Peptide-induced stabilization and intracellular localization of empty HLA class I complexes.

Abstract

SummaryThe human cell line T2 has been reported to be class I assembly deficient, and accordingly expresses reduced amounts of HLA-A2 and no HLA-B5 at the cell surface. By immunoblotting we observe the steady-state class I heavy chain levels of T2 to be near normal when compared with the identical class I alleles of the wild-type cell line T1. In pulse chase experiments, formation of heavy chain/3z-microglobulin complexes is observed for both HLA-A2 and HLA-B5. Culture at reduced temperatures (26 or 20~ does not increase the amount of class I molecules transported, unlike what has been reported for the class I assembly-deficient mouse mutant cell line RMA-S. The HLA-B5 and the HLA-A2 complexes formed by T2 are thermolabile in cell lysates, albeit to different degrees. The thermolability of HLA-B5 can be overcome by addition of HLA-B5-presentable peptides, obtained by trifluoroacetic acid extraction from an HLA-B5-positive cell line, underlining the necessity of peptide for class I stability and indicating that T2-derived class I complexes are devoid of peptide. Cytoplast fusion of T2 cells with RMA-S cells shows the defect in class I assembly of RMA-S to be similar to that of T2. Localization of class I molecules observed by immuno-electron microscopy reveals the accumulation in the T2 cell line of both HLA-B5 and HLA-A2 in the endoplasmic reticulum (ER). Class I molecules are present in all the cisternae of the Golgi complex of T2, but the ratio of HLA-A and -B locus products in the Golgi area differs significantly from that at the cell surface. We conclude that the requirement for peptide in transport of class I molecules manifests itself at a stage beyond the ER, most likely the Golgi area.

Restoration of MHC Class I Surface Expression and Endogenous Antigen Presentation by a Molecular Chaperone

“…The egress of functional MHC class I complexes from the ER to the plasma membrane is dependent on the acquisition of peptide by a chain-b 2 M heterodimers [27][28][29]. Augmented chaperone activity may therefore influence the formation of stable MHC-peptide complexes by increasing the concentration of peptides available for binding MHC class I molecules within the ER.…”

Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment.

“…The observation that in TAP1 ÷ cells some HLA-B27 free heavy chains occur in a sialylated form, is most likely caused by dissociation of complexes formed early on due to a reduced affinity of HLA-B27 for murine self peptides, likely to be of different composition than their human counterparts. Sialylated free HCs are not observed in human cells (Neefjes and Ploegh, 1988;Baas et al, 1992). The murine TAP complex is unable to translocute peptides that terminate in a positively charged residue, yet such residues are abundant among HLA-B27 ligands in human cells ( Heemels and Ploegh, 1995).…”

“…In the T2 cell line these class I molecules appear to be retained in the ER and in the Golgi region (Baas et al, 1992). In another mutant cell line, likely to be (perhaps not completely) TAP-deficient, unassembled class I molecules were shown to recycle from the Golgi to the ER through an intermediate compartment, underscoring the role of the cis-Golgi region in the quality control of unassembled or misfolded molecules (Hsu et al, 1991;Bonifacino and Lippincott-Schwartz, 1991).…”

“…TAP-deficient cell lines such as the T lymphoma cell line RMA-S (Ljunggren and K/arre, 1985;Karre et al, 1986) and the B lymphoblastoid cell lines 721.174 and its derivative T2 (Kavathas et al, 1980;DeMurs et al, 1984) are defective in class I surface expression, although the HC and [32m are synthesized normally and are clearly capable of assembly in the absence of TAP-dependent peptides (Alexander et al, 1989;Ljunggren et al, 1990;Baas et al, 1992). In the T2 cell line these class I molecules appear to be retained in the ER and in the Golgi region (Baas et al, 1992).…”

“…These transgenic animals were then used to generate HLA-B27 animals lacking a functional TAP1 gene (van Santen et al, 1995). They may be used to study the behavior of class I molecules in relation to TAP in different tissues and in particular cell types, especially because several monoclonal and polyclonal antibodies reactive with human class I molecules in ultrathin cryo-sections have been described Peters et al, 1991;Baas et al, 1992).…”