Evolution of the CD163 family and its relationship to the bovine gamma delta T cell co-receptor WC1

Background  

The scavenger receptor cysteine rich (SRCR) domain is an ancient and conserved protein domain. CD163 and WC1 molecules are classed together as group B SRCR superfamily members, along with Spα, CD5 and CD6, all of which are expressed by immune system cells. There are three known types of CD163 molecules in mammals, CD163A (M130, coded for by CD163), CD163b (M160, coded for by CD163L1) and CD163c-α (CD163L1 or SCART), while their nearest relative, WC1, is encoded by a multigene family so far identified in the artiodactyl species of cattle, sheep, and pigs.     

Heterogeneity of the internal structure of PDR1, a family of Ty1/copia-like retrotransposons in pea

We characterised the extent of heterogeneity among PDR1 elements, a Ty1/copia-like retrotransposon family in pea, by restriction mapping and PCR with primers designed to amplify four functional domains. The data suggest that two main subfamilies of PDR1 differ in the size of their 5′-region. There are also sequence variants and rearranged copies which include a wide range of deletions of different sizes and deletions combined with insertions of host DNA, or inversions of various regions of the retrotransposon. A deletion hot-spot has been found at nucleotide position 394, where buffer sequences of 26 bp and 38 bp containing microsatellite motifs have been generated. There is more heterogeneity in the gag domain of PDR1 than in other functional domains, and the extent and pattern of this diversity was assessed among 56 Pisum accessions. We found a higher rate of rearrangement and sequence variation within the gag domain of PDR1 in P. fulvum and P. abyssinicum accessions than would be expected from the degree of insertion site polymorphism. A neighbour-joining phylogenetic tree constructed for gag sequences has a similar branching pattern to the equivalent insertion site tree, implying that the PDR1 family and its gag domain have coevolved with the pea genome. Combining both trees revealed clear and distinct subgroups among the Pisum ssp. Received: 17 March 1999 / Accepted: 20 July 1999     

Basal expression studies of cystatins during specific growth stages of wheat spikes for defining their possible role in differential and stage dependent immunity against Karnal bunt (<Emphasis Type="Italic">Tilletia indica</Emphasis>)

Two genotypes showing differential immunity against Karnal bunt (Tilletia indica) were used to investigate the role of three members of cystatin gene family in growth stage dependent immunity in wheat (Triticum aestivum L.). Three members of cystatin gene family (WC1, WC2, and WC4) were cloned and sequenced. Analysis of sequenced data showed that there was 76–99% nucleotide and protein sequence identity between different genes of the wheat cystatin. In silico amino acid sequence analysis revealed the presence of a conserved signature pattern of residues and also the functional domains were presumed to be actively involved in imparting cysteine protease inhibition capability. The semi-quantitative and quantitative levels of these members were measured by means of RT-PCR, northern blotting, western blotting, and by ELISA techniques. The members of cystatin gene family were expressed in both resistant (HD 29) and susceptible genotypes (WH 542); however, the expression level was significantly (P < 0.001) higher in resistant compared to susceptible genotype at all the stages of wheat spikes. The patterns of expression of WC2, WC4 were similar except in the levels in S₁ and S₂ stages as it remained constant (P > 0.05) in contrary to WC1 family whose expression gradually increased from S_v to S₂ stage. According to the intensity of the detected band in RT PCR, northern blot and western blot, WC1 family seems to be expressed more than the other gene families. The immunoassay results further showed that WC1 protein was abundantly expressed in resistant genotype and high expression was observed at the S2 stage as compared to susceptible genotype (P < 0.001) suggesting that low level of expression of WC1 in S2 stage is responsible for KB infection. The results of the present study clearly indicate the role of cystatin gene family in differential and stage dependent immunity against KB.     

The human lysyl oxidase-like 2 protein functions as an amine oxidase toward collagen and elastin

The lysyl oxidase-like 2 (LOXL2) protein is a human paralogue of lysyl oxidase (LOX) that functions as an amine oxidase for formation of lysine-derived cross-links found in collagen and elastin. In addition to the C-terminal domains characteristic to the LOX family members, LOXL2 contains four scavenger receptor cysteine-rich (SRCR) domains in the N-terminus. In order to assess the amine oxidase activity of LOXL2, we expressed a series of recombinant LOXL2 proteins with deletions in the SRCR domains, using an Escherichia coli expression system. All of the purified recombinant LOXL2 proteins, with or without the SRCR domains in the N-terminus, showed significant amine oxidase activity toward several different types of collagen and elastin in in vitro amine oxidase assays, indicating deletion of the SRCR domains does not interfere with amine oxidase activity of LOXL2. Further, amine oxidase activity of LOXL2 was not susceptible to inhibition by β-aminopropionitrile, an irreversible inhibitor of LOX, suggesting a different enzymatic mechanism between these two paralogues.     

