapasin are still unable to associate with MHC class I in the absence of the MHC class I/calreticulin interaction, confirming the central role of calreticulin in the formation of the MHC class I assembly complex.

ATP-Binding Cassette Transporters↗
PubMed2001

Loss of a glycine in the alpha2 domain affects MHC peptide binding but not chaperone binding.

Prior to the binding of peptide in the endoplasmic reticulum (ER), the major histocompatibility complex (MHC) class I heavy chain associates with an assembly complex that includes the transporter associated with antigen processing (TAP). The proximity of a part of the MHC class I alpha2 domain alpha-helix to areas previously shown to influence assembly complex binding suggests that this region might also be involved in chaperone association. Position 151, found in this part of the alpha2 domain alpha-helix, has a side chain that points up, away from direct contact with peptide, and is occupied by a glycine in all murine MHC class I heavy chains. We found that substitution of this glycine in H-2L(d) with a histidine substantially increased the proportion of peptide-free forms, although TAP binding was not abrogated. Thus, interaction of the heavy chain with peptides, but not with the assembly complex, is influenced by this glycine.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

A region of tapasin that affects L(d) binding and assembly.

Tapasin has been shown to stabilize TAP and to link TAP to the MHC class I H chain. Evidence also has been presented that tapasin influences the loading of peptides onto MHC class I. To explore the relationship between the ability of tapasin to bind to TAP and the MHC class I H chain and the ability of tapasin to facilitate class I assembly, we have created novel tapasin mutants and expressed them in 721.220-L(d) cells. One mutant has a deletion of nine amino acid residues (tapasin Delta334-342), and the other has amino acid substitutions at positions 334 and 335. In this report we describe the ability of these mutants to interact with L(d) and their effects on L(d) surface expression. We found that tapasin Delta334-342 was unable to bind to the L(d) H chain, and yet it facilitated L(d) assembly and expression. Tapasin Delta334-342 was able to bind and stabilize TAP, suggesting that TAP stabilization may be important to the assembly of L(d). Tapasin mutant H334F/H335Y, unlike tapasin Delta334-342, bound to L(d). Expression of tapasin H334F/H335Y in 721.220-L(d) reduced the proportion of cell surface open forms of L(d) and retarded the migration of L(d) from the endoplasmic reticulum. In total, our results indicate that the 334-342 region of tapasin influences L(d) assembly and transport.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Interactions of HLA-B27 with the peptide loading complex as revealed by heavy chain mutations.

MHC class I heavy chains assemble in the endoplasmic reticulum with beta(2)-microglobulin and peptide to form heterotrimers. Although full assembly is required for stable class I molecules to be expressed on the cell surface, class I alleles can differ significantly in their rates of, and dependencies on, full assembly. Furthermore, these differences can account for class I allele-specific disparities in antigen presentation to T cells. Recent studies suggest that class I assembly is assisted by an elaborate complex of proteins in the endoplasmic reticulum, collectively referred to as the peptide loading complex. In this report we take a mutagenesis approach to define how HLA-B27 molecules interact with the peptide loading complex. Our results define subtle differences between how B27 mutants interact with tapasin (TPN) and calreticulin (CRT) in comparison to similar mutations in other mouse and human class I molecules. Furthermore, these disparate interactions seen among class I molecules allow us to propose a spatial model by which all class I molecules interact with TPN and CRT, two molecular chaperones implicated in facilitating the binding of high-affinity peptide ligands.

Antiporters↗
PubMed2000

HLA-B polymorphism affects interactions with multiple endoplasmic reticulum proteins.

To explore the nature of amino acid substitutions that influence association with TAP, we compared a site-directed mutant of HLA-B*0702 (Y116D) to unmutated HLA-B7 in regard to TAP interaction. We found that the mutant had stronger association with TAP, and, in addition, with tapasin and calreticulin. These data confirm the importance of position 116 for TAP association, and indicate that (1) an aspartic acid at the 116 position can facilitate the interaction, and (2) association with tapasin and calreticulin is affected along with TAP. Furthermore, we tested three natural subtypes of HLA-B15, and found that a B15 subtype with a tyrosine at position 116 (B*1510) was strongly associated not only with TAP, but also with tapasin and calreticulin. In contrast, two B15 subtypes with a serine at position 116 (B*1518 and B*1501) exhibited very little or no association with any of these proteins. Thus, very closely related HLA-B subtypes can differ in regard to interaction with the entire assembly complex. Interestingly, when their surface expression was tested by flow cytometry, the HLA-B15 subtypes with little to no detectable intracellular assembly complex association had a slightly, yet consistently, higher level of the open heavy chain form than did the B15 subtype with intracellular assembly complex association. These data suggest that the relatively low strength or short length of interaction between endoplasmic reticulum proteins and natural HLA class I molecules can decrease their surface stability.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

An extensive region of an MHC class I alpha 2 domain loop influences interaction with the assembly complex.

