Alpha Beta Ii Complexes Research Articles

Interaction of MHC class II molecules with the invariant chain: role of the invariant chain (81-90) region.

Association of the invariant chain (Ii) with MHC class II alpha and beta chains is central for their functionality and involves the Ii CLIP(81-104) region. Ii mutants with an antigenic peptide sequence in place of the CLIP region are shown to form alphabetaIi complexes resistant to dissociation by SDS at 25 degrees C. This reflects class II peptide binding site occupancy, since substitution of the major anchor residue within the antigenic peptide sequence of one of these Ii mutants abolishes its capacity to form SDS-stable heterotrimers. Therefore, CLIP location within Ii is compatible with CLIP access to the class II binding groove. However, in wild-type Ii this access does not lead to a tight association, which seems to be affected by the Ii 81-90 region. This region, together with a region C-terminal of CLIP, is shown to contribute to Ii association with HLA-DR1 molecules. Thus, Ii mutants with non-HLA-DR1 binding sequences in place of the CLIP(87-102) region can still associate with HLA-DR1 molecules and inhibit peptide binding.

Selective Modulation of the Major Histocompatibility Complex Class II Antigen Presentation Pathway following B Cell Receptor Ligation and Protein Kinase C Activation

We noticed that B cell receptor ligation or phorbol 12-myristate 13-acetate treatment induced intracellular vesicles containing major histocompatibility complex (MHC) class II and invariant chain (Ii), and increased the amount of transmembrane p12 Ii fragments coimmunoprecipitated with class II molecules. To determine the influence of protein kinase C activation on the MHC class II presentation pathway, we analyzed the subcellular distribution of Ii, the induction of SDS-stable forms of class II molecules, and their ability to present different antigens. Ii chains visualized with luminal and cytoplasmic directed antibodies appeared in early endosomal compartments accessible to transferrin in response to phorbol 12-myristate 13-acetate treatment, whereas transmembrane Ii degradation products equivalent to the p12 Ii fragments were colocalized with the B cell receptors internalized after cross-linking. Protein kinase C activation delayed in parallel the formation of SDS-stable forms of class II molecules and reduced the presentation of antigenic determinants requiring newly synthesized class II alphabeta-Ii complexes. These data indicate that B cell activation affects Ii processing and MHC class II peptide loading in endosomal compartments intersecting the biosynthetic pathway.

Isoforms of the invariant chain regulate transport of MHC class II molecules to antigen processing compartments.

Newly synthesized class II molecules of the major histocompatibility complex must be transported to endosomal compartments where antigens are processed for presentation to class II-restricted T cells. The invariant chain (Ii), which assembles with newly synthesized class II alpha- and beta-chains in the endoplasmic reticulum, carries one or more targeting signals for transport to endosomal compartments where Ii dissociates from alpha beta Ii complexes. Here we show that the transport route of alpha beta Ii complexes is regulated selectively by two forms of Ii (p33 and p35) that are generated by the use of alternative translation initiation sites. Using a novel quantitative surface arrival assay based on labeling with [6-3H]-D-galactose combined with biochemical modification at the cell surface with neuraminidase, we demonstrate that newly synthesized alpha beta Ii molecules containing the Ii-p33 isoform can be detected on the cell surface shortly after passage through the Golgi apparatus/trans-Golgi network. A substantial amount of these alpha beta Ii complexes are targeted to early endosomes either directly from the trans-Golgi network or after internalization from the cell surface before their delivery to antigen processing compartments. The fraction of alpha beta Ii complexes containing the p35 isoform of Ii with a longer cytosolic domain was not detected at the cell surface as determined by iodination of intact cells and the lack of susceptibility to neuraminidase trimming on ice. However, treatment with neuraminidase at 37 degrees C did reveal that some of the alpha beta Ii-p35 complexes traversed early endosomes. These results demonstrate that a fraction of newly synthesized class II molecules arrive at the cell surface as alpha beta Ii complexes before delivery to antigen processing compartments and that class II alpha beta Ii complexes associated with the two isoforms of Ii are sorted to these compartments by different transport routes.

Inhibition of invariant chain (Ii)-calnexin interaction results in enhanced degradation of Ii but does not prevent the assembly of alpha beta Ii complexes.

