Dissociation of β₂‐microglobulin determines the surface quality control of major histocompatibility complex class I molecules

Abstract

Major histocompatibility complex class I proteins, which present antigenic peptides to cytotoxic T lymphocytes at the surface of all nucleated cells, are endocytosed and destroyed rapidly once their peptide ligand has dissociated. The molecular mechanism of this cellular quality control process, which prevents rebinding of exogenous peptides and thus erroneous immune responses, is unknown. To identify the nature of the decisive step in endocytic sorting of class I molecules and its location, we have followed the removal of optimally and suboptimally peptide-loaded murine H-2K(b) class I proteins from the cell surface. We find that the binding of their light chain, β2-microglobulin (β2m), protects them from endocytic destruction. Thus, the extended survival of suboptimally loaded K(b) molecules at 25°C is attributed to decreased dissociation of β2m. Because all forms of K(b) are constantly internalized but little β2m-receptive heavy chain is present at the cell surface, it is likely that β2m dissociation and recognition of the heavy chain for lysosomal degradation take place in an endocytic compartment.

A two-hybrid antibody micropattern assay reveals specific in cis interactions of MHC I heavy chains at the cell surface

“…In STF1 cells, which lack TAP (the transporter associated with antigen processing) and thus cannot load MHC I proteins with high-affinity peptides in the ER (de la Salle et al, 1999), such dimers can be accumulated at the cell surface by incubation at 25°C (Ljunggren et al, 1990; Montealegre et al, 2015). These peptide-receptive K b HC/β 2 m dimers can be detected by subsequent binding of fluorescently labeled peptide (Saini et al, 2013).…”

“…At 37°C, dissociation of β 2 m from K b HC/β 2 m dimers occurs rapidly, whereas at 25°C, the rate of dissociation of β 2 m is significantly reduced (Montealegre et al, 2015; Day et al, 1995). Thus, we repeated the above experiments on anti-HA and Y3 patterns, but before adding the SL8 TAMRA peptide, we shifted the cells to 37°C for two to three hours.…”

“…To further test this conclusion, we co-transfected STF1 cells with HA-K b and scK b -GFP, a single-chain construct in which the K b heavy chain and β 2 m are linked by a peptide linker such that β 2 m cannot dissociate (Montealegre et al, 2015). As in the previous control experiment, no co-patterning was observed (Figure 3B, column 5).…”

“…We used the same STF1 cells co-transfected with HA-K b and K b -GFP and incubated them at 25°C overnight to accumulate both K b constructs at the cell surface. The cells were then shifted to 37°C for 15 min (the half-life of K b HC/β 2 m dimers at the cell surface [Montealegre et al, 2015]) to dissociate β 2 m and initiate co-localization. Then, the cells were trypsinized and lysed, and HA-K b was immunoprecipitated with the anti-HA antibody.…”

“…Optical methods have not provided conclusive evidence which of the three forms are interacting, and biochemical approaches have not clarified where in the cell the binding occurs (Capps et al, 1993; Matko et al, 1994). To add to the complexity, the HC/β 2 m dimers and the free heavy chains have short half-lives in the cell, which complicates their analysis (Montealegre et al, 2015). For the study of the proposed functions of the MHC I protein-protein interactions (endocytosis, synapse architecture, inflammatory response, receptor modulation; see the discussion), knowledge of location and conformation of the associated proteins is essential (Dixon-Salazar et al, 2014; Chen et al, 2017; Burian et al, 2016; Nizsalóczki et al, 2014; Mocsár et al, 2016).…”