The class I α1,2-mannosidases of Caenorhabditis elegans

During the biosynthesis of N-glycans in multicellular eukaryotes, glycans with the compositions Man(5)GlcNAc(2-3) are key intermediates. However, to reach this 'decision point', these N-glycans are first processed from Glc(3)Man(9)GlcNAc(2) through to Man(5)GlcNAc(2) by a number of glycosidases, whereby up to four α1-2-linked mannose residues are removed by class I mannosidases (glycohydrolase family 47). Whereas in the yeast Saccharomyces cerevisiae there are maximally three members of this protein family, in higher organisms there are multiple class I mannosidases residing in the endoplasmic reticulum and Golgi apparatus. The genome of the model nematode Caenorhabditis elegans encodes seven members of this protein family, whereby four are predicted to be classical processing mannosidases and three are related proteins with roles in quality control. In this study, cDNAs encoding the four predicted mannosidases were cloned and expressed in Pichia pastoris and the activity of these enzymes, designated MANS-1, MANS-2, MANS-3 and MANS-4, was verified. The first two can, dependent on the incubation time, remove three to four residues from Man(9)GlcNAc(2), whereas the action of the other two results in the appearance of the B isomer of Man(8)GlcNAc(2); together the complementary activities of these enzymes result in processing to Man(5)GlcNAc(2). With these data, another gap is closed in our understanding of the N-glycan biosynthesis pathway of the nematode worm.     

A class of carbonic anhydrase I – selective activators

A series of ureido and bis-ureido derivatives were prepared by reacting histamine with alkyl/aryl-isocyanates or di-isocyanates. The obtained derivatives were assayed as activators of the enzyme carbonic anhydrase (CA, EC 4.2.1.1), due to the fact that histamine itself has this biological activity. Although inhibition of CAs has pharmacological applications in the field of antiglaucoma, anticonvulsant, anticancer, and anti-infective agents, activation of these enzymes is not yet properly exploited pharmacologically for cognitive enhancement or Alzheimer’s disease treatment, conditions in which a diminished CA activity was reported. The ureido/bis-ureido histamine derivatives investigated here showed activating effects only against the cytosolic human (h) isoform hCA I, having no effect on the widespread, physiologically dominant isoform hCA II. This is the first report in which CA I-selective activators were identified. Such compounds may constitute interesting tools for better understanding the physiological/pharmacological effects connected to activation of this widespread CA isoform, whose physiological function is not fully understood.     

Nonlinkage of major histocompatibility complex class I and class II loci in bony fishes

 In tetrapods, the functional (classical) class I and class II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical class I and class II B loci are in separate linkage groups, and that in at least some of these taxa, the class II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the class I and class II regions are not linked. Received: 30 August 1999 / Revised: 20 October 1999     

Transformation of LMTK− cells with purified HLA class I genes

The expression of two different HLA class I genes was observed after transformation of LMTK- cells. The corresponding class I molecules reacted differentially with monomorphic monoclonal antibodies (m.Ab). Absorption and elution studies of the human alloantibodies reacting with the transformed cells and cellular radioimmunoassay of these cells with polymorphic m.Ab resulted in the identification of HLA-A3 and CW3 molecules. These transformed cells were used to immunize C3H mice and induce the production of xenogeneic antisera, which, following absorption, showed polymorphic reactivity with human cells, suggesting that some of these sera could be used as typing reagents.     

Evidence for secretion of vacuolar α-mannosidase, class I chitinase, and class I β-1,3-glucanase in suspension cultures of tobacco cells

We have investigated the possibility that vacuolar proteins can be secreted into the medium of cultured cells of Nicotiana tabacum L. Time-course and balance-sheet experiments showed that a large fraction, up to ca. 19%, of vacuolar α-mannosidase (EC 3.2.1.24) and vacuolar class I chitinase (EC 3.2.1.14) in suspension cultures accumulated in the medium within one week after subculturing. This effect was most pronounced in media containing 2,4-dichlorophenoxyacetic acid (2,4-D). Under comparable conditions only a small fraction, 1.8–5.1% of the total protein and ca. 1% of malate dehydrogenase (EC 1.1.1.37), which is localized primarily in the mitochondria and cytoplasm, accumulated in the medium. Pulse-chase experiments showed that newly synthesized vacuolar class I isoforms of chitinase and β-1,3-glucanase (EC 3.2.1.39) were released into the medium. Post-translational processing, but not the release of these proteins, was delayed by the secretion inhibitor brefeldin A. Only forms of the proteins present in the vacuole, i.e. mature chitinase and pro-β-1,3-glucanase and mature β-1,3-glucanase, were chased into the medium of tobacco cell-suspension cultures. Our results provide strong evidence that vacuolar α-mannosidase, chitinase and β-1,3-glucanase can be secreted into the medium. They also suggest that secretion of chitinase and β-1,3-glucanase might be via a novel pathway in which the proteins pass through the vacuolar compartment. Received: 3 September 1997 / Accepted: 30 October 1997     