Sequence analysis of pig switch μ, Cμ, and Cμm

 The complete sequence of pig switch μ (Sμ) and the various domains of Cμ, a total of ca 8 kilobases (kb) of sequence, are presented and comparatively analyzed. Sμ starts about 2.5 kb downstream of the single pig J _H gene and ends 0.4 kb upstream of Cμ; the length of Sμ is 3.2 kb. Three major pentameric repeats, gagct, gggct, and gggtt, comprise more than half of the entire Sμ sequence. The two most frequent decamers are gagctgagct and gagctgggct. The longest single repeated sequence is 93 base pairs. The frequency of the various pentamers indicates that pig Sμ is more similar to human Sμ than to mouse Sμ. Received: 23 November 1996 / Revised: 21 April 1997     

Cloning, structural analysis, and expression of the pig IgE ε chain

 As a step in the evolutionary studies of immunoglobulin E (IgE) and for the purpose of developing new reagents that will facilitate a more detailed analysis of IgE-mediated inflammatory reactions in a large animal model, we here present the cloning of the ε chain of IgE in the domestic pig (Sus scrufa). A partial cDNA clone for the ε chain of pig IgE was isolated by polymerase chain reaction (PCR) amplification using degenerate primers directed against conserved regions in the second (CH2) and the fourth (CH4) constant domains of IgE. cDNA derived from mRNA isolated from the spleen and lymph nodes of a pig actively sensitized with a protein extract from the nematode Ascaris suum was used as template. Screening of a spleen cDNA library with the partial cDNA clone as probe resulted in isolation of a clone that contained the entire coding region. The nucleotide sequence was determined and was found to conform with the previously identified mammalian ε-chain sequences. The highest degree of similarity was found to sheep IgE. A DNA construct encoding a baculovirus signal sequence, a histidine hexapeptide, and the CH2-CH3-CH4 domains of the pig IgE ε chain was obtained by PCR amplification. The construct was ligated into the baculovirus expression vector pVL1392. Infection of High Five insect cells with recombinant baculovirus resulted in expression and secretion of a soluble 6 × His-CH2-CH3-CH4 protein product. Received: 9 April 1997 / Revised: 28 May 1997     

The multidomain xylanase A of the hyperthermophilic bacterium Thermotoga neapolitana is extremely thermoresistant

 The nucleotide sequence of the xynA gene, encoding extracellular xylanase A of Thermotoga neapolitana, was determined. The xynA gene was 3264 base pairs (bp) long and encoded a putative polypeptide of 1055 amino acids. Three different domains were identified by sequence comparison and functional analysis of proteins with N- and/or C-terminal deletions. The core domain displayed significant homology to members of the glycosyl hydrolase family 10. N- and C-terminal domains were dispensable for enzymatic activity and seemed to be responsible for thermostability and cellulose binding, respectively. The intact gene and its truncated variants were expressed in Escherichia coli and purified for biochemical characterization. The enzyme was shown to act as an endo-1,4-β-xylanase, but minor activities against lichenan, barley glucan, methylumbelliferyl cellobioside and p-nitrophenyl xyloside were also detected. The specific activity and pH and temperature optima for hydrolysis of oat xylan were 111.3 U⋅mg^-1, 5.5 and 102^°C, respectively. The endoxylanase was stable at 90^°C and retained 50% activity when incubated for 2 h at 100^°C. Received: 19 May 1995/Received revision: 31 July 1995/Accepted: 7 September 1995     

Allelic diversity at class II DRB1 and DQB loci of the pig MHC (SLA)

