Malini Raghavan

MHC class I assembly: out and about

The assembly of major histocompatibility complex (MHC) class I molecules with peptides is orchestrated by several assembly factors including the transporter associated with antigen processing (TAP) and tapasin, the endoplasmic reticulum (ER) oxido-reductases ERp57 and protein disulfide isomerase (PDI), the lectin chaperones calnexin and calreticulin, and the ER aminopeptidase (ERAAP). Typically, MHC class I molecules present endogenous antigens to cytotoxic T lymphocytes (CTLs). However, the initiation of CD8(+) T-cell responses against many pathogens and tumors also requires the presentation of exogenous antigens by MHC class I molecules. We discuss recent developments relating to interactions and mechanisms of function of the various assembly factors and pathways by which exogenous antigens access MHC class I molecules.

Calreticulin in the immune system: ins and outs.

Calreticulin is a calcium-binding chaperone that has several functions in the immune response. In the endoplasmic reticulum (ER), calreticulin facilitates the folding of major histocompatibility complex (MHC) class I molecules and their assembly factor tapasin, thereby influencing antigen presentation to cytotoxic T cells. Although calreticulin is normally ER-resident, it is found at the cell surface of living cancer cells and dying cells. Here, calreticulin promotes cellular phagocytic uptake. In tumor vaccine models, drugs that induce cell surface calreticulin confer enhanced tumor protection in an extracellular calreticulin-dependent manner. Much remains to be understood about the roles of calreticulin in these distinct functions. Further investigations are important towards advancing basic knowledge of glycoprotein-folding pathways, and towards developing new cancer therapeutic strategies.

Walker A Lysine Mutations of TAP1 and TAP2 Interfere with Peptide Translocation but Not Peptide Binding

We generated mutants of the transporter associated with antigen-processing subunits TAP1 and TAP2 that were altered at the conserved lysine residue in the Walker A motifs of the nucleotide binding domains (NBD). In other ATP binding cassette transporters, mutations of the lysine have been shown to reduce or abrogate the ATP hydrolysis activity and in some cases impair nucleotide binding. Mutants TAP1(K544M) and TAP2(K509M) were expressed in insect cells, and the effects of the mutations on nucleotide binding, peptide binding, and peptide translocation were assessed. The mutant TAP1 subunit is significantly impaired for nucleotide binding relative to wild type TAP1. The identical mutation in TAP2 does not significantly impair nucleotide binding relative to wild type TAP2. Using fluorescence quenching assays to measure the binding of fluorescent peptides, we show that both mutants, in combination with their wild type partners, can bind peptides. Since the mutant TAP1 is significantly impaired for nucleotide binding, these results indicate that nucleotide binding to TAP1 is not a requirement for peptide binding to TAP complexes. Peptide translocation is undetectable for TAP1·TAP2(K509M) complexes, but low levels of translocation are detectable with TAP1(K544M)·TAP2 complexes. These results suggest an impairment in nucleotide hydrolysis by TAP complexes containing either mutant TAP subunit and indicate that the presence of one intact TAP NBD is insufficient for efficient catalysis of peptide translocation. Taken together, these results also suggest the possibility of distinct functions for TAP1 and TAP2 NBD during a single translocation cycle.

Endoplasmic Reticulum Calcium Depletion Impacts Chaperone Secretion, Innate Immunity, and Phagocytic Uptake of Cells

A number of immunological functions are ascribed to cell surface-expressed forms of the endoplasmic reticulum (ER) chaperone calreticulin (CRT). In this study, we examined the impact of ER stress-inducing drugs upon cell surface CRT induction and the resulting immunological consequences. We showed that cell surface expression of CRT and secretion of CRT, BiP, gp96, and PDI were induced by thapsigargin (THP) treatment, which depletes ER calcium, but not by tunicamycin treatment, which inhibits protein glycosylation. Surface expression of CRT in viable, THP-treated fibroblasts correlated with their enhanced phagocytic uptake by bone marrow-derived dendritic cells. Incubation of bone marrow-derived dendritic cells with THP-treated fibroblasts enhanced sterile IL-6 production and LPS-induced generation of IL-1β, IL-12, IL-23, and TNF-α. However, extracellular CRT is not required for enhanced proinflammatory responses. Furthermore, the pattern of proinflammatory cytokine induction by THP-treated cells and cell supernatants resembled that induced by THP itself and indicated that other ER chaperones present in supernatants of THP-treated cells also do not contribute to induction of the innate immune response. Thus, secretion of various ER chaperones, including CRT, is induced by ER calcium depletion. CRT, previously suggested as an eat-me signal in dead and dying cellular contexts, can also promote phagocytic uptake of cells subject to ER calcium depletion. Finally, there is a strong synergy between calcium depletion in the ER and sterile IL-6, as well as LPS-dependent IL-1β, IL-12, IL-23, and TNF-α innate responses, findings that have implications for understanding inflammatory diseases that originate in the ER.

