Type analysis of oligosaccharide chains on human and murine MHC class II alpha chains by the lectin-nitrocellulose sheet method

1. The microheterogeneous alpha molecules of class II antigen, DR molecules obtained from human B cell line and I-A molecules from mouse B cell hybridoma cell line, were separated by 2-D PAGE, transferred onto NC sheets and N-linked oligosaccharide types were analyzed by staining with P.O./lectins. 2. This is the first report to show directly the type of oligosaccharide chain corresponding to each spot separated by 2-D PAGE. The glycosylation patterns of class II alpha chains in human and mouse were compared.     

Type analysis of the oligosaccharide chains on microheterogeneous components of bovine pancreatic DNAase by the lectin-nitrocellulose sheet method.

The oligosaccharide chains of microheterogeneous bovine pancreatic DNAases were characterized by the lectin-nitrocellulose sheet method. The active fractions of the DNAases from column chromatography showed four major and several minor spots on a two-dimensional polyacrylamide gel. They were transferred on to nitrocellulose sheets and treated with glycosidases (neuraminidase, endo-beta-N-acetyl glucosaminidase H or F, or peptide N-glycosidase F) and treated with peroxidase-coupled lectins (concanavalin A, Ricinus communis agglutinin or wheat-germ agglutinin). From the results, the most probable oligosaccharide types were proposed to be as follows: the four major spots contained components which had high-mannose type or hybrid-type oligosaccharides, such as those susceptible to endo-beta-N-acetylglucosaminidase H. In addition, spot 1 contained a complex-type biantennary oligosaccharide without sialic acid and spot 3 contained a tri- or tetra-antennary complex-type oligosaccharide with sialic acid. The component corresponding to spot 2 had a hybrid-type oligosaccharide chain with a 'bisecting' acetylglucosamine, linked 1-4 to the beta-mannose residue of the trimannosyl core, and the component corresponding to spot 4 had a high-mannose-type oligosaccharide chain.     

Determinants of the peptide-induced conformational change in the human class II major histocompatibility complex protein HLA-DR1

The human class II major histocompatibility complex protein HLA-DR1 has been shown previously to undergo a distinct conformational change from an open to a compact form upon binding peptide. To investigate the role of peptide in triggering the conformational change, the minimal requirements for inducing the compact conformation were determined. Peptides as short as two and four residues, which occupy only a small fraction of the peptide-binding cleft, were able to induce the conformational change. A mutant HLA-DR1 protein with a substitution in the beta subunit designed to fill the P1 pocket from within the protein (Gly(86) to Tyr) adopted to a large extent the compact, peptide-bound conformation. Interactions important in stabilizing the compact conformation are shown to be distinct from those responsible for high affinity binding or for stabilization of the complex against thermal denaturation. The results suggest that occupancy of the P1 pocket is responsible for partial conversion to the compact form but that both side chain and main chain interactions contribute to the full conformational change. The implications of the conformational change to intracellular antigen loading and presentation are discussed.     

Molecular mapping class II polymorphisms in the human major histocompatibility complex. II. DQ beta

The restriction fragment length polymorphisms have been determined for six restriction enzymes (Bam HI, Bg1 II, Eco RI, Hinc II, Hind III, and Pvu II) and a DQ beta probe on 25 cell lines that are homozygous by consanguinuity at the MHC. These patterns reflect both DR haplotypes and DQ types of the cells tested. At least one non-polymorphic band is present in all the cell lines with every restriction enzyme except Hinc II. This band most probably represents DX beta hybridization. The polymorphic bands indicate that more polymorphism exists in the DQ subregion than is predicted serologically. Each DR haplotype is associated with a unique set of restriction fragments except for DR2 and DR6. The patterns are largely consistent within each DR haplotype. In addition, some bands reflect the established DQ specificities DQw1 and DQw2. Individual bands can be identified that are unique to the haplotypes DR1, DR4, DR5, and DR6 and the DQw1- and DQw2-associated haplotypes. Subdivisions of haplotypes can be identified with this probe. In particular, MVL (DR1), Akiba (DR2), QBL (DR3), FPF (DR5), and APD (DR6) have polymorphisms that distinguish them from other members of their DR haplotype.     

