Primary structure of the gene for the murine Ia antigen-associated invariant chains (Ii). An alternatively spliced exon encodes a cysteine-rich domain highly homologous to a repetitive sequence of thyroglobulin.

The gene for murine Ia-associated invariant (Ii) chains (Ii31 and Ii41) was characterized by sequence analysis. The gene extends over approximately 9 kb and is organized in nine exons. Exon 1 encodes the 5' untranslated region and the cytoplasmic segment, exon 2 the membrane spanning segment and adjacent amino acids and exons 3-8 the extracytoplasmic portion of Ii31. Putative promoter sequences were found upstream of the start of the coding sequence. Between exons 6 and 7 an additional, alternatively spliced exon 6b has been identified. This exon is spliced into the mRNA coding for the Ii-related Ii41 protein. Exon 6b encodes a cysteine-rich domain of 64 amino acids. It shows a remarkably high homology to the repetitive elements in thyroglobulin, a precursor for thyroid hormone. Based on this homology, it is suggested that this domain (TgR) in Tg and in Ii41 may play a role either in hormone formation or as a carrier in the transport of molecules (thyroid hormone or processed antigen respectively) between intracellular compartments.     

Ia-associated invariant chain is fatty acylated before addition of sialic acid

The murine invariant chain (Ii) was found to incorporate radioactive palmitic acid. This binding of fatty acid inhibits the formation of interchain S-S bonds, probably because the cysteine residue in the transmembrane region of the Ii chain is palmitylated. The inhibition of fatty acylation by cerulenin blocks further posttranslational maturation of the invariant chain as shown by two-dimensional gel electrophoresis of Ii immunoprecipitates. In particular, the addition of sialic acid residues is blocked. Thus, it appears that fatty acylation is essential for carbohydrate processing of the Ii chain.     

Immunochemical localisation of cathepsin S, cathepsin L and MHC class II-associated p41 isoform of invariant chain in human lymph node tissue

Antigen presentation by MHC class II molecules requires cysteine proteases (CP) for two convergent proteolytic processes: stepwise degradation of the invariant chain (Ii) and generation of immunogenic peptides. Their activity is controlled by intracellular CP inhibitors, including presumably the p41 isoform of invariant chain (p41 Ii), which is in vitro a potent inhibitor of cathepsin L but not of cathepsin S. In order to evaluate the inhibitory potential of p41 Ii in antigen-presenting cells (APC), these three proteins were stained in lymph node tissue using specific monoclonal and polyclonal antibodies. The most abundant labelling was observed in subcapsular (cortical) and trabecular sinuses of the lymph node. In this area the most frequent APC were macrophages, as confirmed by the CD68 cell marker. Using confocal fluorescence microscopy, co-localisation of p41 Ii with cathepsin S, but not with cathepsin L was found in these cells. Our results are consistent with the hypothesis that cathepsin S participates in degradation of the invariant chain, but they do not support the association between cathepsin L and p41 Ii in APC.     

The role of the Ia-invariant chain complex in the posttranslational processing and transport of Ia and invariant chain glycoproteins

The invariant chain (Ii) is a nonpolymorphic glycoprotein that associates with the Ia alpha- and beta-chains of MHC class II Ag during their transport to the cell surface. Although surface expression of Ia can occur in the absence of Ii, it has not been shown whether the intracellular association of Ia and Ii affects the biosynthetic rate or specificity of posttranslational modifications to the individual molecules. Analysis of transfected cell lines carrying either Ia, Ii, or both Ia and Ii demonstrated efficient assembly of alpha-beta whether or not Ii was present. Pulse-chase studies and two-dimensional gel electrophoresis showed that Ii did not affect the addition of Ia N-linked oligosaccharide chains, or the extent or rate of their conversion to the complex form, nor did it affect the sulfation of alpha and beta glycoproteins. Ii also did not affect the rate of Ia synthesis or appearance of Ia at the cell surface. In contrast, Ia dramatically affected the posttranslational modification of Ii. Although invariant chain was modified by addition of fatty acid, N-linked oligosaccharide, and glycosaminoglycan in the absence of Ia, the processing of Ii-linked oligosaccharide into more acidic, terminally glycosylated forms was significantly less when Ia was absent, and although conversion to proteoglycan did occur, the glycosaminoglycan chains were significantly shorter than normal. The disappearance of radiolabel from the 31,000 Da form of Ii was faster when Ia was present, and the processing of Ii was more rapid when it was associated with Ia. Thus, the rate and manner in which Ii enters and passes through the Golgi is critically affected by Ia.     