Tapasin Interacts with the Membrane-spanning Domains of Both TAP Subunits and Enhances the Structural Stability of TAP1·TAP2 Complexes

The transporter associated with antigen processing (TAP) proteins are involved in transport of peptides from the cytosol into the endoplasmic reticulum. Two subunits, TAP1 and TAP2, are necessary and sufficient for peptide binding and peptide translocation across the endoplasmic reticulum membrane. TAP1 and TAP2 contain an N-terminal hydrophobic membrane-spanning region and a C-terminal nucleotide binding domain. Tapasin is an endoplasmic reticulum resident protein that has been found associated with the TAP subunits and shown to increase expression levels of TAP. Here we investigated TAP-tapasin interactions and their effects on TAP function in insect cells. We show tapasin binding to both TAP1 and TAP2 and to the corresponding nucleotide binding domain-exchanged chimeras as well as to a truncated TAP1·TAP2 complex containing just the membrane-spanning regions of TAP1 and TAP2. However, tapasin interactions with either the truncated TAP construct containing just the nucleotide binding domain are not observed. Tapasin is not required for high affinity peptide binding to TAP1·TAP2 complexes, and in fact, the presence of tapasin slightly reduces the affinity of TAP complexes for peptides. However, at near physiological temperatures, both tapasin and nucleotides stabilize the peptide binding site of TAP1·TAP2 complexes against inactivation, and enhanced thermostability of both TAP subunits is observed in the presence of tapasin. The enhanced structural stability of TAP1·TAP2 complexes in the presence of tapasin might explain the observations that tapasin increases TAP protein expression levels in mammalian cells.

Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I

Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP‐binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane‐spanning helices. TAP1 and TAP2, however, contain additional N‐terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N‐Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild‐type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell‐based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA‐B*2705 processing to similar levels as wild‐type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin‐mediated increase in processing of HLA‐B*2705 and HLA‐B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N‐terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.

Modes of Calreticulin Recruitment to the Major Histocompatibility Complex Class I Assembly Pathway

Major histocompatibility complex (MHC) class I molecules are ligands for T-cell receptors of CD8+ T cells and inhibitory receptors of natural killer cells. Assembly of the heavy chain, light chain, and peptide components of MHC class I molecules occurs in the endoplasmic reticulum (ER). Specific assembly factors and generic ER chaperones, collectively called the MHC class I peptide loading complex (PLC), are required for MHC class I assembly. Calreticulin has an important role within the PLC and induces MHC class I cell surface expression, but the interactions and mechanisms involved are incompletely understood. We show that interactions with the thiol oxidoreductase ERp57 and substrate glycans are important for the recruitment of calreticulin into the PLC and for its functional activities in MHC class I assembly. The glycan and ERp57 binding sites of calreticulin contribute directly or indirectly to complexes between calreticulin and the MHC class I assembly factor tapasin and are important for maintaining steady-state levels of both tapasin and MHC class I heavy chains. A number of destabilizing conditions and mutations induce generic polypeptide binding sites on calreticulin and contribute to calreticulin-mediated suppression of misfolded protein aggregation in vitro. We show that generic polypeptide binding sites per se are insufficient for stable recruitment of calreticulin to PLC substrates in cells. However, such binding sites could contribute to substrate stabilization in a step that follows the glycan and ERp57-dependent recruitment of calreticulin to the PLC.