Presentation of antigenic peptides to CTLs at the cell surface first requires assembly of MHC class I with peptide and beta 2-microglobulin in the endoplasmic reticulum. This process involves an assembly complex of several proteins, including TAP, tapasin, and calreticulin, all of which associate specifically with the beta 2-microglobulin-assembled, open form of the class I heavy chain. To better comprehend at a molecular level the regulation of class I assembly, we have assessed the influence of multiple individual amino acid substitutions in the MHC class I alpha 2 domain on interaction with TAP, tapasin, and calreticulin. In this report, we present evidence indicating that many residues surrounding position 134 in H-2Ld influence interaction with assembly complex components. Most mutations decreased association, but one (LdK131D) strongly increased it. The Ld mutants, with the exception of LdK131D, exhibited characteristics suggesting suboptimal intracellular peptide loading, similar to the phenotype of Ld expressed in a tapasin-deficient cell line. Notably, K131D was less peptide inducible than wild-type Ld, which is consistent with its unusually strong association with the endoplasmic reticulum assembly complex.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Alloreactive and syngeneic CTL are comparably dependent on interaction with MHC class I alpha-helical residues.

The molecular basis for the difference in the strength of T cell responses to self vs alloantigens is unknown, but may reflect how T cells are selected in the thymus. Because T cells with a high affinity for foreign as opposed to self MHC molecules are able to mature, it has been proposed that alloreactive T cells may be more strongly dependent upon interaction with MHC residues than are self-restricted T cells. This study was undertaken to rigorously address this hypothesis. Whereas other studies have compared self vs alloantigen recognition of different MHC alleles by a single T cell clone, we have compared self vs alloantigen recognition of a single MHC allele, H-2Ld, by a large panel of self-restricted and alloreactive T cell clones. Target cells expressing Ld molecules mutated at several different potential TCR contact residues were analyzed to determine which residues are important for recognition by self-restricted vs alloreactive T cells. We unequivocally demonstrate that self-restricted and alloreactive T cells do not differ, but rather are comparably dependent on interaction with MHC residues. Importantly, both self-restricted and alloreactive T cells are dependent upon the same MHC residues as primary contacts and, in addition, share a common recognition pattern of Ld. Furthermore, our analysis enables us to provide a model for allotype-specific T cell recognition of Ld vs Kb class I molecules.

Amino Acid Substitution↗
PubMed1999

Class I MHC molecules: assembly and antigen presentation.

Several years ago, the only factor known to be necessary for the assembly and surface expression of class I MHC was beta 2m; even for beta 2m, it was unclear at what point in class I maturation its role was played. Recent experiments that employed attachment of an endoplasmic reticulum (ER) retention signal to beta 2m have shown that the point of time at which beta 2m is required is while the class I heavy chain is in the ER. Later association between beta 2m and class I is not vital in order for properly folded class I to be expressed at the cell surface. After crystallization of the first class I MHC molecule, it was realized that not only is antigen presented by class I, but that antigen is presented in the form of a peptide that stabilizes the class I structure and allows its transit to the cell surface. Class I allelic differences influence interactions with both peptide and beta 2m, with likely consequences for the ability of the class I heavy chains to present antigen through alternative pathways. Furthermore, it is now also clear that formation of appropriate disulfide bonds in the class I heavy chain is needed before class I can bind peptide antigen securely, a process that may be assisted by an ER chaperone. Many different proteins that are resident in the ER, such as calnexin, transporter associated with antigen processing (TAP), calreticulin, and tapasin, have been found to be integral to class I assembly. TAP, tapasin, and calreticulin bind preferentially to the open form of class I, which can be distinguished with the use of a monoclonal antibody specific for this form. Calreticulin and calnexin contrast in their interactions with class I, despite other similarities between these two chaperones. Overall, class I MHC assembly is now understood to involve the interplay of multiple intra- and intermolecular events in a defined chronological order which ensure continual reporting of cellular contents to cytotoxic T lymphocytes.

Alleles↗
PubMed1998

Calreticulin and calnexin interact with different protein and glycan determinants during the assembly of MHC class I.

Before peptide binding, a variety of endoplasmic reticulum (ER) proteins are associated with class I including calnexin, TAP, calreticulin, and tapasin. Although the selective functions of any one of these ER proteins have been difficult to define, individually or in combination they perform two general chaperone functions for class I. They promote assembly of the class I heterotrimeric molecule (heavy (H) chain, beta2m, and peptide) and they retain incompletely assembled complexes in the ER. In this study, we present evidence that calreticulin clearly differs from calnexin in how it associates with class I. Regarding the structural basis of the association, the oligosaccharide moiety in the alpha1 domain and the amino acid residue at position 227 in the alpha3 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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

The three-dimensional structure of an H-2Ld-peptide complex explains the unique interaction of Ld with beta-2 microglobulin and peptide.