Calnexin is a resident protein of the endoplasmic reticulum (ER) that associates with nascent protein chains. Among the newly synthesized integral membrane proteins known to bind to calnexin is invariant chain (Ii), and Ii release from calnexin coincides with proper assembly with major histocompatibility complex (MHC) class II heterodimers. Although calnexin association with several membrane glycoproteins depends on interactions involving N-linked glycans, we previously reported that a truncation mutant of mouse Ii (mIi1-107) lacking both N-glycosylation sites was highly effective in associating with MHC class II heterodimers and escorting these dimers through the secretory pathway. This could indicate that calnexin, despite binding to both Ii and class II, is not necessary for the proper interaction of these proteins, or that in contrast to most membrane glycoproteins, the N-linked glycans of Ii are not critical to its interaction with this chaperone. To examine this issue, we have directly explored the binding of calnexin to both Ii truncation mutants lacking the typical sites of N-glycosylation or Ii produced in cells treated with tunicamycin to prevent glycan addition. These experiments revealed that either method of eliminating N-linked carbohydrates on Ii also inhibited association with calnexin. A lumenally truncated form of Ii (mIi1-131) that still has N-linked carbohydrates showed a decreased affinity for calnexin compared with intact Ii, however, indicating that calnexin-Ii binding is not determined solely by the sugar moieties. All forms of Ii lacking N-linked sugars and showing defective association with calnexin also had enhanced rates of preendosomal degradation. Despite this effect on degradation rate, tunicamycin treatment did not inhibit the association of class II with glycan-free Ii. These data support the view that calnexin is not an absolute requirement for the proper assembly of class II-Ii nonamers, but rather acts primarily to retain Ii in the ER and to inhibit its degradation. These two properties of calnexin-Ii interaction may help ensure that sufficient intact Ii is available for efficient inactivation of the binding sites of newly synthesized class II molecules, while limiting the ability of excess free Ii to alter the transport properties of the early endocytic pathway.

Transient Aggregation of Major Histocompatibility Complex Class II Chains during Assembly in Normal Spleen Cells

Many cell surface proteins exist as complexes of multiple subunits. It is well established that most such complexes are assembled within the endoplasmic reticulum (ER). However, the mechanistic details of the assembly process are largely unknown. We show here that alpha and beta subunits of major histocompatibility complex class II antigens in spleen cells of normal mice pass through a transiently aggregated phase in the ER prior to assembly with the invariant chain (Ii). Aggregates form immediately after synthesis and disappear concomitantly with assembly of mature alpha beta Ii complexes. In spleen cells lacking Ii, aggregates fail to be efficiently dissociated over time, implicating subunit assembly as a requirement for disaggregation. Two ER chaperones, BiP and calnexin, bind to newly synthesized class II MHC chains but do not contribute appreciably to the large size of the aggregates. Our observations suggest that some subunits of multisubunit complexes pass through a transient, dynamic high molecular weight aggregate phase during the physiological process of assembly. The results further suggest a novel role for Ii in promoting stable dissociation of preformed aggregates containing alpha and beta subunits rather than in preventing their formation.

Association with BiP and aggregation of class II MHC molecules synthesized in the absence of invariant chain.

Class II molecules of the major histocompatibility complex (MHC) are composed of two polymorphic glycoprotein chains (alpha and beta), that associate in the ER with a third, non-polymorphic glycoprotein known as the invariant chain (Ii). We have examined the relationship between the intracellular transport and physico-chemical characteristics of various combinations of murine alpha, beta and Ii chains. Biochemical and morphological analyses of transfected fibroblasts expressing class II MHC chains show that both unassembled alpha and beta chains, as well as a large fraction of alpha+beta complexes synthesized in the absence of Ii chain, are retained in the ER in association with the immunoglobulin heavy chain binding protein, BiP. Analyses by sedimentation velocity on sucrose gradients show that most incompletely assembled class II MHC species exist as high molecular weight aggregates in both transfected fibroblasts and spleen cells from mice carrying a disruption of the Ii chain gene. This is in contrast to the sedimentation properties of alpha beta Ii complexes from normal mice, which migrate as discrete, stoichiometric complexes of M(r) approximately 200,000-300,000. These observations suggest that assembly with the Ii chain prevents accumulation of aggregated alpha and beta chains in the ER, which might relate to the known ability of the Ii chain to promote exit of class II MHC molecules from the ER.