The β2-microglobulin-free heterodimerization of rhesus monkey MHC class I A with its normally spliced variant reduces the ubiquitin-dependent degradation of MHC class I A

The MHC class I (MHC I) molecules play a pivotal role in the regulation of immune responses by presenting antigenic peptides to CTLs and by regulating cytolytic activities of NK cells. In this article, we show that MHC I A in rhesus macaques can be alternatively spliced, generating a novel MHC I A isoform (termed "MHC I A-sv1") devoid of α(3) domain. Despite the absence of β2-microglobulin (β2m), the MHC I A-sv1 proteins reached the cell surface of K562-transfected cells as endoglycosidase H-sensitive glycoproteins that could form disulfide-bonded homodimers. Cycloheximide-based protein chase experiments showed that the MHC I A-sv1 proteins were more stable than the full-length MHC I A in transiently or stably transfected cell lines. Of particular interest, our studies demonstrated that MHC I A-sv1 could form β2m-free heterodimers with its full-length protein in mammalian cells. The formation of heterodimers was accompanied by a reduction in full-length MHC I A ubiquitination and consequent stabilization of the protein. Taken together, these results demonstrated that MHC I A-sv1 and MHC I A can form a novel heterodimeric complex as a result of the displacement of β2m and illustrated the relevance of regulated MHC I A protein degradation in the β2m-free heterodimerization-dependent control, which may have some implications for the MHC I A splice variant in the fine tuning of classical MHC I A/TCR and MHC I A/killer cell Ig-like receptor interactions.     

Expression of HLA class I molecules assembled with structural variants of human β2-microglobulin

 Mouse and human β₂-microglobulin (β₂m), which differ by 30% in their primary sequence, give rise to disparate levels of HLA class I heavy chain expression in transfectants of the β₂m-null FO-1 human melanoma cell line, i.e., mouse β₂m directs expression of HLA class I heavy chains that is only ∼20%–30% of that observed for heavy chains assembled with human β₂m. In this report we describe our efforts to better understand the structural basis of this regulatory phenomenon. Initial insight into the importance of the N-terminus of β₂m on HLA expression came from studies with FO-1 cells transfected with chimeric (human X mouse) B2m genes. Chimeric β₂m molecules containing residues 1–69 from human β₂m and residues 70–99 from mouse β₂m (designated HM- β₂m) induced expression of HLA heavy chains to a significantly greater extent (∼80% of level observed with cognate β₂m) than the reverse chimeric construct (designated MH- β₂m) (10%–15% of level observed with cognate β₂m). These data are consistent with the view that the major determinants of HLA class I heavy chain expression map to the portion of the β₂m molecule which forms the four-stranded β-pleated sheet, comprised of S1, S2, S4, and S5, and one strand of the three-stranded sheet (S3). The mapping of class I regulatory sites to the portion of β₂m containing the four-stranded β-pleated sheet supports the interpretation that the heavy chain contact residues on β₂m play the major role in regulating major histocompatibility (MHC) class I expression. To further dissect β₂m-mediated regulation of HLA class I expression, site-directed mutants of β₂m were prepared by conversion of human β₂m to the mouse sequence at individual amino acid positions within the four-stranded and three-stranded β-pleated sheets. Human to mouse amino acid substitutions were made in each divergent residue between positions 1–66, and as controls for COOH-terminal modification, several residues between positions 75 and 94. Cytofluorometry with HLA class I-specific antibodies indicated that cell surface expression of HLA class I heavy chains was largely insensitive to each of the individual substitutions. It is concluded that a combination of divergent residues mapping to positions of heavy chain contact are responsible for the differences observed in MHC class I expression by heterologous forms of β₂m. Received: 18 March 1997 / Revision: 21 April 1997     

