Major histocompatibility class II genes in rainbow trout (Oncorhynchus mykiss) exhibit temperature dependent downregulation

Major histocompatibility (MH) class II receptors are expressed on the surface of specialized antigen-presenting cells in vertebrate immune systems. Their function is to present peptides derived from exogenous pathogens to CD4+ T cells. Variation in the level of expression of these genes has been linked to pathogenesis in various diseases. Very little has been published on the function of MH class II receptors in teleost fish to date. In this study, we have produced polyclonal antibodies recognizing MH class II alpha and beta proteins of rainbow trout and employed them to characterize the expression pattern of these genes. Deglycosylation using N-glycosidase F and endoglycosidase H showed that MH class II alpha is glycosylated in rainbow trout. MH class II beta was also found to be glycosylated as reported previously. Results from Northern blotting revealed that the expression of these genes was not affected by exposure of rainbow trout to temperature of 5°C. However, at 2°C, downregulation of MH class II alpha and beta genes was evident at both the mRNA and protein levels as assessed by Northern and Western blotting, respectively. Because MH class II antigens play an important role in generating an immune response to bacterial and fungal pathogens, downregulation of these genes at low temperature could account for the susceptibility of fish to low temperature-related diseases such as bacterial cold-water disease and winter saprolegniosis.     

Sequence analysis of OmNramp alpha and quantitative expression of Nramp homologues in different trout strains after infection with Myxobolus cerebralis

Salmonid whirling disease caused by the metazoan parasite Myxobolus cerebralis is an ongoing problem in wild and farmed rainbow trout Oncorhynchus mykiss populations. Rainbow trout from different strains vary in susceptibility to the parasite. Identification of underlying mechanisms could be a starting point for improved control of the disease. We conducted infection trials using 2 rainbow trout strains and brown trout Salmo trutta fario, a species not susceptible to the parasite, to investigate host immune response and resistance mechanisms. We compared expression levels of 2 natural resistance-associated macrophage proteins (Nramp alpha and beta) after infection with M. cerebralis. Total RNA was extracted from skin, muscle, kidney, head and spinal column, and gene expression was quantified by real-time PCR. Significant decreases in expression of both genes were observed at different time points in the infected susceptible rainbow trout compared to the non-infected group. Furthermore, the OmNramp alpha (O. mykiss natural resistance-associated macrophage protein alpha) sequences in 2 resistant and 1 non-resistant rainbow trout strain were analysed and compared for sequence aberrations.     

Comparative analysis of two Nramp loci from rainbow trout.

Innate resistance to intracellular parasites is controlled in part by Nramp1 (Natural resistance-associated macrophage protein 1) in mammals and birds. To isolate Nramp homologs from rainbow trout, a combination of library screening and rapid amplification of cDNA ends was performed. Two closely related Nramp loci, designated OmNramp alpha and OmNramp beta, were cloned and characterized. OmNramp alpha and OmNramp beta encode two highly conserved proteins of 585 and 558 amino acids, respectively. Deduced amino acid seqences showed that the OmNramp alpha and OmNramp beta proteins share 90% of their residues and contain all of the signature features of the Nramp family of proteins: 12 transmembrane domains, two N-linked glycosylation sites, and a conserved transport motif. Phylogenetic analysis supported a close relation to Nramp2 proteins, a related member of the Nramp family. Despite this relation, juvenile trout expressed OmNramp alpha in a manner consistent with an Nramp1 homolog and OmNramp beta similar to an Nramp2 locus. Both trout loci were expressed at relatively high amounts in the ovaries of juveniles, a finding not reported in the investigations of previously characterized mammalian and avian homologs. These results suggest a role for Nramp loci in the follicular development of teleost fishes, as well as in mammals. Because salmonid fishes are ancestral tetraploids, fragments of OmNramp alpha and OmNramp beta were isolated from smelt, a diploid relative, to determine whether the trout loci represent duplicates of a single gene. Homologous sequences for both loci were found in smelt, supporting the hypothesis that OmNramp alpha and OmNramp beta are indeed independent loci that were present before the chromosomal duplication of salmonids. The isolation of Nramp loci from rainbow trout may eventually produce a genetic tool for the control of disease in aquaculture operations. Determining the involvement of trout homologs in innate immunity may also provide insight regarding the evolution of host resistance to pathogens.     