Kinetics of Ii synthesis, processing, and turnover in n-butyrate-treated Burkitt's lymphoma cell lines which express or do not express class II antigens and in hairy leukemic cells

We have sought to understand the role of the electrophoretically invariant chain (Ii) in class II antigen functions, particularly in certain transformed cells in which we have previously demonstrated hyperexpression of Ii. Molecular structures and relative kinetics of Ii synthesis, processing and turnover were compared in paired, Ia+ and Ia- Burkitt's lymphoma (BL) cell lines and in hairy cell leukemia (HCL) cells. Cells were metabolically labeled with [35S] methionine for 15 min (with or without a cold methionine chase to 3 hr) or were continuously labeled for 3 hr. One- and two-dimensional gel electrophoresis resolved immunoprecipitates formed with a) a heteroantiserum to purified class II antigen (demonstrating alpha and beta chains and Ii associated with that complex), b) a heteroantiserum to hairy cell leukemia (HCL) membranes (demonstrating principally the dominant, basic form of Ii molecules, class I antigens, and some additional proteins), and c) a monoclonal antibody to human Ii. Treatment of Ia+ Jijoye and its daughter, Ia- P3HR-1, BL cells with 4 mM butyrate for 48 hr enhanced the synthesis of the dominant, basic form of Ii but did not affect apparent turnover rates of that pool of Ii chains in either cell line. In Ia+ Jijoye cells but not in Ia- P3HR-1 cells Ii was terminally processed to more acidic, sialic acid-derivatized forms. Butyrate treatment did not alter the relative turnover rate of terminally processed Ii in Jijoye cells. The level of the dominant, basic form of Ii in HCL cells equaled that in butyrate-treated Jijoye cells, and relative turnover rates of this terminally unprocessed Ii pool were similar in HCL and Jijoye cells. However, HCL Ia-associated Ii was not terminally processed, as was Ia-associated Ii in Jijoye cells. The expression of Ia auxiliary proteins, p41 and p25, was also enhanced in Jijoye cells by butyrate treatment and was prominent in HCL cells. From these experiments, we may hypothesize the following. In lymphoblastoid cells, two pathways for Ii turnover could exist. One is through association with Ia complexes and progressive terminal processing of carbohydrate side chains and a second is not associated with Ia or, apparently, with such processing. Because Ii is not found to be terminally processed in the absence of class II antigen, Ia might be considered to direct the processing of a subset of Ii towards some function (rather than vice versa).(ABSTRACT TRUNCATED AT 400 WORDS)     

Murine class II (Ia) molecules associate with human invariant chain

Polymorphic class II (Ia) major histocompatibility complex (MHC) gene products associate intracytoplasmically with a third nonpolymorphic class II molecule, the invariant chain (Ii), which is encoded by gene(s) unlinked to the MHC. Although the role of the Ii chain in the expression of cell surface Ia molecules is unclear, it has been suggested that the Ii chain helps in the assembly and intracellular transport of class II antigens. In this study, we demonstrate that the murine polymorphic class II antigens of an interspecies mouse-human hybrid, which has segregated the murine invariant chain gene, associates with the human invariant chain gene intracytoplasmically. The murine Ia antigens are expressed on the cell surface and can function as restriction elements in antigen presentation to T cells. The biochemical analysis demonstrates that the regions of the Ii gene that are critical to its interaction with Ia molecules are conserved between species.     

One gene encodes two distinct Ia-associated invariant chains

Murine immune response region-encoded Ia antigens are associated not only with invariant (Ii) chain but also with several other polypeptides, most notably a 41K protein. The present report demonstrates a striking similarity between Ii and 41K. These polypeptides were found to be serologically cross-reactive, and they also display a similar peptide composition on partial enzymatic digestion. Both polypeptides are derived from distinct unglycosylated precursors, as demonstrated by tunicamycin treatment and cellfree translation. Hybrid selection of mRNA, as well as Northern blotting with an Ii cDNA, shows the presence of two mRNA coding for the Ii chain and the 41K protein. Rat fibroblasts transfected with a mouse genomic Ii clone synthesize both the murine Ii chain and the 41K protein. These observations demonstrate that a single Ii gene encodes two distinct but closely related proteins.     

