The human HLA-B27 class I molecule exhibits a strong association with the inflammatory arthritic disorder ankylosing spondylitis and other related arthropathies. Major histocompatibility complex class I heavy chains normally associate with ␤ 2 -microglobulin and peptide in the endoplasmic reticulum before transit to the cell surface. However, an unusual characteristic of HLA-B27 is its ability to form heavy chain homodimers through an unpaired cysteine at position 67 in the peptide groove. Homodimers have previously been detected within the ER and at the cell surface, but their mechanism of formation and role in disease remain undefined. Here we demonstrate, in the rat C58 thymoma cell line and in human HeLa cells transfected with HLA-B27, that homodimer formation involves not only cysteine at position 67 but also the conserved structural cysteine at position 164. We also show that homodimer formation can be induced in the non-disease-associated HLA class I allele HLA-A2 by slowing its assembly rate by incubation of cells at 26°C, suggesting that homodimer formation in the endoplasmic reticulum may occur as a result of the slower folding kinetics of HLA-B27. Finally, we report an association between unfolded HLA-B27 molecules and immunoglobulin-binding protein at the cell surface.

The oxidoreductase ERp57 is an integral component of the peptide loading complex of major histocompatibility complex (MHC) class I molecules, formed during their chaperone-assisted assembly in the endoplasmic reticulum. Misfolded MHC class I molecules or those denied suitable peptides are retrotranslocated and degraded in the cytosol. The presence of ERp57 during class I assembly suggests it may be involved in the reduction of intrachain disul®des prior to retrotranslocation. We have studied the ability of ERp57 to reduce MHC class I molecules in vitro. Recombinant ERp57 speci®cally reduced partially folded MHC class I molecules, whereas it had little or no effect on folded and peptide-loaded MHC class I molecules. Reductase activity was associated with cysteines at positions 56 and 405 of ERp57, the N-terminal residues of the active CXXC motifs. Our data suggest that the reductase activity of ERp57 may be involved during the unfolding of MHC class I molecules, leading to targeting for degradation. Keywords: chaperone/endoplasmic reticulum/MHC class I/oxidoreductase ERp57/protein folding IntroductionMajor histocompatibility complex (MHC) class I molecules present short peptides to CD8 + T lymphocytes, permitting the detection and elimination of pathogeninfected cells. MHC class I molecules meet and bind peptides during assembly within the endoplasmic reticulum (ER) in an assembly process known to involve a series of interactions with the ER resident chaperones calnexin and calreticulin, the accessory molecule tapasin and the oxidoreductase ERp57 (Lehner and Trowsdale, 1998;Pamer and Cresswell, 1998). An early interaction of the MHC class I heavy chain with calnexin is normally rapidly replaced by the formation of a peptide loading complex containing calreticulin, ERp57, tapasin and the transporter associated with antigen processing (TAP) (Solheim et al., 1997;Diedrich et al., 2001). The MHC class I-speci®c accessory molecule tapasin is critical for the formation of this complex. Tapasin physically links MHC class I± chaperone complexes to TAP and is also involved in editing the peptide repertoire bound by MHC class I molecules Grandea and Van Kaer, 2001;Purcell et al., 2001).ERp57 is a member of the protein disul®de isomerase (PDI) family, whose functions include disul®de bond oxidation, reduction and isomerization (Gilbert, 1997;High et al., 2000;Ellgaard and Helenius, 2001). The active cysteine residues are contained within two CXXC motifs located within N-and C-terminal thioredoxin-like domains (Hirano et al., 1995;Urade et al., 1997). ERp57 forms complexes with the ER chaperones calnexin and calreticulin (Oliver et al., , 1999High et al., 2000;Molinari and Helenius, 2000). These chaperone complexes possess speci®city for monoglucosylated polypeptides, and it has been postulated that through consecutive rounds of deglucosylation and reglucosylation of nonnative glycoprotein substrates, cycles of folding occur until a native protein conformation has been adopted (Elliott et al., 1997;Zapun et al., 1998;Oliver ...

SUMMARYThe transporter associated with antigen processing (TAP) supplies peptides into the lumen of the endoplasmic reticulum (ER) for binding by major histocompatibility complex (MHC) class I molecules. TAP comprises two polypeptides, TAP1 and TAP2, each a 'half-transporter' encoding a transmembrane domain and a nucleotide-binding domain. Immunoprecipitation of rat TAP1 and TAP2 expressed individually in the human TAP-deficient cell line, T2, revealed that both bound the endogenously expressed HLA-A2 and -B51 class I molecules. Using HLA-encoding recombinant vaccinia viruses HLA-A*2501, -B*2704, -B*3501 and -B*4402, alleles also associated with both TAP1 and TAP2. Thus, TAP1 and TAP2 do not appear to differ in their ability to interact with MHC class I alleles. Single TAP polypeptide subunits also formed MHC class I peptideloading complexes, and their nucleotide-binding domains retained the ability to interact with ATP, and may permit the release of peptide-loaded MHC class I molecules in the absence of a peptide transport cycle. It is also demonstrated by chemical cross-linking that TAP2, but not TAP1, has the ability to form a homodimer complex both in whole cells and in detergent lysates. Together these data indicate that single TAP polypeptide subunits possess many of the features of the TAP heterodimer, demonstrating them to be useful models in the study of ATP-binding cassette (ABC) transporters.

Major histocompatibility complex (MHC) class I molecules load peptides in the endoplasmic reticulum in a process during which the peptide cargo is normally optimized in favor of stable MHC-peptide interactions. A dynamic multimolecular assembly termed the peptideloading complex (PLC) participates in this process and is composed of MHC class I molecules, calreticulin, ERp57, and tapasin bound to the transporter associated with antigen processing (TAP) peptide transporter. We have exploited the observation that the rat MHC class I allele RT1-A a , when expressed in the rat C58 thymoma cell line, effectively competes and prevents the endogenous RT1-A u molecule from associating with TAP. However, stable RT1-A u molecules are assembled efficiently in competition with RT1-A a , demonstrating that cargo optimization can occur in the absence of TAP association. Defined mutants of RT1-A a , which do not allow formation of the PLC, fail to become thermostable in C58 cells. Wild-type RT1-A a , which does allow PLC formation, also fails to become thermostable in this cell line, which carries the rat TAPB transporter that supplies peptides incompatible for RT1-A a binding. Full optimization of RT1-A a requires the presence of the TAP2A allele, which is capable of supplying suitable peptides. Thus, formation of the PLC alone is not sufficient for optimization of the MHC class I peptide cargo.