Linkage arrangement of Na,K-ATPase genes in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss)

As part of our efforts to characterize Na,K-ATPase isoforms in salmonid fish, we investigated the linkage arrangement of genes coding for the alpha and beta-subunits of the enzyme complex in the tetraploid-derived genome of the rainbow trout (Oncorhynchus mykiss). Genetic markers were developed from four of five previously characterized alpha-subunit isoforms (alpha1b, alpha1c, alpha2 and alpha3) and four expressed sequence tags derived from yet undescribed beta-subunit isoforms (beta1a, beta1b, beta3a and beta3b). Sex-specific linkage analysis of polymorphic loci in a reference meiotic panel revealed that Na,K-ATPase genes are generally dispersed throughout the rainbow trout genome. A notable exception was the colocalization of two alpha-subunit genes and one beta-subunit gene on linkage group RT-12, which may thus share a conserved orthologous segment with linkage group 1 in zebrafish (Danio rerio). Consistent with previously reported homeologous relationships among the chromosomes of the rainbow trout, primers designed from the alpha3-isoform detected a pair of duplicated genes on linkage groups RT-27 and RT-31. Similarly, the evolutionary conservation of homeologous regions on linkage groups RT-12 and RT-16 was further supported by the map localization of gene duplicates for the beta1b isoform. The detection of homeologs within each gene family also raises the possibility that novel isoforms may be discovered as functional duplicates.     

Identification and characterization of a variant of the third component of complement (C3) in rainbow trout (Salmo gairdneri) serum

A rainbow trout serum protein that is cross-reactive with the third complement component of rainbow trout (C3-1) was purified to homogeneity and its structural and functional properties compared with those of C3-1. This protein (termed C3-related protein: C3-2) bears a close structural resemblance to C3-1, although C3-2 apparently shows no hemolytic activity. Like C3-1, C3-2 consists of two disulfide-linked polypeptide chains (128,000 alpha and 72,000 beta) and retains the unique thiol ester site in the alpha-chain. C3-2 shares some antigenicity with C3-1, but it also displays distinctive antigenic determinants of its own. Comparison of tryptic peptide maps revealed that about 20% of the peptides was specific to either C3-1 or C3-2, and about 80% of the peptides were common to both proteins. Amino acid compositions of the alpha- and beta-chains of C3-2 were similar to those of C3-1. Furthermore, amino acid sequence analysis of the NH2 termini of the alpha- and beta-chains of C3-2 revealed a high degree of homology with those of C3-1, 24 of 26 residues in the alpha-chain and all 20 in the beta-chain of C3-2 were identical with those found in C3-1. Both C3-1 and C3-2 were detected in all the adult rainbow trout tested and in first generation offspring randomly bred from them.     

On high- and low-affinity agonist sites in GABAA receptors

GABAA receptors are activated via low-affinity binding sites for the agonists GABA or muscimol. Evidence has been provided that the amino acid residue alpha 1F64 located at the beta2(+)/alpha1(-) subunit interface forms part of this binding site. In radioactive ligand binding studies the agonist [3H]muscimol has been found to interact with the receptor via a high-affinity binding site. This site has been interpreted as a conformational variant of the low-affinity site. Alternatively, the high-affinity binding site has been located to the alpha1(+)/beta2(-) interface and the homologous residue to alpha 1F64, beta 2Y62 has been proposed to constitute an important part of this site. Here we investigated the effect of the point mutation alpha 1F64L and the homologous mutation beta 2Y62L on agonist and antagonist binding and functional properties in alpha 1 beta 2 gamma 2 GABAA receptors. While the mutation in the alpha1 subunit had drastic consequences on all studied properties, including desensitization, the mutation in the beta2 subunit had little consequence. Our observations are relevant for the relative location of high- and low-affinity agonist sites in GABAA receptors.     

An asymmetric contribution to gamma-aminobutyric type A receptor function of a conserved lysine within TM2-3 of alpha1, beta2, and gamma2 subunits

