Showing papers by "Beatriz M. Carreno published in 1995"

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

Abstract: 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.

Aglycosylated and phosphatidylinositol-anchored MHC class I molecules are associated with calnexin. Evidence implicating the class I-connecting peptide segment in calnexin association.

Abstract: The endoplasmic reticulum resident protein calnexin interacts with several glycoproteins including class I MHC molecules. Calnexin is thought to retain free class I heavy chains and/or promote their folding and assembly with beta 2-microglobulin and peptide ligand. Whereas with other glycoproteins, Asn-linked glycans seem to be involved in calnexin association, with class I molecules the transmembrane region has been implicated. To critically define the structures on class I molecules that determine their interaction with calnexin, we have studied carbohydrate-deficient and transmembrane-variant class I molecules. Carbohydrate-deficient class I molecules were found to accumulate intracellularly in an open, non-beta 2-microglobulin-associated conformation. However, open as well as conformed class I molecules showed significant calnexin association whether they were aglycosylated or fully glycosylated. Thus, carbohydrate moieties may be necessary for efficient class I folding, but are not required for calnexin association. Calnexin was also found associated with a soluble class I molecule that has a truncated transmembrane segment, demonstrating that membrane attachment of class I is not required for interaction with calnexin. Finally, two isoforms of the class Ib molecule Q7b were compared. Unexpectedly, the glycosylphosphatidylinositol-anchored Q7b isoform was found associated with calnexin, whereas the soluble Q7b isoform was not calnexin associated. These comparisons of Q7b isoforms implicate the class I-connecting peptide segment and not the transmembrane region as a site of interaction with calnexin.

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

Abstract: 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.

β2‐microglobulin with an endoplasmic reticulum retention signal increases the surface expression of folded class I major histocompatibility complex molecules

Abstract: 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.