A role for calnexin (IP90) in the assembly of class II MHC molecules.

Major histocompatibility complex (MHC) class II antigens consist of alpha and beta chains that associate intracellularly with the invariant (I) chain. The HLA-DR alpha beta I complex assembles in the endoplasmic reticulum (ER) into a nonameric structure via progressive addition of three alpha beta dimers to a core invariant chain trimer. We have examined intracellular association of alpha beta I complexes with the resident ER protein calnexin. Calnexin associates rapidly (within 3 min) with newly synthesized alpha, beta and I chains, and remains associated with the assembling alpha beta I complex until the final alpha beta dimer is added, forming the complete nonamer. Dissociation of calnexin parallels egress of alpha beta I from the ER. These results suggest that calnexin retains and stabilizes both free class II subunits and partially assembled class II-I chain complexes until assembly of the nonamer is complete.     

Both alpha and beta chains of HLA-DC class II histocompatibility antigens display extensive polymorphism in their amino-terminal domains

At least three class II antigens, all composed of an alpha and a beta subunit, are encoded in the human major histocompatibility complex, i.e., DR, DC and SB. Two cDNA clones, encoding a DC alpha and a DC beta chain, respectively, were isolated from a cDNA library of the lymphoblastoid cell line Raji (DR3,w6). The two polypeptides predicted from the nucleotide sequences of these clones are each composed of a signal peptide, two extracellular domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Comparison of the DC alpha sequence with two previously published partial sequences shows that the majority of the differences is located in the amino-terminal domain. The differences are not randomly distributed; a cluster of replacements is present in the central portion of the amino-terminal domain. Likewise, the allelic polymorphism of the DC beta chains occurs preferentially in the amino-terminal domain, where three minor clusters of replacements can be discerned. The non-random distribution of the variability of DC alpha and beta chains may be due to phenotypic selection against replacement substitutions in the second domains of the polypeptides.     

Chemical cross-linking of Ia alloantigen alpha and beta chains with dimethyl 3,3'-dithiobispropionimidate.

We have examined the polypeptide chain composition of membrane-bound and detergent-solubilized Ia antigens using the chemical cross-linking reagent dimethyl 3,3'-dithiobispropionimidate (DTBP). Products of the I-E/C subregion of the major histocompatibility complex, which were solubilized from spleen cells with the detergent NP-40 and partially purified by affinity chromatography on lentil lectin-agarose, could be almost completely cross-linked by DTBP. Thus, the characteristic 33,000 m.v. (alpha) and 28,000 (beta) polypeptide chains seen on sodium dodecylsulfate polyacrylamide gels disappeared and a major new species of 60,000 m.w. appeared after cross-linking. When isolated and reduced with 2-mercaptoethanol, the 60,000 m.w. peak was found to be comprised to alpha and beta chains. Similar results were obtained when I-E/C, as well as I-A, alpha and beta chains were crosslinked on the cell surface. These data demonstrate that the alpha and beta chains of the Ia antigens exist primarily in the form of a dimer both in detergent solution and in situ.     

Translation and assembly of HLA-DR antigens in Xenopus oocytes injected with mRNA from a human B-cell line.

HLA-DR antigens are polymorphic cell surface glycoproteins, expressed primarily in B lymphocytes and macrophages, which are thought to play an important role in the immune response. Two polypeptide chains, alpha and beta, are associated at the cell surface, and a third chain associates with alpha and beta intracellularly. RNA isolated from the human B-cell line Raji was injected in Xenopus laevis oocytes. Immunoprecipitates of translation products with several monoclonal antibodies revealed the presence of HLA-DR antigens similar to those synthesized in Raji cells. One monoclonal antibody was able to bind the beta chain after dissociation of the three polypeptide chains with detergent. The presence of all three chains was confirmed by two-dimensional gel electrophoresis. The glycosylation pattern of the three chains was identical to that observed in vivo, as evidenced in studies using tunicamycin, an inhibitor of N-linked glycosylation. The presence of alpha chains assembled with beta chains in equimolar ratio was further demonstrated by amino-terminal sequencing. An RNA fraction enriched for the three mRNAs, encoding alpha, beta, and intracellular chains, was isolated. This translation-assembly system and the availability of monoclonal antibodies make it possible to assay for mRNA encoding specific molecules among the multiple human Ia-like antigens.     

Inhibition of endosomal proteolytic activity by leupeptin blocks surface expression of MHC class II molecules and their conversion to SDS resistance alpha beta heterodimers in endosomes.