Mutations that impair the expression and/or function of gamma-aminobutyric acid type A (GABAA) receptors can lead to epilepsy. The familial epilepsy gamma2(K289M) mutation affects a basic residue conserved in the TM2-3 linker of most GABAA subunits. We investigated the effect on expression and function of the Lys --> Met mutation in mouse alpha1(K278M), beta2(K274M), and gamma2(K289M) subunits. Compared with cells expressing wild-type and alpha1beta2gamma2(K289M) receptors, cells expressing alpha1(K278M)beta2gamma2 and alpha1beta2(K274M)gamma2 receptors exhibited reduced agonist-evoked current density and reduced GABA potency, with no change in single channel conductance. The low current density of alpha1beta2(K274M)gamma2 receptors coincided with reduced surface expression. By contrast the surface expression of alpha1(K278M)beta2gamma2 receptors was similar to wild-type and alpha1beta2gamma2(K289M) receptors suggesting that the alpha1(K278M) impairs function. In keeping with this interpretation GABA-activated channels mediated by alpha1(K278M)beta2gamma2 receptors had brief open times. To a lesser extent gamma2(K289M) also reduced mean open time, whereas beta2(K274M) had no effect. We used propofol as an alternative GABAA receptor agonist to test whether the functional deficits of mutant subunits were specific to GABA activation. Propofol was less potent as an activator of alpha1(K278M)beta2gamma2 receptors. By contrast, neither beta2(K274M) nor gamma2(K289M) affected the potency of propofol. The beta2(K274M) construct was unique in that it reduced the efficacy of propofol activation relative to GABA. These data suggest that the alpha1 subunit Lys-278 residue plays a pivotal role in channel gating that is not dependent on occupancy of the GABA binding site. Moreover, the conserved TM2-3 loop lysine has an asymmetric function in different GABAA subunits.     

The carboxy-terminal lysine of alpha B-crystallin is an amine-donor substrate for tissue transglutaminase.

A hexapeptide, corresponding to the sequence around the glutamine in beta A3-crystallin that functions as amine-acceptor for transglutaminase, was synthesized. This peptide was biotinylated and used as a probe to identify amine-donor substrates for transglutaminase among lens proteins. It was found that Ca(2+)-activated transglutaminase linked this peptide not only to several beta-crystallins but, unexpectedly, also to alpha B-crystallin. The C-terminal lysine residue of alpha B-crystalline could be identified as the site of linkage. This strengthens the notion that, at least in crystallins, all transglutaminase substrate residues are located in terminal extensions of the polypeptides. It was shown that in lens homogenate, alpha B-crystallin can be covalently crosslinked to beta-crystallins by transglutaminase. The transglutaminase-mediated crosslinking of alpha B-crystallin may have implications for its involvement in normal and pathological processes in lens and other tissues.     

Recognition of the N-terminal modules of thrombospondin-1 and thrombospondin-2 by alpha6beta1 integrin

In addition to its recognition by alpha3beta1 and alpha4beta1 integrins, the N-terminal pentraxin module of thrombospondin-1 is a ligand for alpha6beta1 integrin. alpha6beta1 integrin mediates adhesion of human microvascular endothelial and HT-1080 fibrosarcoma cells to immobilized thrombospondin-1 and recombinant N-terminal regions of thrombospondin-1 and thrombospondin-2. alpha6beta1 also mediates chemotaxis of microvascular cells to thrombospondin-1 and thrombospondin-2. Using synthetic peptides, LALERKDHSG was identified as an alpha6beta1-binding sequence in thrombospondin-1. This peptide inhibited alpha6beta1-dependent cell adhesion to thrombospondin-1, thrombospondin-2, and the E8 fragment of murine laminin-1. The Glu residue in this peptide was required for activity, and the corresponding residue (Glu90) in the N-terminal module of thrombospondin-1 was required for its recognition by alpha6beta1, but not by alpha4beta1. alpha6beta1 was also expressed in human umbilical vein endothelial cells; but in these cells, only certain agonists could activate the integrin to recognize thrombospondins. Selective activation of alpha6beta1 integrin in microvascular endothelial cells by the anti-beta1 antibody TS2/16 therefore accounts for their adhesion responses to thrombospondins and explains the distinct functions of alpha4beta1 and alpha6beta1 integrins as thrombospondin receptors in microvascular and large vessel endothelial cells.     

Proteomic identification of rainbow trout seminal plasma proteins

In the study, the combination of protein fractionation by 1DE and HPLC‐ESI‐MS/MS was used to characterize the rainbow trout seminal plasma proteome. Our results led to the creation of a catalogue of rainbow trout seminal plasma proteins (152 proteins) and significantly contributed to the current knowledge regarding the protein composition of fish seminal plasma. The major proteins of rainbow trout seminal plasma, such as transferrin, apolipoproteins, complement C3, serum albumin, and hemopexin‐, alpha‐1‐antiproteinase‐, and precerebellin‐like protein, were recognized as acute‐phase proteins (proteins that plasma concentration changes in response to inflammation). This study provides the basis for further functional studies of fish seminal plasma proteins, as well as for the identification of novel biomarkers for sperm quality. The MS data have been deposited in the ProteomeXchange with identifier PXD000306 ( http://proteomecentral.proteomexchange.org/dataset/PXD000306 ).     

