Complex Class II-associated Invariant Chain Research Articles

MHC Class II-Associated Invariant Chain-Induced Enlarged Endosomal Structures: A Morphological Study

The major histocompatibility complex class II-associated invariant chain is believed to direct newly synthesized class II to endocytic compartments. Invariant chain synthesized at high levels in transiently transfected cells induces formation of large vesicular structures. We have examined the effect of stable expression of invariant chain in human fibroblasts by light and electron microscopy. Invariant chain expression dramatically modified endocytic compartments and induced the formation of greatly enlarged structures. These modifications were not lethal. Ultrastructurally, at least three morphologically distinct enlarged compartments could be discerned in the cells. These three compartments may represent early and late endosomes and lysosomes. Internalization of anti-invariant chain antibodies shows that invariant chain may reach the large endosomes via rapid internalization from the plasma membrane. Internalized protein remained in the enlarged vesicles for 4–6 h, indicating an invariant chain-induced delay in the pathway to lysosomes. Although the large invariant chain-induced vesicles have not yet been seen in professional antigen-presenting cells, the invariant chain-induced effects may play a role in regulating the endocytic pathway, creating a special environment for MHC class II to bind antigen.

Equistatin, a New Inhibitor of Cysteine Proteinases fromActinia equina, Is Structurally Related to Thyroglobulin Type-1 Domain

It is well known that the activities of the lysosomal cysteine proteinases are tightly regulated by their endogenous inhibitors, cystatins. Here we report a new inhibitor of cysteine proteinases isolated from sea anemone Actinia equina. The inhibitor, equistatin, is an acidic protein with pI 4.7 and molecular weight of 14,129. It binds tightly and rapidly to cathepsin L (ka = 5.7 x 10(7) M-1 s-1, Ki = 0.051 nM) and papain (ka = 1.2 x 10(7) M-1 s-1, Ki = 0.57 nM). The lower affinity for cathepsin B (Ki = 1.4 nM) was shown to be due mainly to a lower second order association rate constant (ka = 0.04 x 10(6) M-1 s-1). The inhibitor is composed of 128 amino acids forming two repeated domains with 48% identity. Neither of the domains shows any sequence homology to cystatins, but they do show a significant homology to thyroglobulin type-1 domains. A highly conserved consensus sequence motif of Cys-Trp-Cys-Val together with conserved Cys, Pro, and Gly residues is present in major histocompatibility complex class II-associated p41 invariant chain, nidogen, insulin-like growth factor proteins, saxiphilin domain a, pancreatic carcinoma marker proteins (GA733), and chum salmon egg cysteine proteinase inhibitor. In each of the domains of the equistatin, the three residues are similarly conserved, and the sequences Val-Trp-Cys-Val and Cys-Trp-Cys-Val are present in domains a and b, respectively. We suggest that equistatin belongs to a new superfamily of protein inhibitors of cysteine proteinases named thyroglobulin type-1 domain inhibitors. This superfamily currently includes equistatin, major histocompatibility complex class II- associated p41 invariant chain fragment, and chum salmon egg cysteine proteinase inhibitor.

GTP-bound forms of rab6 induce the redistribution of Golgi proteins into the endoplasmic reticulum.

rab6 is a ubiquitous ras-like GTPase involved in intra-Golgi transport. We have studied at both morphological and biochemical levels the behavior of Golgi resident proteins in HeLa cells overexpressing wild-type rab6 and GTP- and GDP-bound mutants of rab6 (rab6 Q72L and rab6 T27N, respectively). We show that wild-type rab6 and rab6 Q72L overexpression induces the redistribution of the trans-Golgi protein beta-1,4-galactosyltransferase into the endoplasmic reticulum (ER) and allows the addition of sialylated O-glycans on an ER-retained protein, the major histocompatibility complex class II-associated invariant chain. Remarkably, rab6 Q72L effects, which require the integrity of microtubules, were almost indistinguishable from those induced by brefeldin A, a fungic metabolite that causes a mixing of Golgi and ER membranes. In contrast, overexpression of rab6 T27N does not cause the redistribution of Golgi proteins, but inhibits basal O-glycosylation of the major histocompatibility complex class II-associated invariant chain.

Shared promoter elements between a viral superantigen and the major histocompatibility complex class II-associated invariant chain.