The biosynthesis of MHC Class II molecules starts with the assembly of the alpha and beta subunits and the invariant chain. Intracellular transport of Class II molecules was followed in pulse-chase experiments of a human Epstein-Barr virus-transformed B lymphoblastoid cell line. Entry of Class II molecules into the endocytotic pathway and their cell surface appearance were monitored using neuraminidase as a fluid endocytotic marker and as a surface probe, respectively. In the course of intracellular transport, the Class II associated invariant chain is removed by proteases located in the endosomal pathway. Here, we show that leupeptin inhibits not only invariant chain breakdown, but also surface deposition of newly synthesized Class II molecules. Class II molecules display remarkable resistance to SDS at ambient temperature when occupied by peptide. We exploit this property to show that peptide binding precedes surface expression, and takes place in the course of intracellular transport through an endosomal compartment. Leupeptin blocks the conversion of Class II molecules to an SDS resistant complex.     

Coordinate induction of Ia alpha, beta, and Ii mRNA in a macrophage cell line

We demonstrated a tightly coordinated timing in the appearance of mRNA for the four class II (Ia) MHC chains, A alpha, A beta, E alpha, and E beta, and the Ia-associated invariant chain in a murine macrophage cell line after the addition of immune interferon (IFN-gamma) or of IFN-gamma-containing supernatants from Con A-stimulated spleen cells. The marked increase in mRNA levels for these molecules at approximately 8 hr after IFN-gamma addition contrasts sharply with the earlier, more gradual kinetics observed for class I (H-2) and beta 2-microglobulin mRNA. The difference in kinetics of IFN-gamma induction of class I and class II mRNA suggests differential regulation of the expression of Ia and H-2 antigens. The long lag period preceding detection of Ia mRNA raises the possibility that IFN-gamma may not directly mediate the increase in mRNA expression, but may act through an additional cellular intermediate.     

Separation of three class II antigens from a homozygous human B cell line

Three class II molecules were isolated from a homozygous DRw6 human B lymphoblastoid cell line using the monoclonal antibodies L243 (L203), L227, LKT 111, and Genox 3.53. Two of the antigens appeared to employ the same heavy chain but expressed different light chains. The two light chains were separated after denaturation using L227 and LKT 111. One or both of these two molecules carried the DRw6 and MT2 determinants. The third class II antigen expressed the DC1 determinant. It was composed of a heavy and light chain different from the DR-like antigen subunits. The antibodies L243, L227, and LKT 111 did not preclear the cell lysate of the DC1 antigen recognized by Genox 3.53. However, a xenoanti-DR serum immunoprecipitated both the DR-like and the DC1 antigens. Thus, in total, one cell line can express at least two class II heavy chains and three class II light chains. This observation was not unique to this cell line.     

Differential regulation of HLA-DR mRNAs and cell surface antigens by interferon

Human interferons-alpha, -beta and -gamma enhance HLA-DR mRNAs in all the human lymphoblastoid and melanoma cell lines studied. The increase concerns both alpha and beta chain mRNAs. Moreover, we show that immune interferon-gamma preferentially enhances class II MHC mRNA. This effect of IFN-gamma on the synthesis of alpha and beta HLA-DR chains has been also analysed by immunoprecipitation. It is abolished by a monoclonal antibody directed against human IFN-gamma. The effect of interferon on the cell surface level of HLA-DR molecules does not always correspond to the enhancement of HLA-DR mRNA. Our experiments suggest that this discrepancy between the enhancement of HLA-DR mRNA and cell surface antigen might be due to a constitutively high level of the corresponding antigens on several of the human cells studied.     

Assembly and transport properties of invariant chain trimers and HLA-DR-invariant chain complexes.

The MHC class II-associated invariant chain behaves as a resident endoplasmic reticulum protein in the absence of class II molecules. In humans, two predominant forms exist; one, p35, differs from the other, p33, by an N-terminal cytoplasmic extension of 16 amino acids that contains a strong endoplasmic reticulum-retention signal. Here we show that one mechanism for retention of p33 is its association with p35 in mixed invariant chain trimers. However, even for p33 homotrimers transport from the endoplasmic reticulum is inefficient. In an MHC class II-positive B cell line, the formation of invariant chain trimers is rapid and is the first intermediate in the assembly of a nine-chain alpha beta-invariant chain complex. With time, three higher molecular weight complexes are progressively formed. These correspond to an invariant chain trimer with one alpha beta dimer, two alpha beta dimers, and three alpha beta dimers, respectively. No free alpha beta dimers are detectable early in biosynthesis. However, beginning at 2 h of chase, alpha beta dimers begin to appear concomitant with the disappearance of the completely assembled alpha beta-invariant chain complex. This conversion is virtually complete by 4 h, and presumably reflects the proteolytic degradation of the invariant chain component of the alpha beta-invariant chain complex and the generation of endosomal alpha beta dimers capable of binding antigenic peptides.     