 The loci encoding the β chain of the pig major histocompatibility complex (MHC) class II antigens, SLA-DR and -DQ, have been known to exhibit a remarkable degree of allelic polymorphism. Here, to understand the generation of SLA class II polymorphism, 25 SLA-DRB1 and 24 SLA-DQB genes including newly identified 12 SLA-DRB1 and 7 SLA-DQB genes obtained from miniature pigs were analyzed based on the nucleotide and deduced amino acid sequences. Most of the allelic diversity was attributed to the variable sequences which encode a β₁ domain consisting of a β-pleated sheet followed by an α helix. In the β₁ domain coding region, there were four GC-rich sequences, which have been considered to involve the intra-exon sequence exchange also in other gene evolutions. The first and second GC-rich sequences were χ-like sequences, which have been shown to be a putative recombination signal, and were stably conserved among SLA-DRB1 and DQB genes. These χ-like sequences identified in SLA-DRB1 and SLA-DQB were found to encode the first turning point of the β-pleated sheet and the boundary between the β-pleated sheet and the α helix. Analysis of clustered sequence variation also suggested intra-exon gene conversions in which the χ-like sequences act as putative breakpoints. In addition to point mutations and selection mechanism, intra-exon gene conversions must be an important mechanism in the generation of allelic polymorphism at the SLA-DRB1 and SLA-DQB. Received: 3 December 1998 / Revised: 29 June 1999     

BTL-II : a polymorphic locus with homology to the butyrophilin gene family, located at the border of the major histocompatibility complex class II and class III regions in human and mouse

 Comparison of human and mouse genomic sequence at the border of the major histocompatibility complex (MHC) class II and class III regions revealed a locus encoding six exons with homology to the butyrophilin gene family and the location of a previously decribed gene, testis-specific basic protein (TSBP). We named the new locus BTL-II, for butyrophilin-like MHC class II associated. The six discernable exons of the BTL-II locus encode a small hydrophobic amino acid sequence (which may be a signal peptide), two immunoglobulin domains, a small 7-amino acid, heptad repeat-like exon, and a further two immunoglobulin domains. In mouse, an additional butyrophilin-like gene (NG10) is situated adjacent to BTL-II. Expression studies of the BTL-II locus in mouse showed that it is expressed in a range of gut tissues. We demonstrate that like many other genes from the MHC, BTL-II is polymorphic in a selection of diverse HLA haplotypes. In the light of the newly discovered locus, we revisit and discuss the possible origin of the butyrophilin gene family Received: 20 September 1999 / Revised: 28 December 1999     

Molecular characterization of xynX, a gene encoding a multidomain xylanase with a thermostabilizing domain from Clostridium thermocellum

A Clostridium thermocellum gene, xynX, coding for a xylanase was cloned and the complete nucleotide sequence was determined. The xylanase gene of Clostridium thermocellum consists of an ORF of 3261 nucleotide encoding a xylanase (XynX) of 1087 amino acid residues (116 kDa). Sequence analysis of XynX showed a multidomain structure that consisted of four different domains: an N-terminal thermostabilizing domain homologous to sequences found in several thermophilic enzymes, a catalytic domain homologous to family 10 glycosyl hydrolases, a duplicated cellulose-binding domain (CBD) homologous to family IX CBDs, and a triplicated S-layer homologous domain. A deletion mutant of xynX having only the catalytic region produced a mutant enzyme XynX-C which retained catalytic activity but lost thermostability. In terms of half-life at 70 °C, the thermostability of XynX-C was about six times lower than that of the other mutant enzyme, XynX-TC, produced by a mutant containing both the thermostabilizing domain and the catalytic domain. The optimum temperature of XynX-C was about 5–10 °C lower than that of XynX-TC. Received: 12 January 2000 / Received revision: 24 April 2000 / Accepted: 1 May 2000     

Differences in distribution of single nucleotide polymorphisms among intracellular pattern recognition receptors in pigs

Pathogens localized extracellularly or incorporated into endosomes are recognized mainly by Toll-like receptors, whereas pathogens and pathogen-derived molecules that invade into the cytoplasm of host cells typically are recognized by intracellular pattern recognition receptors (PRRs), such as retinoic acid-inducible gene (RIG)-like helicases (RLHs) and nucleotide-binding oligmerization domain (NOD)-like receptors (NLRs). RIG-I and melanoma differentiation-associated gene 5 (MDA5), which belong to the RLH family, recognize viral genomic RNA, whereas NOD2, a member of the NLR family, responds to microbial peptidoglycans. These receptors may play an important role in pig opportunistic infectious diseases, such as pneumonia and diarrhea, which markedly impair livestock productivity, such that polymorphisms of these receptor genes are potential targets of pig breeding to increase disease resistance. Here, we report single nucleotide polymorphisms (SNPs) in porcine DDX58, IFIH1, and NOD2, which encode RIG-I, MDA5, and NOD2, respectively. Interestingly, compared with DDX58 and IFIH1, NOD2 abounded in nonsynonymous SNPs both throughout the coding sequence and in sequences encoding domains important for ligand recognition, such as helicase domains for RIG-I and MDA5 and leucine-rich repeats in NOD2. These differences in the distribution of SNPs in intracellular PRRs may parallel the diversity of their ligands, which include nucleic acids and peptidoglycans. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification of tandemly repeated type VI cellulose-binding domains in an endoglucanase from the aerobic soil bacterium Cellvibrio mixtus