Superantigens have the ability to stimulate subsets of T lymphocytes bearing particular T-cell receptor Vbeta chains. The best-known viral superantigen is Mls, a product of the murine mammary tumor virus (MMTV) sag gene. The MMTV superantigen is not displayed by the virus itself; however, after infection of B lymphocytes, the superantigen is expressed. The resulting immune stimulation is essential for viral transmission. We have analyzed the transcriptional elements which control Mls-1 expression. Here we present evidence that a region at the 3' end of Mtv-7 env, Penv2, controls B-cell-specific expression of sag. Penv2 has elements homologous with promoters of immunoglobulin H chain, the invariant chain, and major histocompatibility complex class II, suggesting a coordinate regulation of expression of these various B-cell-specific genes and indicating a possible eukaryotic origin of MMTV sag. We have determined that both an IgH heptamer element and a Y box are essential for Penv2 promoter activity and that tandem octamer motifs in the U3 region of the 3' MMTV long terminal repeat function as enhancers. We propose that Penv2 controls constitutive Mls expression in B lymphocytes.

Major histocompatibility complex class II-associated p41 invariant chain fragment is a strong inhibitor of lysosomal cathepsin L.

The invariant chain (Ii) is associated with major histocompatibility complex class II molecules during early stages of their intracellular transport. In an acidic endosomal/lysosomal compartment, it is proteolytically cleaved and removed from class II heterodimers. Participation of aspartic and cysteine proteases has been observed in in vitro degradation of Ii, but the specific enzymes responsible for its in vivo processing are as yet undefined. We have previously isolated a noncovalent complex of the lysosomal cysteine protease cathepsin L with a peptide fragment derived from the p41 form of Ii from human kidney. Here we show that this Ii fragment, which is identical to the alternatively spliced segment of p41, is a very potent competitive inhibitor of cathepsin L (equilibrium inhibition constant Ki = 1.7 X 10(-12) M). It inhibits two other cysteine proteases, cathepsin H and papain, but to much lesser extent. Cysteine proteases cathepsins B, C, and S, as well as representatives of serine, aspartic, and metalloproteases, are not inhibited at all. These findings suggest a novel role for p41 in the regulation of various proteolytic activities during antigen processing and presentation. The Ii inhibitory fragment shows no sequence homology with the known cysteine protease inhibitors, and may, therefore, represent a new class.

Proteolysis of major histocompatibility complex class II-associated invariant chain is regulated by the alternatively spliced gene product, p41.

Invariant chain (Ii) is an intracellular type II transmembrane glycoprotein that is associated with major histocompatibility complex class II molecules during biosynthesis. Ii exists in two alternatively spliced forms, p31 and p41. Both p31 and p41 facilitate folding of class II molecules, promote egress from the endoplasmic reticulum, prevent premature peptide binding, and enhance localization to proteolytic endosomal compartments that are thought to be the sites for Ii degradation, antigen processing, and class II-peptide association. In spite of the dramatic and apparently equivalent effects that p31 and p41 have on class II biosynthesis, the ability of invariant chain to enhance antigen presentation to T cells is mostly restricted to p41. Here we show that degradation of Ii leads to the generation of a 12-kDa amino-terminal fragment that in p41-positive, but not in p31-positive, cells remains associated with class II molecules for an extended time. Interestingly, we find that coexpression of the two isoforms results in a change in the pattern of p31 degradation such that endosomal processing of p31 also leads to extended association of a similar 12-kDa fragment with class II molecules. These data raise the possibility that p41 may have the ability to impart its pattern of proteolytic processing on p31 molecules expressed in the same cells. This would enable a small number of p41 molecules to modify the post-translational transport and/or processing of an entire cohort of class II-Ii complexes in a manner that could account for the unique ability of p41 to enhance antigen presentation.

Direct observation of disordered regions in the major histocompatibility complex class II-associated invariant chain.

Invariant chain (Ii) is a trimeric membrane protein which binds and stabilizes major histocompatibility complex class II heterodimers in the endoplasmic reticulum and lysosomal compartments of antigen-presenting cells. In concert with an intracellular class II-like molecule, HLA-DM, Ii seems to facilitate loading of conventional class II molecules with peptides before transport of the class II-peptide complex to the cell surface for recognition by T cells. The interaction of Ii with class II molecules is thought to be mediated in large part through a region of 24 amino acids (the class II-associated Ii peptide, CLIP) which binds as a cleaved moiety in the antigenic peptide-binding groove of class II molecules in HLA-DM-deficient cell lines. Here we use nuclear magnetic resonance techniques to demonstrate that a soluble recombinant Ii ectodomain contains significant disordered regions which probably include CLIP.