Invariant chain associates with HLA class II antigens via its extracytoplasmic region

Complexes of HLA class II alpha- and beta-chains with invariant chain were proteolytically digested to study domain interactions between these molecules. Detergent extracts of metabolically labeled monensin-treated B lymphoblastoid cells (B-LCL) were digested with proteinase K and immunoprecipitated with anti-HLA-DR or anti-invariant chain antibodies. Subsequent two-dimensional polyacrylamide amide gel electrophoresis showed that proteinase K treatment results in the sequential generation of three polypeptides of approximately 21,500, 19,500, and 18,000 daltons respectively. All are proteolytic fragments derived from invariant chain, and all remain associated with class II antigens. Two-dimensional gels of endoglycosidase H-treated immunoprecipitates showed that all three fragments contain two N-linked oligosaccharides. Neuraminidase treatment of immunoprecipitates and Bandeiraea simplicifolia lectin binding of cell extracts showed that the largest fragment, but not the smallest fragment, also contains O-linked oligosaccharides. None of the fragments possess the transmembrane region; fragments were released in soluble form when biosynthetically labeled B-LCL were ruptured by freezing and thawing and intact membranes were separated from aqueous components by ultracentrifugation. Lack of the transmembrane sequence was confirmed on the 18,000 dalton fragment by demonstrating through specific peptide cleavage at tryptophanyl residues that this fragment retains a substantial portion of the C-terminal region of I chain beyond trp162. Retention of the C-terminal region excludes the presence of the transmembrane region when m.w. are considered. Our data, taken in context of the amino acid sequence of the invariant chain predicted by the cDNA clone, demonstrate that invariant chain interacts with class II antigens via its extracytoplasmic region.     

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.     

Purification and partial amino acid sequence of papain-solubilized class II transplantation antigens

Papain-solubilized human class II (HLA-DR) antigens have been purified from cadaveric spleens by ion-exchange chromatography, gel chromatography, and immunosorbent purification. The isolated papain-solubilized antigens comprised two subunits with apparent molecular weights of 23 000 and 30 000, respectively. The circular dichroism spectrum for the isolated class II antigens was similar to spectra recorded for HLA-A, -B, and -C antigens, immunoglobulins, and immunoglobulin fragments. Thus, class II antigens contain a considerable amount of beta structure. The small subunit (beta chain) exhibited extensive charge heterogeneity on two-dimensional isoelectric focusing polyacrylamide gel electrophoresis, whereas the large subunit (alpha chain) was more homogeneous. The structural heterogeneity of beta chains remained after neuraminidase treatment. The NH2-terminal amino acid sequence of the beta chains displayed multiple residues in several positions in accordance with the genetic polymorphism displayed by this chain. The alpha chain also displayed multiple residues in some positions, suggesting either that some of the genetic polymorphism of the class II antigens may be endowed in this chain or that multiple loci control the expression of several alpha chains. Papain-solubilized class II antigen subunits were homologous in their amino acid sequences with HLA-DR antigens of defined antigenic specificity as well as with murine I-E/C antigens.     

Isolation of chicken major histocompatibility complex class II (B-L) beta chain sequences: comparison with mammalian beta chains and expression in lymphoid organs

By cross-hybridization in low stringency conditions, using a probe derived from an HLA-DQ beta cDNA clone, we have isolated several chicken genomic DNA clones. These clones were mapped to the major histocompatibility complex (MHC) of the chick (B complex) by virtue of their ability to detect restriction enzyme length polymorphisms between congenic lines of chicken. Evidence was obtained for the presence of at least three B-L beta genes in the chicken genome. The B-L beta genes are transcribed specifically in tissues containing cells of the B lymphocyte and myeloid lineages and expressing the B-L antigens. Exons encoding the beta 1, beta 2 and transmembrane domains of a B-L beta chain have been identified with 63, 66 and 62% similarity with the HLA-DQ beta sequence. This first isolation of an MHC class II gene outside of the mammalian class provides insight into the evolution of MHC genes based on the comparison of avian and mammalian class II beta chain amino acid and nucleotide sequences.     