Transfection of murine LMTK− cells with purified HLA class I genes

The individual contributions of the first two external domains of the HLA-B7 heavy chain to the expression of allele-specific (B7) and locus-specific (B and C) antigenic determinants were investigated using hybrid class I genes. Hybrid genes were constructed in vitro by exon shuffling between the parent genes HLA-B7, HLA-Cw3, HLA-A3, and H-2K ^d, and their expression was monitored following transfection into mouse L cells. The results show that most allele-specific antigenic determinants are associated with the first external domain of the 137 heavy chain, whereas all the locus-specific antigenic determinants tested map to the second external domain.Abbreviations used in this paper BSA bovine serum albumin - FCS fetal calf serum - mAb monoclonal antibody - PBL peripheral blood lymphocytes - PBS phosphate-buffered saline     

Characterization of a divergent non-classical MHC class I gene in sharks

Sharks are the most ancient group of vertebrates known to possess members of the major histocompatibility complex (MHC) gene family. For this reason, sharks provide a unique opportunity to gain insight into the evolution of the vertebrate immune system through comparative analysis. Two genes encoding proteins related to the MHC class I gene family were isolated from splenic cDNA derived from spiny dogfish shark ( Squalus acanthias). The genes have been designated MhcSqac-UAA*01 and MhcSqac-UAA*NC1. Comparative analysis demonstrates that the Sqac-UAA*01 protein sequence clusters with classical MHC class I of several shark species and has structural elements common to most classical MHC class I molecules. In contrast, Sqac-UAA*NC1 is highly divergent from all vertebrate classical MHC class I proteins, including the Sqac-UAA *01 sequence and those of other shark species. Although Sqac-UAA*NC1 is clearly related to the MHC class I gene family, no orthologous genes from other species were identified due to the high degree of sequence divergence. In fact, the Sqac NC1 protein sequence is the most divergent MHC class-I-like protein identified thus far in any shark species. This high degree of divergence is similar in magnitude to some of the MHC class-I-related genes found in mammals, such as MICA or CD1. These data support the existence of a class of highly divergent non-classical MHC class I genes in the most primitive vertebrates known to possess homologues of the MHC and other components of the adaptive immune system.     

Genomic analysis of common chimpanzee major histocompatibility complex class I genes

To investigate how MHC class I genes have changed in the approximately 5 million years since chimpanzees and humans diverged, we characterized six genomic fragments ranging in size from 5.1 to 6.1 kb, each containing the complete coding region, introns, and flanking regions of one of the following chimpanzee class I genes: Patr-A, Patr-E, Patr-F, Patr-G, Patr-H, and Patr-J. In humans, these genes are closely linked within the class I region and are representatives of three distinct functional categories of class I genes: the highly polymorphic Ia genes (HLA-A), the conserved Ib genes (HLA-E, HLA-F, and HLA-G), and the class I pseudogenes (HLA-H and HLA-J). Southern blot analysis of chimpanzee and human class I genes produced nearly identical patterns, suggesting that the organization and linkage of these genes differs little in the two species. Comparison of the chimpanzee fragment sequences with their human orthologues revealed structural conservation of these genes yet differences in their degree of functional constraint. This is apparent in the location and nature of the amino acid changes between species and the substantial differences in levels of divergence at functional and nonfunctional sites. Additionally, there is no correlation between patterns of divergence at these sites and intraspecific variation, an observation explained by either appreciable gene conversion or high levels of recombination, the latter unlikely given the observed strong linkage disequilibrium of these loci.     

Physical mapping and evolution of the centromeric class I gene-containing region of the rat MHC

We physically mapped the centromeric part of the BN rat MHC (RT1n haplotype) in a contig of overlapping P1-derived artificial chromosome (PAC) clones encompassing about 300 kb. The following genes were identified and ordered as: (Syngap, Hset, Daxx, Bing1)-Tapbp-Rgl2-Ke2-Bing4-B3galt4- Rps18-Sacm2l-RT1-A1-RT1-A2-RT1-A3-Ring1-Ring2-++ +Ke4-Rxrb-Col11a2-RT1-Hb-Ring3-RT1-DMb. Thus, in contrast to other RT1 haplotypes, RT1n contains three class I genes, RT1-A1, RT1-A2, and RT1-A3, mapping between the Sacm2l and Ring1 genes. Comparisons of the sequences flanking the Sacm2L and Ring1 genes in rat, human, and mouse suggest that the class I gene-containing region was inserted between these genes in rat and mouse at a similar position. Thus, this insertion is likely to have occurred in a common ancestor of these rodents, although the presence of a site particularly permissive for insertions cannot be excluded.     