Major Histocompatibility Complex Class II-associated Invariant Chain Gene Expression Is Up-regulated by Cooperative Interactions of Sp1 and NF-Y

Expression of the major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is required for efficient and complete presentation of antigens by MHC class II molecules and a normal immune response. The Ii gene is generally co-regulated with the MHC class II molecules at the level of transcription and a shared SXY promoter element has been described. This report defines the proximal promoter region of Ii which may regulate Ii transcription distinct from MHC class II. In vivo genomic footprinting identified an occupied, imperfect CCAAT box and an adjacent GC box in the proximal region. These sites are bound in Ii-ositive cell lines and upon interferon-gamma induction of Ii transcription. In contrast, both sites are unoccupied in Ii-egative cell lines and in inducible cell lines prior to interferon-gamma treatment. Together these two sites synergize to stimulate transcription. Independently, the transcription factor NF-Y binds poorly to the imperfect CCAAT box with a rapid off rate, while Sp1 binds to the GC box. Stabilization of NF-Y binding occurs upon Sp1 binding to DNA. In addition, the half-life of Sp1 binding also increased in the presence of NF-Y binding. These findings suggest a mechanism for the complete functional synergy of the GC and CCAAT elements observed in Ii transcription. Furthermore, this report defines a CCAAT box of imperfect sequence which binds NF-Y and activates transcription only when stabilized by an adjacent factor, Sp1.

Binding of major histocompatibility complex class II to the invariant chain-derived peptide, CLIP, is regulated by allelic polymorphism in class II.

Major histocompatibility complex class II-associated invariant chain (Ii) provides several important functions that regulate class II expression and function. One of these is the ability to inhibit class II peptide loading early in biosynthesis. This allows for efficient class II folding and egress from the endoplasmic reticulum, and protects the class II peptide binding site from loading with peptides before entry into endosomal compartments. The ability of Ii to interact with class II and interfere with peptide loading has been mapped to Ii exon 3, which encodes amino acids 82-107. This same region of Ii has been described as a nested set of class II-associated Ii peptides (CLIPs) that are transiently associated with class II in normal cells and accumulate in human histocompatibility leukocyte antigen-DM-negative cell lines. Currently it is not clear how CLIP and the CLIP region of Ii blocks peptide binding. CLIP may bind directly to the class II peptide binding site, or may bind elsewhere on class II and modulate class II peptide binding allosterically. In this report, we show that CLIP can interact with many different murine and human class II molecules, but that the affinity of this interaction is controlled by polymorphic residues in the class II chains. Likewise, structural changes in CLIP also modulate class II binding in an allele-dependent manner. Finally, the specificity and kinetics of CLIP binding to class II molecule is similar to antigenic peptide binding to class II. These data indicate that CLIP binds to class II in an analogous fashion as conventional antigenic peptides, suggesting that the CLIP segment of Ii may actually occupy the class II peptide binding site.

Major Histocompatibility Complex Class II-associated Invariant Chain Gene Expression Is Up-regulated by Cooperative Interactions of Sp1 and NF-Y

Major Histocompatibility Complex Class II-associated Invariant Chain Gene Expression Is Up-regulated by Cooperative Interactions of Sp1 and NF-Y

The invariant chain is required for intracellular transport and function of major histocompatibility complex class II molecules.

The major histocompatibility complex (MHC) class II-associated invariant chain (Ii) is thought to act as a chaperone that assists class II during folding, assembly, and transport. To define more precisely the role of Ii chain in regulating class II function, we have investigated in detail the biosynthesis, transport, and intracellular distribution of class II molecules in splenocytes from mice bearing a deletion of the Ii gene. As observed previously, the absence of Ii chain caused significant reduction in both class II-restricted antigen presentation and expression of class II molecules at the cell surface because of the intracellular accumulation of alpha and beta chains. Whereas much of the newly synthesized MHC molecules enter a high molecular weight aggregate characteristic of misfolded proteins, most of the alpha and beta chains form dimers and acquire epitopes characteristic of properly folded complexes. Although the complexes do not bind endogenously processed peptides, class II molecules that reach the surface are competent to bind peptides added to the medium, further demonstrating that at least some of the complexes fold properly. Similar to misfolded proteins, however, the alpha and beta chains are poorly terminally glycosylated, suggesting that they fail to reach the Golgi complex. As demonstrated by double label confocal and electron microscope immunocytochemistry, class II molecules were found in a subcompartment of the endoplasmic reticulum and in a population of small nonlysosomal vesicles possibly corresponding to the intermediate compartment or cis-Golgi network. Thus, although alpha and beta chains can fold and form dimers on their own, the absence of Ii chain causes them to be recognized as "misfolded" and retained in the same compartments as bona fide misfolded proteins.

Human major histocompatibility complex class II-associated invariant chain gene promoter. Functional analysis and in vivo protein/DNA interactions of constitutive and IFN-gamma-induced expression.