Biosynthetic intermediates of HLA class II antigens from B lymphoblastoid cell lines

Detergent extracts of B lymphoblastoid cell lines (B-LCL) were subjected to immunoaffinity chromatography and gel filtration to purify HLA class II antigens. Class II antigens purified from B-LCL cultured for 24 hr in 10 microM monensin, in which glycoproteins are trapped in transit through the Golgi apparatus, exist in a large macromolecular complex composed of the alpha- and beta-subunits of class II molecules associated with the invariant (I) chain and a sulfated macromolecule that appears to be a proteoglycan. Gel filtration experiments on Sephacryl S-300 reveal that the complex has a Stokes radius corresponding to a globular protein of approximately 270,000 m.w. Analysis of radiolabeled preparations by two-dimensional gel electrophoresis suggests that the complex contains the alpha-, the beta-, and I chain subunits in a 1:1:1 ratio. Dissociation of the protein components followed by gel filtration of the proteoglycan indicates that the proteoglycan contributes approximately 180,000 m.w. to the complex. These results suggest that the complex contains one copy each of the alpha-, the beta-, and the I subunits associated with a proteoglycan molecule. This complex appears to represent a biosynthetic intermediate in the expression of class II molecules which is induced to accumulate intracellularly by monensin treatment of B-LCL.     

Biochemical characterization of Ia antigens. I. Characterization of the 31K polypeptide associated with I-A subregion Ia antigens

Procedures are presented for the preparative isolation of murine Ia antigens directly from splenocyte detergent extracts with monoclonal immunoadsorbents. Utilizing these procedures, three Ia (I-A subregion) polypeptides (alpha, 31K, beta) were isolated and their m.w. and pI values characterized. Evidence is presented that indicates that: 1) the 31K polypeptide probably does not associate with the Ia alpha and beta chain complex during the Ia isolation procedure; 2) the 31K polypeptide is not tightly bound to the alpha/beta Ia complex and can be selectively removed by freezing and thawing and by washing the Ia-immunoadsorbent with buffers containing pyrrolidinone (a polar solvent); and (3) unlike the alpha and beta chains, the 31K polypeptide is not intrinsically radiolabeled with 3H fucose and 3H glucosamine, indicating that the 31K polypeptide either contains a carbohydrate structure that is different from that of the alpha and beta chains or it is not a glycopeptide. These data suggest that although Ia antigens are probably comprised of three polypeptides in the intact cell, only two (alpha and beta) are required to maintain alloantigenic determinants.     

Separation of four class II molecules from DR2 and DRw6 homozygous B lymphoid cell lines

Four human class 11 molecules, one FA, one DC1, and two DR-like molecules, were isolated from DR2 and DRw6 homozygous cell lines by means of a variety of monoclonal antibodies and were compared with each other by two-dimensional (2-D) gel electrophoresis. Anti-DR2 or anti-DR3 + 5 + w6 sera immunoprecipitated two distinct light chains (L1 and L2) and one heavy chain (H1) from a DR2 or DRw6 homozygous cell line, respectively. One or both of these two class II molecules were also immunoprecipitated by DR-specific monoclonal antibodies and were considered to constitute a DR family of molecules. Three DC1-specific monoclonal antibodies, SDR4.1, Tu22, and PLM5, immunoprecipitated a set of heavy (H2) and light (L3) chains distinct from those of two DR-like molecules. The heavy chains of the DC1 antigens from DR2 and DRw6 cell lines were indistinguishable, whereas the light chains were structurally distinct from each other. A fourth molecule, FA, was identified by a novel monoclonal antibody and was also detected by two additional antibodies, Tu39 and SG171, that blocked the SB-specific T-cell proliferative response. The FA light chain (L4) was distinct from those of the former three antigens on both cell lines, whereas the FA heavy chain was indistinguishable from the DC1 heavy chain (H2) in this 2-D gel analysis. Thus, four light chains and two heavy chains were isolated from both DR2 and DRw6 homozygous cell lines. A possible gene-antigen organization of the DC-like antigens was also discussed.     

Structural comparison of I-A antigens produced by a cloned murine T suppressor cell line with B-Cell-Derived I-A

A cloned, antigen-specific T suppressor cell line derived from a CBA mouse expresses large amounts of I-A and I-E antigens. Comparative two-dimensional polyacrylamid gel electrophoresis of biosynthetically labeled I-A antigens immunoprecipitated with a variety of monoclonal I-Ak-specific antibodies suggested that alpha, beta and Ii polypeptide chains are identical with B-cell-derived I-A. Dimeric complexes formed by I-A chains derived from B or T suppressor cells were also similar with two major exceptions. Pulse-labeled T-cell-derived Ia antigen was complexed with two additional unknown components of about 31K. These components were not visible in pulse-chased (processed) materials. In addition, T suppressor-cell-derived I-A antigens did not contain S-S linked dimers consisting of processed alpha and beta chains, which are usually formed during solubilization of B cells. We consider the possibility that in T cells these chains are associated with other structures, thus preventing S-S linkage between alpha and beta chains.     