Differential expression of Ia and Ia-associated invariant chain in mouse tissues after in vivo treatment with IFN-gamma

B10.BR mice were injected i.v. with varying doses of recombinant IFN-gamma on three consecutive days. In tissue sections of 13 organs, the distribution of Ia antigens and Ia-associated invariant chain (Ii) was studied by using an immunoperoxidase technique. In the control animal, Ia and Ii were shown to be co-expressed in most tissues. However, on Kupffer cells, a small number of hepatocytes, and a subset of lymphocytes in lymph nodes and in the splenic red pulp only Ii, and no Ia, was detectable. In contrast, strongly Ia+ interdigitating reticulum cells of T-dependent areas of lymph nodes and spleen were only weakly stained for Ii. IFN-gamma treatment resulted in a dramatic increase of MHC antigen expression throughout the body, with striking differences in the inducibility of certain tissues for Ia and Ii: Bronchial epithelium was clearly induced to express the invariant chain, whereas Ia antigens remained entirely absent. Moreover, in kidney tubules and colon epithelium, Ii was induced more broadly than Ia. In contrast to the induction of Ii on endothelial cells of larger vessels in kidney, heart, and lungs, no de novo expression of Ia or Ii in capillary endothelial cells was observed. The number of detectable Ia+/Ii+ interstitial dendritic cells considerably increased upon exposure to IFN-gamma. Neither neurons nor glial cells were induced to MHC antigen expression. Our data demonstrate that IFN-gamma applied i.v. is a potent inducer or enhancer of Ia antigens and invariant chain in a variety of cell types.     

The p41 isoform of invariant chain is a chaperone for cathepsin L

The p41 splice variant of major histocompatibility complex (MHC) class II-associated invariant chain (Ii) contains a 65 aa segment that binds to the active site of cathepsin L (CatL), a lysosomal cysteine protease involved in MHC class II-restricted antigen presentation. This segment is absent from the predominant form of Ii, p31. Here we document the in vivo significance of the p41-CatL interaction. By biochemical means and electron microscopy, we demonstrate that the levels of active CatL are strongly reduced in bone marrow-derived antigen-presenting cells that lack p41. This defect mainly concerns the mature two-chain forms of CatL, which depend on p41 to be expressed at wild-type levels. Indeed, pulse-chase analysis suggests that these mature forms of CatL are degraded by endocytic proteases when p41 is absent. We conclude that p41 is required for activity of CatL by stabilizing the mature forms of the enzyme. This suggests that p41 is not merely an inhibitor of CatL enzymatic activity, but serves as a chaperone to help maintain a pool of mature enzyme in late-endocytic compartments of antigen-presenting cells.     

The cytoplasmic tail of invariant chain regulates endosome fusion and morphology

The major histocompatibility complex class II associated invariant chain (Ii) has been shown to inhibit endocytic transport and to increase the size of endosomes. We have recently found that this property has a significant impact on antigen processing and presentation. Here, we show in a cell-free endosome fusion assay that expression of Ii can increase fusion after phosphatidylinositol 3-kinase activity is blocked by wortmannin. In live cells wortmannin was also not able to block formation of the Ii-induced enlarged endosomes. The effects of Ii on endosomal transport and morphology depend on elements within the cytoplasmic tail. Data from mutagenesis analysis and nuclear magnetic resonance-based structure calculations of the Ii cytoplasmic tail demonstrate that free negative charges that are not involved in internal salt bridges are essential for both interactions between the tails and for the formation of enlarged endosomes. This correlation indicates that it is interactions between the Ii cytoplasmic tails that are involved in endosome fusion. The combined data from live cells, cell-free assays, and molecular dynamic simulations suggest that Ii molecules on different vesicles can promote endosome docking and fusion and thereby control endosomal traffic of membrane proteins and endosomal content.     

MHC class II-associated invariant chain contains a sorting signal for endosomal compartments

The invariant chain (Ii) is a transmembrane protein that associates with the MHC class II molecules in the endoplasmic reticulum. Expression of Ii in MHC class II-negative CV1 cells showed that it acquired complex-type oligosaccharide side chains and was retained in endosomal compartments. To search for a sorting signal, we made progressive deletions from the cytoplasmic N-terminus of Ii. Deleting 11 amino acid residues resulted in a protein that was still sorted and retained in endosomal vesicles, whereas deletion of 15 or more amino acid residues resulted in a protein that became resident in the plasma membrane. Amino acids 12-15 are thus essential for intracellular transport to endosomal compartments. As Ii is intracellularly associated with the MHC class II molecules, it is proposed that Ii determines the intracellular transport route of these molecules.     