Major histocompatibility complex (MHC) class II-associated invariant chain (Ii) gene and the MHC class II molecules are physically and functionally associated within class II-expressing cell types. These genes are generally co-expressed in various cell types and coordinately induced by cytokines such as interferon-gamma (IFN-gamma). Human Ii gene shares a regulatory mechanism with MHC class II genes via the X and Y cis-acting elements. Ii X and Y are required for constitutive expression in B lymphoid cell lines and for induction by IFN-gamma in a glioblastoma cell line. The Servenius element (S) contributes 5-fold transcriptional activity to class II gene HLA-DRA, and an S homologue has been implied in Ii gene regulation. Now we report that along with X and Y, the Ii S box functions in a positive manner to regulate Ii transcription in B cell lines but not in T cell lines. In addition, S,X, and Y are all necessary for induced expression of Ii in an IFN-gamma-regulated glioblastoma cell line and in primary untransformed glial cells. Transcriptional activity of Ii X and Y elements correlates with the presence of in vivo protein/DNA interactions in Ii-expressing cells. Most interestingly, in vivo interactions are induced upon IFN-gamma induction in a time-dependent fashion. Thus, the Ii promoter contains elements that coordinately regulate expression with the MHC class II genes, and these functional sites are contacted in vivo emphasizing their importance in Ii gene transcription.

Enhanced antigen presentation in the absence of the invariant chain endosomal localization signal.

The cytosolic tail of the major histocompatibility complex class II-associated invariant chain (Ii) molecule is thought to contain the endosomal localization signal that directs and/or retains newly synthesized class II within the endosomal antigen processing compartment. To determine the role of this signal in class II transport and antigen presentation we have generated class II-positive L cell transfectants that coexpress wild type or truncated forms of Ii. Deletion of the endosomal localization signal from Ii results in rapid transport of class II-Ii complexes to the cell surface. Once at the cell surface, the complex is efficiently internalized, Ii is degraded, and class II free of Ii is recycled back to the plasma membrane. Interestingly, the truncated form of Ii is still able to increase the efficiency of antigen presentation to T cells. These data suggest that the ability of Ii to enhance antigen presentation is not limited to Golgi apparatus-endosomal sorting and raise the possibility that endocytosed class II can form immunogenic complexes with newly processed antigen.

Human major histocompatibility complex class II-associated invariant chain gene promoter : functional analysis and in vitro protein/DNA interactions of constitutive and IFN-γ-induced expression

Human major histocompatibility complex class II-associated invariant chain gene promoter : functional analysis and in vitro protein/DNA interactions of constitutive and IFN-γ-induced expression

Uncoupling of chondroitin sulfate glycosaminoglycan synthesis by brefeldin A.

Brefeldin A has dramatic, well-documented, effects on the structural and functional organization of the Golgi complex. We have examined the effects of brefeldin A (BFA) on the Golgi-localized synthesis and addition of chondroitin sulfate glycosaminoglycan carbohydrate side chains. BFA caused a dose-dependent inhibition of chondroitin sulfate glycosaminoglycan elongation and sulfation onto the core proteins of the melanoma-associated proteoglycan and the major histocompatibility complex class II-associated invariant chain. In the presence of BFA, the melanoma proteoglycan core protein was retained in the ER but still acquired complex, sialylated, N-linked oligosaccharides, as measured by digestion with endoglycosidase H and neuraminidase. The initiation of glycosaminoglycan synthesis was not affected by BFA, as shown by the incorporation of [6-3H]galactose into a protein-carbohydrate linkage region that was sensitive to beta-elimination. The ability of cells to use an exogenous acceptor, p-nitrophenyl-beta-D-xyloside, to elongate and sulfate core protein-free glycosaminoglycans, was completely inhibited by BFA. The effects of BFA were completely reversible in the absence of new protein synthesis. These experiments indicate that BFA effectively uncouples chondroitin sulfate glycosaminoglycan synthesis by segregating initiation reactions from elongation and sulfation events. Our findings support the proposal that glycosaminoglycan elongation and sulfation reactions are associated with the trans-Golgi network, a BFA-resistant, Golgi subcompartment.

Tumor necrosis factor alpha regulates expression of the major histocompatibility complex class II-associated invariant chain by binding of an NF-kappa B-like factor to a promoter element.