Class II histocompatibility molecules associate with calnexin during assembly in the endoplasmic reticulum

Class II histocompatibility antigens are composed of polymorphic alpha and beta polypeptides which associate in the endoplasmic reticulum (ER) with a third, non-polymorphic invariant polypeptide (Ii). The alpha beta Ii complexes are subsequently transported through the Golgi to the endosomes, where the Ii chain dissociates before the alpha beta complex is transported to the cell surface. Results from transport-defective class II expression variant studies suggest that class II intracellular transport is regulated in multiple intracellular compartments. Consistent with this, a large number of studies have demonstrated that protein folding and/or oligomerization is facilitated in the ER by a class of proteins collectively known as molecular chaperones. In this report, we show that the ER-resident protein calnexin associates with human and murine class II antigens. Specifically, calnexin associates in the ER with free Ii polypeptides and partially assembled wild-type class II complexes, including A alpha and/or A alpha Ii complexes, as well as with alpha beta dimers isolated from class II transport-defective cells. Calnexin also physically associates with alpha beta Ii complexes, but not with mature alpha beta dimers. These results suggest that calnexin may regulate class II intracellular transport by facilitating the production of transport competent molecules out of the ER. In addition, we report that the nucleotide sequence of the gene encoding murine calnexin shows a high degree of homology to human IP90 and dog calnexin at both the nucleotide and deduced amino acid sequence level. The isolation of cDNA fragments encoding murine calnexin will allow us to further evaluate the functional consequences of calnexin-class II interaction.

Distinct binding sites on HLA-DR for invariant chain and staphylococcal enterotoxins.

During biosynthesis, class II molecules of the major histocompatibility complex exist as complexes of the polymorphic alpha and beta chains in association with trimers of the invariant chain (Ii). The nonpolymorphic Ii contains sequences necessary for proper targeting of class II to endosomal compartments, where Ii is degraded. Ii also prevents the premature association of antigenic peptides with class II molecules. It is not known whether the effect of Ii on peptide binding extends to other ligands of class II, specifically exogenous superantigens. Cells expressing a mutant Ii molecule stably associated with HLA-DR at the cell surface were tested for their ability to interact with staphylococcal toxins. Most toxins (staphylococcal enterotoxins A-E and exfoliative toxin) were found to bind to cells expressing this alpha beta Ii complex with levels comparable to cells expressing only alpha beta chains at the cell surface. Cells expressing surface alpha beta Ii complexes stimulated polyclonal populations of peripheral blood T cells in association with these toxins. Binding of toxic shock syndrome toxin (TSST) and subsequent stimulation of T cells were reduced by the presence of the Ii. This reduction was not due to an alteration in the repertoire of T cells responding to TSST in the presence of Ii. Data from experiments with a T-cell clone suggest that interactions between class II molecules and T-cell antigen receptors occur during staphylococcal enterotoxin-mediated stimulation and that surface Ii does not interfere with such interactions.

Stable surface expression of invariant chain prevents peptide presentation by HLA-DR.

Class II major histocompatibility complex (MHC) molecules are cell surface glycoproteins that bind and present immunogenic peptides to T cells. Intracellularly, class II molecules associate with a polypeptide referred to as the invariant (Ii) chain. Ii is proteolytically degraded and dissociates from the class II complex prior to cell surface expression of the mature class II alpha beta heterodimer. Using human fibroblasts transfected with HLA-DR1 and Ii cDNAs, we now demonstrate that truncation of the cytoplasmic domain of Ii results in the failure of Ii to dissociate from the alpha beta Ii complex and leads to stable expression of class II alpha beta Ii complexes on the cell surface. Furthermore, biochemical analysis and peptide presentation assays demonstrated that transfectants with stable surface alpha beta Ii complexes expressed very few free alpha beta heterodimers at the surface and were very inefficient in their ability to present immunogenic peptides to T cells. These results support the hypothesis that the cytoplasmic domain of Ii is responsible for endosomal targeting of alpha beta Ii and directly demonstrate that association with Ii interferes with the antigen presentation function of class II molecules.