Role of the conserved lysine residue in the middle of the predicted extracellular loop between M2 and M3 in the GABA(A) receptor.

In alpha1, beta2, and gamma2 subunits of the gamma-aminobutyric acid A (GABA(A)) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in alpha1 and beta2 subunits resulted each in about a sixfold shift of the concentration-response curve for GABA to higher concentrations, no significant effect by mutation in the gamma subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the alpha1 subunit or the beta2 subunit. Concentration-response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the alpha and beta subunits. Binding of [3H]Ro 15-1788 was unaffected by the mutation in the alpha subunit, whereas the binding of [3H]muscimol was shifted to lower affinity. Mutation of the residue in the alpha1 subunit to E, Q, or R resulted in an about eight-, 10-, or fivefold shift, respectively, to higher concentrations of the concentration-response curve for GABA. From these observations, it is concluded that the corresponding residues on the alpha1 and beta2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.     

Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits

Previous studies on the membrane-cytoplasm interphase of human integrin subunits have shown that a conserved lysine in subunits alpha(2), alpha(5), beta(1), and beta(2) is embedded in the plasma membrane in the absence of interacting proteins (Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (1999) in J. Biol. Chem. 274, 37030-37034). Using a glycosylation mapping technique, we here show that alpha(10) and beta(8), two subunits that deviate significantly from the integrin consensus sequences in the membrane-proximal region, were found to have the conserved lysine at a similar position in the lipid bilayer. Thus, this organization at the C-terminal end of the transmembrane (TM) domain seems likely to be general for all 24 integrin subunits. Furthermore, we have determined the N-terminal border of the TM domains of the alpha(2), alpha(5), alpha(10), beta(1), and beta(8) subunits. The TM domain of subunit beta(8) is found to be 22 amino acids long, with a second basic residue (Arg(684)) positioned just inside the membrane at the exoplasmic side, whereas the lipidembedded domains of the other subunits are longer, varying from 25 (alpha(2)) to 29 amino acids (alpha(10)). These numbers implicate that the TM region of the analyzed integrins (except beta(8)) would be tilted or bent in the membrane. Integrin signaling by transmembrane conformational change may involve alteration of the position of the segment adjacent to the conserved lysine. To test the proposed "piston" model for signaling, we forced this region at the C-terminal end of the alpha(5) and beta(1) TM domains out of the membrane into the cytosol by replacing Lys-Leu with Lys-Lys. The mutation was found to not alter the position of the N-terminal end of the TM domain in the membrane, indicating that the TM domain is not moving as a piston. Instead the shift results in a shorter and therefore less tilted or bent TM alpha-helix.     

A putative beta2-adrenoceptor from the rainbow trout (Oncorhynuchus mykiss). Molecular characterization and pharmacology.

Extensive molecular characterization of mammalian beta-adrenoceptors has revealed complex modes of regulation and interaction. Relatively little attention, however, has focused on adrenoceptors from early branching vertebrates such as fish. Using an RT-PCR approach we have cloned a rainbow trout beta2-adrenoceptor gene that codes for a 409-amino-acid protein with the same seven transmembrane domain structure as its mammalian counterparts. This rainbow trout beta2-adrenoceptor shares a high degree of amino-acid sequence conservation with other vertebrate beta2-adrenoceptors. The conclusion that this sequence is a rainbow trout beta2-adrenoceptor is further supported by phylogenetic analysis of vertebrate beta-adrenoceptor sequences and competitive pharmacological binding data. RNase protection assays demonstrate that the rainbow trout beta2-adrenoceptor gene is highly expressed in the liver and red and white muscle, with lower levels of expression in the gills, heart, kidney and spleen of the rainbow trout. The lack of regulatory phosphorylation sites within the G-protein-binding domain of the rainbow trout beta2-adrenoceptor sequence suggests that the in vivo control of trout beta2-adrenoceptor signaling differs substantially from that of mammals.     