Expression of the major histocompatibility complex (MHC) class I and class II antigens and the class II-associated invariant chain (Ii) is strongly increased by treatment of cells with tumor necrosis factor alpha (TNF-alpha) and gamma interferon. We investigated elevation of expression of the invariant chain gene by TNF-alpha. Rat fibroblast cells transfected with the mouse Ii gene containing 802 base pairs of 5' sequences could be stimulated for Ii expression by treatment with TNF-alpha. Analysis of 5'-deleted Ii gene promoter-CAT constructs provided evidence for the presence of a TNF-alpha response box (TRB). Cloning of TRB in front of a non-TNF-alpha-responsive promoter could transfer the TNF-alpha stimulatory effect. We demonstrate binding of a TNF-alpha-induced factor to a kappa B-like motif within TRB. Mutations introduced into the kappa B element of the Ii promoter-CAT plasmid abolished the TNF-alpha-mediated stimulatory effect. Comparison of the TNF-alpha-induced factor and lipopolysaccharide-induced NF-kappa B in gel mobility shift assays upon partial protease digestion suggests similar DNA-binding protein cores. Further support for the NF-kappa B-like nature of the TNF-alpha-induced factor was obtained in methylation interference assays. The TNF-alpha-induced nuclear factor comprises DNA contact sites that are identical to those described for NF-kappa B. This TNF-alpha-induced factor also interacts with kappa B-like sequences of the MHC Kb, Ek alpha, and beta 2-microglobulin promoter, suggesting a common TNF-alpha-mediated regulatory signal for expression of MHC antigens and Ii.

Tumor Necrosis Factor α Regulates Expression of the Major Histocompatibility Complex Class II-Associated Invariant Chain by Binding of an NF-κB-Like Factor to a Promoter Element

Expression of the major histocompatibility complex (MHC) class I and class II antigens and the class II-associated invariant chain (Ii) is strongly increased by treatment of cells with tumor necrosis factor alpha (TNF-alpha) and gamma interferon. We investigated elevation of expression of the invariant chain gene by TNF-alpha. Rat fibroblast cells transfected with the mouse Ii gene containing 802 base pairs of 5' sequences could be stimulated for Ii expression by treatment with TNF-alpha. Analysis of 5'-deleted Ii gene promoter-CAT constructs provided evidence for the presence of a TNF-alpha response box (TRB). Cloning of TRB in front of a non-TNF-alpha-responsive promoter could transfer the TNF-alpha stimulatory effect. We demonstrate binding of a TNF-alpha-induced factor to a kappa B-like motif within TRB. Mutations introduced into the kappa B element of the Ii promoter-CAT plasmid abolished the TNF-alpha-mediated stimulatory effect. Comparison of the TNF-alpha-induced factor and lipopolysaccharide-induced NF-kappa B in gel mobility shift assays upon partial protease digestion suggests similar DNA-binding protein cores. Further support for the NF-kappa B-like nature of the TNF-alpha-induced factor was obtained in methylation interference assays. The TNF-alpha-induced nuclear factor comprises DNA contact sites that are identical to those described for NF-kappa B. This TNF-alpha-induced factor also interacts with kappa B-like sequences of the MHC Kb, Ek alpha, and beta 2-microglobulin promoter, suggesting a common TNF-alpha-mediated regulatory signal for expression of MHC antigens and Ii.

Interferon-gamma, mitomycin C, and cycloheximide as regulatory agents of MHC class II-associated invariant chain expression.

The murine and human major histocompatibility complex class II-associated invariant chain genes are expressed in mature B cells and in antigen-presenting cells. Several pre-B cell lines and fibroblasts do not naturally contain invariant chain mRNA. Expression is inducible, however, by interferons and other agents interfering with proliferation. Mitomycin C induces the transcription of the gene in pre-B cells, but not in fibroblasts. Interferon-gamma acts in both types of cells. Cycloheximide inhibits the induction of the invariant chain mRNA by interferon-gamma, suggesting that protein synthesis is required. In fact, cycloheximide itself increases the transcriptional rate at the invariant chain gene, suggesting the existence of a labile repressor or an indirect action through cycloheximide arrest of the cell cycle. Lipopolysaccharide (LPS) activation of B lymphocytes causes a rapid decrease of the invariant chain mRNA level and of the amount of invariant chain protein due to rapid turnover. Also class II alpha and beta mRNA expression decreases after LPS treatment. The decrease of invariant chain protein is accompanied by increased surface expression of alpha and beta. The murine invariant chain gene transfected into human fibroblasts is regulated by the same agents and the same dose of agents as is the endogenous gene. The differentiation marker invariant chain thus seems to be transcribed from a gene that is accessible to regulation even in nonlymphoid cells and the expression of which is linked to states of nonproliferation. The sequence responsible for these responses is contained within the cloned genomic fragment and is conserved between mouse and man.