Endocytosis of interleukin 2 receptors in human T lymphocytes: distinct intracellular localization and fate of the receptor alpha, beta, and gamma chains

Members of the cytokine receptor family are composed of several noncovalently linked chains with sequence and structure homologies in their extracellular domain. Receptor subfamily members share at least one component: thus the receptors for interleukin (IL) 2 and IL15 have common beta and gamma chains, while those for IL2, 4, 7, and 9 have a common gamma chain. The intracellular pathway followed by IL2 receptors after ligand binding and endocytosis was analyzed by immunofluorescence and confocal microscopy in a human T lymphocytic cell line. Surprisingly, the alpha, beta, and gamma chains had different intracellular localizations after being endocytosed together. The alpha chain was always in transferrin-positive compartments (early/recycling endosomes), both at early and late internalization times, but was never detected in rab7-positive compartments (late endosomes). On the other hand, at late internalization times, the beta and gamma chains were excluded from transferrin-positive organelles and did not colocalize with alpha. Furthermore, beta could be found in rab7-positive vesicles. These differences suggest that the alpha chain recycles to the plasma membrane, while the beta and gamma chains are sorted towards the degradation pathway. The half-lives of these three chains on the cell surface also reflect their different intracellular fates after endocytosis. The beta and gamma chains are very short-lived polypeptides since their half-life on the surface is only approximately 1 h, whereas alpha is a much more stable surface protein. This shows for the first time that components of a multimeric receptor can be sorted separately along the endocytic pathway.     

Peptide and beta 2-microglobulin regulation of cell surface MHC class I conformation and expression.

We have examined the roles of peptide and beta 2-microglobulin (beta 2m) in regulating the conformation and expression level of class I molecules on the cell surface. Using a cell line synthesizing H-2Dd H chain and mouse beta 2m but defective in endogenous peptide loading, we demonstrate the ability of either exogenous peptide or beta 2m alone to increase surface H-2Dd expression at both 25 degrees C and 37 degrees C. Peptide and beta 2m show marked synergy in their abilities to increase surface class I expression, with minimal increases promoted by peptide in the absence of free beta 2m. Low temperature-induced molecules have indistinguishable rates of loss of beta 2m and alpha 1/alpha 2 domain conformational epitopes during culture at 37 degrees C. However, the rate of alpha 3 epitope loss is much slower, indicating a minimum of two steps in class I loss from the cell surface: 1) loss of beta 2m binding to H chain and unfolding of the alpha 1/alpha 2 region; then 2) denaturation, degradation, or internalization of the free H chains possessing alpha 3 epitopes. These data show for the first time that free H chains survive for a finite time on the membrane in a form capable of refolding into alpha 1/alpha 2 epitope positive molecules upon addition of beta 2m and peptide. This refolding in the presence of beta 2m and peptide can explain the reported requirement for both components in sensitizing cells for class I-dependent CTL lysis. It also indicates that such conformational changes in class I molecules are not strictly dependent on either newly synthesized H chains or on intracellular chaperons. The study of H chain-peptide-beta 2m interaction on the cell surface may be relevant to understanding intracellular peptide loading events.     

Cloning,expression, and crystallization of the V delta domain of a human gamma delta T-cell receptor.

T-lymphocytes recognize a wide variety of antigens through highly diverse cell-surface glycoproteins known as T-cell receptors (TCRs). These disulfide-linked heterodimers are composed of alpha and beta or gamma and delta polypeptide chains consisting of variable (V) and constant (C) domains non-covalently associated with at least four invariant chains to form the TCR-CD3 complex. It is well established that alpha beta TCRs recognize antigen in the form of peptides bound to molecules of the major histocompatibility complex (MHC); furthermore, information on the three-dimensional structure of alpha beta TCRs has recently become available through X-ray crystallography. In contrast, the antigen specificity of gamma delta TCRs is much less well understood and their three-dimensional structure is unknown. We have cloned the delta chain of a human TCR specific for the MHC class I HLA-A2 molecule and expressed the V domain as a secreted protein in the periplasmic space of Escherichia coli. Following affinity purification using a nickel chelate adsorbent, the recombinant V delta domain was crystallized in a form suitable for X-ray diffraction analysis. The crystals are orthorhombic, space group P2(1)2(1)2 with unit cell dimensions a = 69.9, b = 49.0, c = 61.6 A. and diffract to beyond 2.3 A resolution. The ability of a V delta domain produced in bacteria to form well-ordered crystals strongly suggests that the periplasmic space can provide a suitable environment for the correct in vivo folding of gamma delta TCRs.