Inhibitory fragment from the p41 form of invariant chain can regulate activity of cysteine cathepsins in antigen presentation

Cysteine cathepsins play an indispensable role in proteolytic processing of the major histocompatibility complex class II-associated invariant chain (Ii) and foreign antigens in a number of antigen presenting cells. Previously it was shown that a fragment of 64 residues present in the p41 form of the Ii (p41 fragment) selectively inhibits the endopeptidase cathepsin L, whereas the activity of cathepsin S remains unaffected. Comparison of structures indicated that the selectivity of interactions between cysteine cathepsins and the p41 fragment is far from being understood and requires further investigation. The p41 fragment has now been shown also to inhibit human cathepsins V, K, and F (also, presumably, O) and mouse cathepsin L with K(i) values in the low nanomolar range. These K(i) values are sufficiently low to ensure complex formation at physiological concentrations. In addition we have found that the p41 fragment can inhibit cathepsin S too. These findings suggest that regulation of the proteolytic activity of most of the cysteine cathepsins by the p41 fragment is an important and widespread control mechanism of antigen presentation.     

The HLA-D-associated invariant chain binds palmitic acid at the cysteine adjacent to the membrane segment

The highly polymorphic HLA-D antigens are associated with a nonpolymorphic polypeptide chain, designated invariant chain. This invariant chain is shown to incorporate fatty acid. Invariant chain metabolically labeled with [3H]palmitic acid releases its label after treatment with hydroxylamine indicating an ester linkage of the palmitic acid. The binding of fatty acid to the invariant chain inhibits the formation of S-S-linked dimers. This suggests that the sole cysteine residue of the invariant chain is blocked by binding of fatty acid. A peptide shared by [3H]palmitic acid- or [35S]cysteine-labeled invariant chain digests supports the hypothesis that the palmitic acid binds to the cysteine which is located close to the membrane-spanning domain on the cytoplasmic site. Inhibition of N-glycosylation with tunicamycin demonstrates binding of the fatty acid to the nonglycosylated precursor of the invariant chain. Additionally, blocking of fatty acylation by cerulenin inhibits further maturation of the invariant chain, as sialylation.     

Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells

Dendritic cells (DC) play a pivotal role as antigen presenting cells (APC) and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70) during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii.     

Assembly of major histocompatibility complex class II subunits with invariant chain

The highly polymorphic major histocompatibility complex class II (MHCII) polypeptides assemble in the ER with the assistance of invariant chain (Ii) chaperone. Ii binds to the peptide-binding pocket of MHCII heterodimers. We explored the mechanism how MHCII subunits attach to Ii. Expression with single alpha or beta subunits from three human HLA and two mouse H2 class II isotypes revealed that Ii co-isolates predominantly with the alpha polypeptide. Co-isolation with alpha chain requires the groove binding Ii-segment and depends on M91 of Ii. Immunoprecipitation of Ii from pulse chase labeled cells showed sequential assembly of alpha and beta chains.     

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.     

A tyrosine-based motif in the cytoplasmic domain of the alphavirus envelope protein is essential for budding.

The budding of enveloped viruses from cellular membranes is believed to be dependent on the specific interaction between transmembrane spike proteins and cytoplasmic core components of the virus. We found that the cytoplasmic domain of the E2 transmembrane spike glycoprotein of Semliki Forest virus contains two essential determinants which are absolutely needed for budding. The first constitutes a single tyrosine residue in the context of a direct pentapeptide repeat. The tyrosine could only partially be substituted for other residues with aromatic or bulky hydrophobic side chains, although these immediately reverted to the original genotype. The second determinant involves palmitylated cysteine residues flanking the tyrosine repeat motif. The function of these is probably to anchor the tail against the inner surface of the membrane so that the tyrosine-containing motif is properly presented to the nucleocapsid. This is the first example where a membrane virus employs a tyrosine signal for the selective incorporation of spike proteins into budding structures.     