Cellulose-binding domains (CBD) play a pivotal role during plant cell wall hydrolysis by cellulases and xylanases from aerobic soil bacteria. Recently we␣have reported the molecular characterisation of a single-domain endoglucanase from Cellvibrio mixtus, suggesting that some cellulases produced by this aerobic bacterium preferentially hydrolyse soluble cellulosic substrates. Here we describe the complete nucleotide sequence of a second cellulase gene, celB, from the soil bacterium C. mixtus. It revealed an open reading frame of 1863 bp that encoded a polypeptide, defined as cellulase B (CelB), with a predicted M _r of 66 039. CelB contained a glycosyl hydrolase family 5 catalytic domain at its N terminus followed by two repeated domains, which exhibited sequence identity with type VI CBD previously found in xylanases. Full-length CelB bound to cellulose while catalytically active truncated cellulase derivatives were unable to bind the polysaccharide, confirming that CelB is a modular enzyme and that the type VI CBD homologues were functional. Analysis of the biochemical properties of CelB revealed that the enzyme hydrolyses a range of cellulosic substrates, although it was unable to depolymerise Avicel. We propose that type VI CBD, usually found in xylanases, provide an additional mechanism by which cellulases can accumulate on the surface of the plant cell wall, although they do not potentiate cellulase activity directly. These results demonstrate that C. mixtus, in common with other aerobic bacteria, is able to produce cellulases that are directed to the hydrolysis of cellulose in its natural environment, the plant cell wall. Received: 6 October 1997 / Received revision: 22 December 1997 / Accepted: 2 January 1998     

The spalt-related gene of Drosophila melanogaster is a member of an ancient gene family,defined by the adjacent,region-specific homeotic gene spalt

 We report the full coding sequence of a new Drosophila gene, spalt-related, which is homologous and adjacent to the region-specific homeotic gene, spalt. Both genes have three widely spaced sets of C₂H₂ zinc finger motifs, but spalt-related encodes a fourth pair of C-terminal fingers resembling the Xenopus homologue, Xsal-1. The degrees of sequence divergence among all three members of this family are comparable, suggesting that the Drosophila genes originated from an ancient gene duplication. The spalt-related gene is expressed with quantitative variations from mid-embryogenesis (8–12 h) to the adult stage, but not in ovaries or early embryos. Expression is localized to limited parts of the body, including specific cell populations in the nervous system. In the wing disc, spalt and spalt-related are expressed in indistinguishable domains; in the nervous system and some other organs the expression patterns extensively overlap but are not identical, indicating that the genes have partially diverged in terms of developmental regulation. A characteristic central set of zinc fingers specifically binds to an A/T-rich consensus sequence, defining some DNA binding properties of this ancient family of nuclear factors. Received: 31 July 1996 / Accepted: 4 September 1996     

Possible roles for a non-modular, thermostable and proteinase-resistant cellulase from the mesophilic aerobic soil bacterium Cellvibrio mixtus

The widespread presence of cellulose-binding domains in cellulases from aerobic bacteria and fungi suggests the existence of a strong selective pressure for the retention of these non-catalytic modules. The complete nucleotide sequence of the cellulase gene, celA, from the aerobic soil bacterium Cellvibrio mixtus, was determined. It revealed an open reading frame of 1089 bp that encoded a polypeptide, defined as cellulase A (CelA), of M _r 41 548. CelA displayed features characteristic of an endo-β-1,4-glucanase, rapidly decreasing the viscosity of the substrate while releasing only moderate amounts of reducing sugar. Deletion studies in celA revealed that removal of 78 nucleotides from the 5′ end or 75 from the 3′ end of the gene led to the complete loss of cellulase activity of the encoded polypeptides. The deduced primary structure of CelA revealed an N-terminal signal peptide followed by a region that exhibited significant identity with the catalytic domains of cellulases belonging to glycosyl hydrolase family 5. These data suggest that CelA is a single-domain endoglucanase with no distinct non-catalytic cellulose-binding domain. Analysis of the biochemical properties of CelA revealed that the enzyme hydrolyses a range of soluble cellulosic substrates, but was inactive against Avicel, xylan or any other hemicellulose. CelA was resistant to proteolytic inactivation by pancreatic proteinases and surprisingly, in view of its mesophylic origin, was shown to be thermostable. The significance of these findings in relation to the role of single-domain cellulases in plant cell wall hydrolysis by aerobic microorganisms is discussed. Received: 26 May 1997 / Received revision: 4 July 1997 / Accepted: 4 July 1997     