Characterization of the MHC class I region of the Japanese pufferfish (Fugu rubripes)

A BAC map of the Japanese pufferfish (Fugu) MHC class I region was constructed using a mixture of sequence scanning and sequence-tagged site mapping methodologies. The Fugu MHC class Ia genes are linked to genes which are found within the human classical MHC class II and extended class II regions, a situation which has been found in the MHC of all teleosts mapped so far. The 300-kb contig comprises 24 MHC-related genes and is bounded by six non-MHC genes, which are thought to represent an evolutionary breakpoint within the region. Comparative analysis with both human and zebrafish MHC maps indicates two blocks of genes (KNSL2, ZNF297, DAXX, TAPBP, FLOTILLIN; and PSMB8, PSMB10, PSMB9, ABCB3, FABGL, BRD2, COL11A2, RXRB) which have remained linked over 400 million years and may represent an ancestral arrangement of the vertebrate MHC. Zebrafish and Fugu diverged between 100-200 million years ago and differences exist between these two fish species. The position and number of MHC class Ia genes is not conserved between species, there is an inversion of a block of nine genes centering on the PSMB cluster, and additional genes are present in zebrafish coding for a transport-associated protein and a beta proteasome subunit. The extent of these differences has implications for the extrapolation of fish model organism data to commercial aquaculture species. The data presented here represent the most extensive analysis of a fish MHC class Ia region described so far and clearly delimit the extent of this region in Fugu and, potentially, all teleosts.     

Molecular and functional characterization of genes encoding horse MHC class I antigens

Sequence and functional analyses were undertaken on two cDNAs and a genomic clone encoding horse major histocompatibility complex (MHC) class I molecules. All of the clones were isolated from a single horse that is homozygous for all known horse MHC class I and class II antigens. The two cDNAs (clones 8-9 and 1-29) were isolated from a lymphocyte library and encode polymorphic MHC antigens from two loci. The genomic cosmid clone, isolated from a sperm library, contains the 8-9 gene. All three genes were expressed in mouse L-cells and were recognized by alloantisera and, for the cDNAs, by alloreactive cytotoxic T lymphocytes. A total of 3815 bp of the genomic clone were sequenced, extending from 429 bp upstream (5') of the leader peptide through the 3' untranslated region. Promoter region motifs and an intron-exon structure characteristic of MHC class I genes of other species were found. A subclone containing 407 bp of the promoter region was inserted into a chloramphenicol acetyl transferase reporter plasmid, tested in transient transfection assays, and found to have promoter activity in heterologous cells. This genomic clone will enable detailed studies of MHC class I gene regulation in horse trophoblasts, and in horse retroviral infections.     

Expression of a class I MHC transgene: effects of in vivo α/β-interferon treatment

Transgenic mice containing a swine class I major histocompatibility complex (MHC) gene,PD1, express swine MHC (SLA) antigen. The tissue distribution of PD1 RNA parallels that observed in the swine, indicating that the expression ofPD1 is regulated and thattrans-acting factors involved in this regulation have been conserved between the species. Although PD1 RNA levels were much greater in transgenic spleen than in thymus, no difference in the chromatin organization of thePD1 gene was detected. In both tissues, a single DNase I hypersensitive site mapped within the 5′ flanking region. In vivo treatment of the transgenics with mouse α, β-interferon increases PD1 expression in a number of tissues. In the spleen, this increase parallels that observed for the endogenous transplantation antigen, K^b, but differs markedly from the differentiation antigen, Qa-2. Increases in cell surface expression of both PD1 and K^b occurred equally in splenic T- and B-cell populations following α,β-interferon treatment. In contrast, Qa-2 expression in B cells was enhanced by α,β-interferon, whereas it was unaffected in T cells and thymocytes.     