Human immunodeficiency virus type 1 nef expression prevents AP-2-mediated internalization of the major histocompatibility complex class II-associated invariant chain

The lentiviral Nef protein has been studied extensively for its ability to induce the downregulation of several immunoreceptors on the surfaces of infected cells. However, Nef expression is unique in inducing highly effective upregulation of the major histocompatibility complex class II-associated chaperone invariant (Ii) chain complexes in different cell types. Under normal conditions, endocytosis of the Ii chain and other molecules, like the transferrin receptor and CD4, is rapid and AP-2 dependent. Human immunodeficiency virus type 1 (HIV-1) Nef expression strongly reduces the internalization of the Ii chain, enhances that of CD4, and does not modify transferrin uptake. The mutation of AP-2 binding motifs LL164 and DD174 in Nef leads to the inhibition of Ii chain upregulation. In AP-2-depleted cells, surface levels of the Ii chain are high and remain unmodified by Nef expression, further indicating that Nef regulates Ii chain internalization via the AP-2 pathway. Immunoprecipitation experiments revealed that the Ii chain can interact with Nef in a dileucine-dependent manner. Importantly, we have shown that Nef-induced CD4 downregulation and Ii chain upregulation are genetically distinguishable. We have identified natural nef alleles that have lost one of the two functions but not the other one. Moreover, we have characterized Nef mutant forms possessing a similar phenotype in the context of HIV-1 infection. Therefore, the Nef-induced accumulation of Ii chain complexes at the cell surface probably results from a complex mechanism leading to the impairment of AP-2-mediated endocytosis rather than from direct competition between Nef and the Ii chain for binding AP-2.     

Endoplasmic reticulum-associated degradation-induced dissociation of class II invariant chain complexes containing a glycosylation-deficient form of p41

The quality control system in the secretory pathway can identify and eliminate misfolded proteins through endoplasmic reticulum-associated degradation (ERAD). ERAD is thought to occur by retrotranslocation through the Sec61 complex into the cytosol and degradation by the proteasome. However, the extent of disassembly of oligomeric proteins and unfolding of polypeptide chains that is required for retrotranslocation is not fully understood. In this report we used a glycosylation mutant of the p41 isoform of invariant chain (Ii) to evaluate the ability of ERAD to discriminate between correctly folded and misfolded subunits in an oligomeric complex. We show that loss of glycosylation at position 239 of p41 does not detectably affect Ii trimerization or association with class II but does result in a defect in endoplasmic reticulum export of Ii that ultimately leads to its degradation via the ERAD pathway. Although class II associated with the mutated form of p41 is initially retained in the endoplasmic reticulum, it is subsequently released and traffics through the Golgi to the plasma membrane. ERAD-mediated degradation of the mutant p41 is dependent on mannose trimming and inhibition of mannosidase I stabilizes Ii. Interestingly, inhibition of mannosidase I also results in prolonged association between the mutant Ii and class II, indicating that complex disassembly and release of class II is linked to mannosidase-dependent ERAD targeting of the misfolded Ii. These results suggest that the ERAD machinery can induce subunit disassembly, specifically targeting misfolded subunits to degradation and sparing properly folded subunits for reassembly and/or export.     

MHC class II-associated invariant chain isoforms regulate pulmonary immune responses

Asthma, a chronic inflammatory disease of the lung, is characterized by reversible airway obstruction and airway hyperresponsiveness (AHR), and is associated with increased production of IgE and Th2-type cytokines (IL-4, IL-5, and IL-13). Development of inflammation within the asthmatic lung depends on MHC class II-restricted Ag presentation, leading to stimulation of CD4(+) T cells and cytokine generation. Conventional MHC class II pathways require both MHC-associated invariant chain (Ii) and HLA-DM (H2-M in mice) chaperone activities, but alternative modes of Ag presentation may also promote in vivo immunity. In this study, we demonstrate that Ii(-/-) and H2-M(-/-) mice fail to develop lung inflammation or AHR following sensitization and challenge with OVA in a mouse model of allergic inflammation. To assess potentially distinct contributions by Ii chain isoforms to lung immunity, we also compared allergen-induced lung inflammation, eosinophilia, IgE production, and AHR in mice genetically altered to express either p31 Ii or p41 Ii isoform alone. Sole expression of either Ii isoform alone facilitates development of allergen-induced lung inflammation and eosinophilia. However, animals expressing only the p31 Ii isoform exhibit abrogated IgE and AHR responses as compared with p41 Ii mice in this model of allergen-induced lung inflammation, suggesting that realization of complete immunity within the lung requires expression of p41 Ii. These findings reveal a crucial role of Ii and H2-M in controlling the immune response within the lung, and suggest that p31 Ii and p41 Ii manifest nonredundant roles in development of immunity.