Asparagine deamidation perturbs antigen presentation on class II major histocompatibility complex molecules

Post-translational protein modifications can be recognized by B and T lymphocytes and can potentially make "self"-proteins appear foreign to the immune system. Such modifications may directly affect major histocompatibility complex-restricted T cell recognition of processed peptides or may perturb the processing events that generate such peptides. Using the tetanus toxin C fragment protein as a test case, we show that spontaneous deamidation of asparagine residues interferes with processing by the enzyme asparagine endopeptidase (AEP) and contributes to diminished antigen presentation. Deamidation inhibits AEP action either directly, when asparagine residues targeted by AEP are modified, or indirectly, when adjacent Asn residues are deamidated. Thus, deamidation of long-lived self-proteins may qualitatively or quantitatively affect the spectrum of self-peptides displayed to T cells and may thereby contribute to the onset or exacerbation of autoimmune disease.     

Inhibition of HLA-DR assembly,transport, and loading by human cytomegalovirus glycoprotein US3: a novel mechanism for evading major histocompatibility complex class II antigen presentation

Human cytomegalovirus (HCMV) establishes persistent lifelong infections and replicates slowly. To withstand robust immunity, HCMV utilizes numerous immune evasion strategies. The HCMV gene cassette encoding US2 to US11 encodes four homologous glycoproteins, US2, US3, US6, and US11, that inhibit the major histocompatibility complex class I (MHC-I) antigen presentation pathway, probably inhibiting recognition by CD8(+) T lymphocytes. US2 also inhibits the MHC-II antigen presentation pathway, causing degradation of human leukocyte antigen (HLA)-DR-alpha and -DM-alpha and preventing recognition by CD4(+) T cells. We investigated the effects of seven of the US2 to US11 glycoproteins on the MHC-II pathway. Each of the glycoproteins was expressed by using replication-defective adenovirus vectors. In addition to US2, US3 inhibited recognition of antigen by CD4(+) T cells by a novel mechanism. US3 bound to class II alpha/beta complexes in the endoplasmic reticulum (ER), reducing their association with Ii. Class II molecules moved normally from the ER to the Golgi apparatus in US3-expressing cells but were not sorted efficiently to the class II loading compartment. As a consequence, formation of peptide-loaded class II complexes was reduced. We concluded that US3 and US2 can collaborate to inhibit class II-mediated presentation of endogenous HCMV antigens to CD4(+) T cells, allowing virus-infected cells to resist recognition by CD4(+) T cells.     

Molecular mapping of class II polymorphisms in the human major histocompatibility complex. I. DR beta

We have studied 27 cell lines homozygous by consanguinity for the major histocompatibility complex to establish the restriction fragment length polymorphism (RFLP) patterns seen with six different restriction enzymes (Bam HI, Bg1 II, Eco RI, Hinc II, Hind III, Pvu II) and DR beta chain probes. The probes used were a full-length cDNA DR beta probe and a probe specific for the 3' untranslated region. The RFLP obtained represent the first standard patterns for the individual haplotypes DR1 through 7 and DR9 as defined by genetically homozygous lines. The patterns obtained reflect the DR specificities closely, as well as the DRw52 and DRw53 specificities. These latter specificities are associated with the most prominent patterns of RFLP. Bands are present which are unique for the haplotypes DR1, DR2, DR4, DR7, DRw52, and DRw53, and could be used for typing these haplotypes in heterozygotes. Subtypes can be identified for all of the haplotypes except DR1. These subtypes indicate that there is an extensive amount of polymorphism in the DR subregion that has not been identified serologically.     

Domain structures of cell surface glycopeptides encoded by class I and class II beta genes of the major histocompatibility complex

Published sequence data of MHC genes, cDNAs and MHC products were analyzed for their sequence homologies. Alignment statistics revealed that class I gene products consist of four mutually homologous domains, and that class II beta gene products is composed of three mutually homologous domains. Not only extracellular domains but also newly discovered C-terminal shorter domains of class I and class II beta gene products were found to have evolved from a one-domain-long beta 2-microglobulin-like protein by repeated exon duplications and splittings.     