Solution at 2.5-A resolution of the three-dimensional structure of H-2Ld with a single nine-residue peptide provides a structural basis for understanding its unique interaction with beta-2 microglobulin (beta2m) and peptide. Consistent with the biological data that show an unusually weak association of Ld with beta2m, a novel orientation of the alpha1/alpha2 domains of Ld relative to beta2m results in a dearth of productive contacts compared with other class I proteins. Characteristics of the Ld antigen-binding cleft determine the unique motif of peptides that it binds. Ld has no central anchor residue due to the presence of several bulky side chains in its mid-cleft region. Also, its cleft is significantly more hydrophobic than that of the other class I molecules for which structures are known, resulting in many fewer H-bonds between peptide and cleft residues. The choice of Pro as a consensus anchor at peptide position 2 appears to be related to the hydrophobicity of the B pocket, and to the unique occurrence of Ile (which mirrors Pro in its inability to form H-bonds) at position 63 on the edge of this pocket. Thus, the paucity of stabilizing H-bonds combined with poor complementarity between peptide postion 2 Pro and the B pocket contribute to the weak association between Ld and its peptide antigen. The unique structural interactions of Ld with beta2m and peptide could make Ld more suited than other classical class I molecules to play a role in alternative pathways of antigen presentation.

Animals↗
PubMed1997

Prominence of beta 2-microglobulin, class I heavy chain conformation, and tapasin in the interactions of class I heavy chain with calreticulin and the transporter associated with antigen processing.

Newly synthesized class I heavy (H) chain/beta 2m heterodimers awaiting peptides in the endoplasmic reticulum are associated with the transporter associated with Ag processing (TAP). We present evidence here that calreticulin, but not calnexin, displays steady state association with class I/TAP complexes. To separate the ability of beta 2m to bind with TAP and calreticulin from that of H chain, we studied human cell lines that lack expression of beta 2m or H chain. Little if any H chain was detected in association with TAP and calreticulin in the beta 2m- cell line Daudi. By contrast, high levels of beta 2m are found with TAP and calreticulin in the H chain-deficient cell line LCL 721.221, even after preclearance of the trace amount of class IB protein expressed by this cell line. Thus, beta 2m appears to bind TAP in the absence of H chain, providing an elegant mechanism to retain beta 2m in the endoplasmic reticulum at the site of peptide loading. To investigate whether other molecules participate in the binding of beta 2m and H chain to TAP and calreticulin, we analyzed the deletion mutant cell line LCL 721.220, which lacks tapasin. In 721.220, TAP and calreticulin are not associated with each other. Also, in these cells, H chain/beta 2m are not associated with TAP, but H chain and a low level of beta 2m are associated with calreticulin. These results suggest that tapasin is an obligatory mediator of the assemblage of calreticulin/H chain/beta 2m with TAP.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Are transporter associated with antigen processing (TAP) and tapasin class I MHC chaperones?

Class I MHC heavy chains associate with many proteins in the endoplasmic reticulum, including TAP, calnexin, calreticulin, and the newly defined tapasin molecule. Recent studies have begun to resolve the nature of how these proteins interact with class I as well as the functional significance of each of these interactions. We propose here that TAP and tapasin are leading candidates to be highly specific chaperones for the class I molecule.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

TAP associates with a unique class I conformation, whereas calnexin associates with multiple class I forms in mouse and man.

To define the rules governing de novo assembly of the trimeric class I complex, we have identified the class I folding/assembly intermediates associated with calnexin or TAP, using both human and mouse cell lines. To better characterize the class I H chain structure associated with TAP, mouse mAb that distinguish open (64-3-7+) vs folded (30-5-7+) Ld heavy (H) chains were used. We report here that open forms of Ld are uniquely and specifically associated with TAP and that the conformational change in the class I H chain coincident with peptide binding induces TAP release. Chimeric Ld/Q10 displayed TAP association, demonstrating that soluble class I molecules can bind TAP. As previously reported, beta 2m was found to be required for H chain association with TAP. Interestingly, beta 2m was associated with TAP in the human class I-negative cell line LCL 721.221, suggesting that beta 2m can bind to TAP before class I H chain. In contrast to TAP, which binds a specific class I conformation, calnexin was detected in association with multiple forms of both mouse and human class I. Most significantly, we show for the first time that beta 2m-assembled forms of human as well as mouse class I molecules interact with calnexin. Based on these findings, we propose a model for the sequential assembly of class I heterotrimers and their respective interactions with TAP and calnexin.