Identification of novel Tapasin polymorphisms and linkage disequilibrium to MHC class I alleles

Tapasin is a Mr 48,000 glycoprotein and has a specialized role in MHC class I-restricted antigen presentation. It is encoded by a gene which maps centromeric to the MHC class II region of human Chromosome 6 within 200 kb of HLA-DP. There is variable dependence upon tapasin for MHC class I expression among different MHC class I alleles. HLA-B*4402 and to a lesser extent HLA-A1 and B8 are tapasin dependent, whereas HLA-B27, A2 and to a lesser extent B7 and A3 are tapasin independent. We investigated whether tapasin is polymorphic and whether these Tapasin alleles are in linkage with any MHC class I alleles. We identified three new mutations within intron 4, which are in a particular linkage with the previously described exon 4 (G16003C) dimorphism. The intronic mutations are G16146T, G16232A, and T16317A (numbering according to cosmid clone F0811; GenBank accession number Z97184). The allele frequency of Tapasin*01 (G16003) was 0.47 and Tapasin*02 (C16003) was 0.53 in this UK population. Four of the eight possible intronic haplotypes were identified and their cis linkage with the tapasin dimorphism ascertained. Tapasin*01 was associated with all the identified haplotypes, while Tapasin*02 was only associated with the wild-type intronic sequence (GGT). There was no significant linkage (P>0.01) of the Tapasin dimorphism or new Tapasin alleles to any of the MHC class I A, B, or C alleles studied or to the extended A1 B8 DR3 haplotype.     

Sequence of the pig major histocompatibility region containing the classical class I genes

A segment comprising 307,078 nucleotides of the pig major histocompatibility complex (SLA) was completely sequenced. The segment corresponded to the entire SLA classical class I-containing region of the serologically defined SLA H01 haplotype. In all, 11 genes were characterized, comprising 7 class I genes located on the centromeric part of the sequence (SLA-1, 2, 3, 4, 5, 9, and 11) and 4 ring finger-related family genes located on its telomeric part. No member of one family was intermingled with a member of the other or with any third-party gene. All class I genes except SLA-11 were similarly orientated. The SLA-1, 2, and 3 genes displayed both promoter and overall coding regions compatible with normal functions. The SLA-4, 11, and 9 genes were considered pseudogenes because they exhibited marked anomalies. Although the SLA-5 gene had a complete coding region, it displayed mutations in promoter elements which could modify its expression. The great molecular similarity observed among the class I genes extended far outside them, and resulted from segmental duplications. The ring finger genes exhibited great homology with their human counterparts. In pig, one of these genes appeared to correspond to a complete gene which in humans is probably a pseudogene. In all, the 11 genes characterized span about 20% of the total sequence. The remaining 80% consists of interspersed repeat elements. The present results, together with the sequence previously reported involving the SLA class I-related genes, open the way for a better understanding of pig MHC organization.     

Dynamics of free versus complexed β2-microglobulin and the evolution of interfaces in MHC class I molecules

In major histocompatibility complex (MHC) class I molecules, monomorphic β₂-microglobulin (β₂m) is non-covalently bound to a heavy chain (HC) exhibiting a variable degree of polymorphism. β₂M can stabilize a wide variety of complexes ranging from classical peptide binding to nonclassical lipid presenting MHC class I molecules as well as to MHC class I-like molecules that do not bind small ligands. Here we aim to assess the dynamics of individual regions in free as well as complexed β₂m and to understand the evolution of the interfaces between β₂m and different HC. Using human β₂m and the HLA–B*27:09 complex as a model system, a comparison of free and HC-bound β₂m by nuclear magnetic resonance spectroscopy was initially carried out. Although some regions retain their flexibility also after complex formation, these studies reveal that most parts of β₂m gain rigidity upon binding to the HC. Sequence analyses demonstrate that some of the residues exhibiting flexibility participate in evolutionarily conserved β₂m–HC contacts which are detectable in diverse vertebrate species or characterize a particular group of MHC class I complexes such as peptide- or lipid-binding molecules. Therefore, the spectroscopic experiments and the interface analyses demonstrate that β₂m fulfills its role of interacting with diverse MHC class I HC as well as effector cell receptors not only by engaging in conserved intermolecular contacts but also by falling back upon key interface residues that exhibit a high degree of flexibility.