New genes in the class II region of the human major histocompatibility complex

A detailed map of the class II region of the human major histocompatibility complex has been constructed by pulsed-field gel electrophoresis. This map revealed clusters of sites for enzymes that cut preferentially in unmethylated CpG-rich DNA often found at the 5' ends of genes. Three of these clusters have been cloned by cosmid walking and chromosome jumping. Analysis of the clones encompassing these regions through the use of zoo blots, Northern blots, and cDNA libraries resulted in the discovery of four novel genes. The D6S111E and D6S112E genes are centromeric to the HLA-DPB2 gene, while D6S113E and D6S114E are between HLA-DNA and HLA-DOB. Preliminary characterization of the new genes indicates that they are unrelated to the class II genes themselves, although D6S114E expression, like class II expression, is inducible with interferon. In addition, the HLA-DNA gene has been accurately positioned and oriented for the first time.     

Acidification of internalized class I major histocompatibility complex antigen by T lymphoblasts

It has previously been shown that activated murine T lymphocytes express intracellular vesicles containing the class I major histocompatibility complex (MHC) antigen H-2K. Evidence has also been provided that such vesicles may be part of a cellular pathway of spontaneous H-2K antigen internalization and recycling, which is specific to T-lymphoid cells. Dual fluorescence flow cytometry has now been used to establish that H-2K antigen is acidified upon internalization in concanavalin A-stimulated but not lipopolysaccharide-stimulated murine splenocytes, thus providing further support that in T lymphoblasts this class I MHC antigen may travel intracellular routes similar to those reported for other cell surface receptors.     

Cloning of the bovine major histocompatibility complex class II genes

Summary. Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage Λ vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Cloning of the bovine major histocompatibility complex class II genes

Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.     

Class II genes of the human major histocompatibility complex. The DO beta gene is a divergent member of the class II beta gene family

A novel class II beta chain gene is described. This gene, tentatively called DO beta, displays considerably less polymorphism than beta genes of the DP, DQ, and DR loci. The nucleotide sequence of the DO beta gene is strikingly similar to that of the previously identified murine A beta 2 gene. The DO beta gene displays the same exon/intron organization as other beta genes although the fifth exon and the translated portion of the sixth exon are longer than in other genes. A striking feature of the amino acid sequence deduced from the DO beta gene sequence is the pronounced hydrophobicity of the NH2-terminal region. This feature distinguishes the putative DO beta chain from other class II beta chains and raises the possibility that DO beta chains may interact with an alpha chain that is structurally different from those of the DP, DQ, and DR loci. It further suggests that the putative DO molecule may have a function different from those of other class II antigens.     

A reductionist cell-free major histocompatibility complex class II antigen processing system identifies immunodominant epitopes

Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4(+) T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: human leukocyte antigen-DR1 (HLA-DR1), HLA-DM and cathepsins. Our reductionist system successfully identified the physiologically selected immunodominant epitopes of two model antigens: hemagglutinin-1 (HA1) from influenza virus (A/Texas/1/77) and type II collagen (CII). When applied for identification of new epitopes from a recombinant liver-stage antigen of malaria falciparum (LSA-NRC) or HA1 from H5N1 influenza virus ('avian flu'), the system selected single epitopes from each protein that were confirmed to be immunodominant by their capacity to activate CD4(+) T cells from H5N1-immunized HLA-DR1-transgenic mice and LSA-NRC-vaccinated HLA-DR1-positive human volunteers. Thus, we provide a new tool for the identification of physiologically relevant helper T cell epitopes from 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.     

Involvement of major histocompatibility complex class II antigen in Epstein-Barr virus-mediated B cell proliferation.

Five MHC class II monoclonal antibodies costimulated proliferation of cord blood leukocytes with Epstein-Barr virus. These agonistic antibodies were of different isotypes, but all of them were either specific for or cross-reacting with HLA-DR. The other MHC class II antibodies, including three that were specific for HLA-DQ and one that was specific for HLA-DP and also those that were specific for MHC class I or leukocyte common antigen, were not costimulatory. The agonistic effect of different MHC class II antibodies was additive, such that costimulation by different antibodies combined significantly exceeded that achieved by either of these antibodies alone. Spent culture media of B cell lines also costimulated B cell proliferation with the virus. Although MHC class II antibodies augmented the effects of suboptimal concentration of the conditioned media, their combined effects did not exceed the maximum costimulation achieved by either the antibodies or the spent culture media alone. These results raised the possibility that MHC class II antigen may contain distinct functional domains involved in the regulation of B cell progression.