ATP-Binding Cassette Transporters↗
PubMed1995

Peptide-induced rescue of serologic epitopes on class I MHC molecules.

To monitor conformational changes in MHC class I structure induced by interaction with peptide or beta 2-microglobulin (beta 2-m), we have taken a serologic approach. Previous studies by us and others have defined circumstances wherein specific peptides can decrease serologic recognition of class I molecules. However, such blocking of serologic epitopes has often been interpreted as steric hindrance by peptide side chains. In this paper, we describe peptide-induced gains in recognition by mAbs 30-5-7, 34-1-2, and B22/249. In experiments with mAb 30-5-7, impaired reactivity, which resulted from an Ld loop mutation, was specifically rescued by the binding of a beta-galactosidase-derived peptide to the Ld mutant. In studies with mAb 34-1-2, poor Ld detection was enhanced by mutations in Ld at beta 2-m interaction sites or by changes within the peptide-binding groove. To evaluate whether known peptides in the Ld groove could influence 34-1-2 recognition, we tested six peptide ligands, four of which increased the reactivity of 34-1-2 with the Ld-expressing cell to various degrees (up to 14-fold). It is of interest that Ld mutations at position 9 and 95/97 made significant differences in the ranking of the peptides in regard to their ability to increase recognition by 34-1-2 and B22/249. This finding suggests that mutations in the binding groove can alter peptide conformation and result in secondary changes in class I structure. On the basis of the cumulative serologic data, we propose that the class I molecule displays considerable fluidity, and is structurally influenced by both beta 2-m and peptide.

Amino Acid Sequence↗
PubMed1995

Induction of peptide-specific CD8+ CTL clones in beta 2-microglobulin-deficient mice.

We have examined the ability of beta 2-m- mice to produce CD4-8+ T cells by generating CD8+ CTLs to a defined ligand. We report here the first demonstration of peptide-specific, self-class I MHC-restricted CTLs from beta 2-m-deficient mice. We have used the KOD mouse, an H-2d beta 2-m- strain, to generate CTLs that recognize the class I MHC molecule Ld in association with one of two Ld-binding immunogenic peptides. Testing of these CTLs on a panel of Ld-binding peptides reveals a high degree of peptide specificity. Peptide-specific CTL bulk cultures from KOD mice differ from those generated in beta 2-m+ mice in that they possess altered affinities for their peptide ligands. In addition, we show that CTLs generated from beta 2-m- mice in the presence of beta 2-m+ stimulator cells and exogenous peptide are specific either for the exogenous peptide or for endogenous peptides that are present in association with Ld on the surface of beta 2-m+ cells, but are not present at detectable levels on beta 2-m- cells. These results demonstrate that positive selection of CD8+ CTLs can occur in vivo on the very low levels of class I MHC found in the KOD mouse. Furthermore, CTLs from the KOD mouse maintain a high degree of peptide specificity despite reduced levels of class I MHC.

Amino Acid Sequence↗
PubMed1995

Beta 2-microglobulin with an endoplasmic reticulum retention signal increases the surface expression of folded class I major histocompatibility complex molecules.

With beta 2-microglobulin- (beta 2m-) cell lines such as R1E/Db, the surface expression of class I major histocompatibility complex molecules is greatly impaired, and class I molecules that are on the surface are generally misfolded. To determine whether beta 2m must be continually present with the class I heavy chain for the class I molecule to reach the surface in a folded conformation, a sequence encoding an endoplasmic reticulum (ER) retention signal (KDEL) was attached onto the 3' end of a beta 2m cDNA. After this chimeric cDNA was transfected into R1E/Db cells, beta 2m-KDEL protein was detectable by an anti-beta 2m serum within the cells but not at the cell surface. Interestingly, R1E/Db cells transfected with beta 2m-KDEL were found to express a high level of conformationally correct Db molecules at the cell surface. This observation implies that beta 2m has a critical and temporal role in the de novo folding of the class I heavy chain. We propose that the critical time for beta 2m association is when the class I molecule is docked with the transporter associated with antigen processing (TAP) and first interacts with peptide.

Amino Acid Sequence↗
PubMed1995

Conformational changes induced in the MHC class I molecule by peptide and beta 2-microglobulin.

Assembly of the class I MHC molecule is inextricably linked to the antigen presentation function of the class I molecule. Association of the class I MHC molecule with beta 2-microglobulin (beta 2m) is a prerequisite for association with the heterodimeric protein TAP, and once peptide is acquired, the class I molecule folds and begins its sojourn to the cell surface. To maintain its folded conformation, class I MHC requires peptide but not beta 2m, and the sequence of the peptide bound exercises a subtle influence on the structure of the class I molecule that is likely to be a factor in T cell receptor discrimination of MHC/peptide complexes.

Animals↗