HLA-B44 Polymorphisms at Position 116 of the Heavy Chain Influence TAP Complex Binding via an Effect on Peptide Occupancy

A single residue polymorphism distinguishes HLA-B*4402(D116) from HLA-B*4405(Y116), which was suggested to allow HLA-B*4405 to acquire peptides without binding to tapasin-TAP complexes. We show that HLA-B*4405 is not inherently unable to associate with tapasin-TAP complexes. Under conditions of peptide deficiency, both allotypes bound efficiently to TAP and tapasin, and furthermore, random nonamer peptides conferred higher thermostability to HLA-B*4405 than to HLA-B*4402. Correspondingly, under conditions of peptide sufficiency, more rapid peptide-loading, dissociation from TAP complexes, and endoplasmic reticulum exit were observed for HLA-B*4405, whereas HLA-B*4402 showed greater endoplasmic reticulum retention and enhanced tapasin-TAP binding. Together, these studies suggest that position 116 HLA polymorphisms influence peptide occupancy, which in turn determines binding to tapasin and TAP. Relative to HLA-B*4405, inefficient peptide loading of HLA-B*4402 is likely to underlie its stronger tapasin dependence for cell surface expression and thermostability, and its enhanced susceptibility to pathogen interference strategies.

Use of chimeric proteins to investigate the role of transporter associated with antigen processing (TAP) structural domains in peptide binding and translocation

The transporter associated with antigen processing (TAP) comprises two subunits, TAP1 and TAP2, each containing a hydrophobic membrane-spanning region (MSR) and a nucleotide binding domain (NBD). The TAP1/TAP2 complex is required for peptide translocation across the endoplasmic reticulum membrane. To understand the role of each structural unit of the TAP1/TAP2 complex, we generated two chimeras containing TAP1 MSR and TAP2 NBD (T1MT2C) or TAP2 MSR and TAP1 NBD (T2MT1C). We show that TAP1/T2MT1C, TAP2/T1MT2C, and T1MT2C/T2MT1C complexes bind peptide with an affinity comparable to wild-type complexes. By contrast, TAP1/T1MT2C and TAP2/T2MT1C complexes, although observed, are impaired for peptide binding. Thus, the MSRs of both TAP1 and TAP2 are required for binding peptide. However, neither NBD contains unique determinants required for peptide binding. The NBD-switched complexes, T1MT2C/T2MT1C, TAP1/T2MT1C, and TAP2/T1MT2C, all translocate peptides, but with progressively reduced efficiencies relative to the TAP1/TAP2 complex. These results indicate that both nucleotide binding sites are catalytically active and support an alternating catalytic sites model for the TAP transport cycle, similar to that proposed for P-glycoprotein. The enhanced translocation efficiency of TAP1/T2MT1C relative to TAP2/T1MT2C complexes correlates with enhanced binding of the TAP1 NBD-containing constructs to ATP-agarose beads. Preferential ATP interaction with TAP1, if occurring in vivo, might polarize the transport cycle such that ATP binding to TAP1 initiates the cycle. However, our observations that TAP complexes containing two identical TAP NBDs can mediate translocation indicate that distinct properties of the nucleotide binding site per se are not essential for the TAP catalytic cycle.

Distinct Assembly Profiles of HLA-B Molecules

MHC class I polymorphisms are known to influence outcomes in a number of infectious diseases, cancers, and inflammatory diseases. Human MHC class I H chains are encoded by the HLA-A, HLA-B, and HLA-C genes. These genes are highly polymorphic, with the HLA-B locus being the most variable. Each HLA class I protein binds to a distinct set of peptide Ags, which are presented to CD8+ T cells. HLA-disease associations have been shown in some cases to link to the peptide-binding characteristics of individual HLA class I molecules. In this study, we show that polymorphisms at the HLA-B locus profoundly influence the assembly characteristics of HLA-B molecules and the stabilities of their peptide-deficient forms. In particular, dependence on the assembly factor tapasin is highly variable, with frequent occurrence of strongly tapasin-dependent or independent allotypes. Several polymorphic HLA-B residues located near the C-terminal end of the peptide are key determinants of tapasin-independent assembly. In vitro refolded forms of tapasin-independent allotypes assemble more readily with peptides compared to tapasin-dependent allotypes that belong to the same supertype, and, during refolding, reduced aggregation of tapasin-independent allotypes is observed. Paradoxically, in HIV-infected individuals, greater tapasin-independent HLA-B assembly confers more rapid progression to death, consistent with previous findings that some HLA-B allotypes shown to be tapasin independent are associated with rapid progression to multiple AIDS outcomes. Together, these findings demonstrate significant variations in the assembly of HLA-B molecules and indicate influences of HLA-B–folding patterns upon infectious disease outcomes.