Desulforubrerythrin from <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, a novel multidomain protein

A novel multidomain metalloprotein from Campylobacter jejuni was overexpressed in Escherichia coli, purified, and extensively characterized. This protein is isolated as a homotetramer of 24-kDa monomers. According to the amino acid sequence, each monomer was predicted to contain three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix bundle domain harboring a non-sulfur μ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV–vis, EPR, and resonance Raman spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein contains two FeCys₄ centers with reduction potentials of +240 mV (desulforedoxin-like center) and +185 mV (rubredoxin-like center). These centers are in the high-spin configuration in the as-isolated ferric form. The protein further accommodates a μ-oxo-bridged diiron site with reduction potentials of +270 and +235 mV for the two sequential redox transitions. The protein is rapidly reoxidized by hydrogen peroxide and has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 μmol H₂O₂ min⁻¹ mg⁻¹. Owing to its building blocks and its homology to the rubrerythrin family, the protein is named desulforubrerythrin. It represents a novel example of the large diversity of the organization of domains exhibited by this enzyme family.     

Identification of new HLA class I region genes by sample sequencing

 Although many human major histocompatibility genes have been identified, relatively few have been localized to the class I region. We searched for new class I region genes by sample sequencing, a process in which short stretches of random genomic sequence are generated from cosmids and then compared with sequences deposited in nucleotide databases. Four class I region cosmids were isolated for sample sequencing by screening a chromosome 6 specific cosmid library with probes derived from specific class I region genes or with overlapping class I region yeast artificial chromosomes. Cosmids were sonnicated to produce fragments of 0.5 – 1 kilobases, subcloned, and sequenced using an automated sequencer. Sequences were then compared with nucleotide sequences deposited in the GenBank databases using the BLASTN algorithm. A number of potential new class I region genes were identified, including a cDNA with similarity to the tre oncogene, the trans-activating factor SC1 (TCF19), and a member of the interferon inducible 1 – 8 gene family. These observations suggest that sample sequencing is an efficient method for identifying new class I region genes, which can be applied to other regions of the genome and to other species, and support previous observations that the class I region contains a variety of genes other than those encoding HLA antigens. Received: 10 December 1996 / Revised: 7 January 1997     

Genomic organization of the sheep immunoglobulin JH segments and their contribution to heavy chain variable region diversity

 The sheep immunoglobulin heavy chain Igh-J locus has been characterized in order to determine the genomic organization of JH segments and their contribution to heavy chain diversity. The locus contains six segments, of which two are functional and four are apparently pseudogenes. These segments span a 1.8 kilobase (kb) region. The distance between JH-ps4 (the 3′-most segment) and the first domain of the μ-chain encoding constant gene is about 5 kb. The two functional JH segments have a standard upstream recombination signal sequence, including heptamer and nonamer sequences separated by a 22–23 nucleotide spacer, and end with a RNA donor splice site. These two segments possess all the characteristic JH invariant residues and are found in expressed μ heavy chain variable regions. The 5′ functional JH1 segment is used in more than 90% of the cDNAs sequenced to date. The contribution of JH segment germline multiplicity to variable regions diversity appears therefore to be minimal. Comparison with other mammalian JH segments shows that all loci are very closely related and probably have evolved from a common ancestral locus. Received: 19 November 1996 / Revised: 17 March 1997     

Characterization of Repetitive DNA Elements in Arabidopsis

We have applied computational methods to the available database and identified several families of repetitive DNA elements in the Arabidopsis thaliana genome. While some of the elements have features expected of either miniature inverted-repeat transposable elements (MITEs) or retrotransposons, the most abundant class of repetitive elements, the AthE1 family, is structurally related to neither. The AthE1 family members are defined by conserved 5′ and 3′ sequences, but these terminal sequences do not represent either inverted or direct repeats. AthE1 family members with greater than 98% identity are easily identified on different Arabidopsis chromosomes. Similar to nonautonomous DNA-based transposon families, the AthE1 family contains members in which the conserved terminal domains flank unrelated sequences. The primary utility of characterizing repetitive sequences is in defining, at least in part, the evolutionary architecture of specific Arabidopsis loci. The repetitive elements described here make up approximately 1% of the available Arabidopsis thaliana genomic sequence. Received: 13 October 1998 / Accepted: 30 December 1998