Cell surface expression of hybrid murine/human MHC class II beta alpha dimers. Key influence of residues in the amino-terminal portion of the beta 1 domain

To aid in the identification of key residues responsible for the control of class II MHC beta-alpha dimer assembly and expression, a series of cotransfections of human plus mouse beta- and alpha-genes was performed. The resulting expression data were correlated with the sequences of the relevant proteins to identify residues that played critical roles in these processes. For the I-E/DR homologues good expression was seen for both E beta DR alpha and DR beta E alpha combinations involving several allelically variable beta-chains of each species. These results are consistent with the sequence conservation seen for I-E and DR gene products, and indicate that the species-specific differences that do exist play little role in controlling dimer formation or transport. For A beta chains, a more complex picture was seen. A beta d, but not A beta k or A beta b, was found to coexpress with human alpha-chains. Not only did A beta d show expression with the homologous DQ alpha-chain, but it also was expressed with DR alpha and DP alpha. These data indicate that species-specific residues do not control dimer expression under these conditions and confirm that allelically polymorphic residues have a crucial role in this process. Mapping studies using recombinant A beta genes established the importance of the residues in the amino-terminal half of the beta 1 domain in the differences observed among the A beta alleles. Sequence comparison of DR beta, DP beta, DQ beta, E beta, and A beta chains in this region revealed a single residue (position 12) conserved in most chains and differing in a nonconservative fashion between A beta d vs A beta b or k. A beta d has the conserved lysine at this position, whereas A beta b has methionine and A beta k has glutamine. To test whether this residue actually was important physiologically, a lysine codon was created in a recombinant A beta gene possessing the amino-terminal sequence of the kappa haplotype, and the ability of this mutant chain to be expressed with various mouse A alpha-chains was examined. This mutant chain was shown to gain the ability to be efficiently expressed with A alpha d without losing its ability to be expressed with A alpha k. These data reemphasize the special role played by allelically polymorphic residues in Ia expression and identify one such polymorphic site as position 12.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reversal of agonist selectivity by mutations of conserved amino acids in the binding site of nicotinic acetylcholine receptors

Homomeric alpha7 and heteromeric alpha4beta2 nicotinic acetylcholine receptors (nAChR) can be distinguished by their pharmacological properties, including agonist specificity. We introduced point mutations of conserved amino acids within the C loop, a region of the receptor critical for agonist binding, and we examined the expression of the mutant receptors in Xenopus oocytes. Mutation of either a conserved C loop tyrosine (188) to phenylalanine or a nearby conserved aspartate (197) to alanine resulted in alpha7 receptors for which the alpha7-selective agonist 3-(4-hydroxy, 2-methoxybenzylidene) anabaseine (4OH-GTS-21) had roughly the same potency as for wild-type receptors, whereas the physiologic agonist acetylcholine (ACh) showed drastically reduced potency for these mutant receptors. Corresponding mutations in alpha4 receptors co-expressed with beta2 resulted in alpha4beta2 receptors for which ACh potency was relatively unchanged, although the efficacy of the alpha7-selective agonist 4OH-GTS-21 was increased greatly relative to that of ACh. We also investigated the significance of a conserved lysine (145 in alpha7), proposed to form a stable salt bridge with Asp-197 in the resting state of the receptor. Mutations of this residue in both alpha7 and alpha4 resulted in receptors that were largely unresponsive to both ACh and 4OH-GTS-21. Our results suggest that initiation of gating depends both on specific interactions between residues in the C loop domain and, depending on receptor subtype, the physiochemical properties of the agonist, so that in the altered environment of the alpha4Y190F-binding site, large hydrophobic benzylidene anabaseines may close the C loop and initiate channel gating more effectively than the polar agonist ACh.     

Activation of p53 by conjugation to the ubiquitin-like protein SUMO-1

The growth-suppressive properties of p53 are controlled by posttranslational modifications and by regulation of its turnover rate. Here we show that p53 can be modified in vitro and in vivo by conjugation to the small ubiquitin-like protein SUMO-1. A lysine residue at amino acid position 386 of p53 is required for this previously undescribed modification, strongly suggesting that this lysine residue serves as the major attachment site for SUMO-1. Unlike ubiquitin, attachment of SUMO-1 does not appear to target proteins for rapid degradation but rather, has been proposed to change the ability of the modified protein to interact with other cellular proteins. Accordingly, we provide evidence that conjugation of SUMO-1 to wild-type p53 results in an increased transactivation ability of p53. We suggest that posttranslational modification of p53 by SUMO-1 conjugation provides a novel mechanism to regulate p53 activity.     

Polysialoglycoproteins of Salmonidae fish eggs. Complete structure of 200-kDa polysialoglycoprotein from the unfertilized eggs of rainbow trout (Salmo gairdneri)

Polysialoglycoproteins (PSGP) we first isolated from the unfertilized eggs of rainbow trout (Salmo gairderi) and now found to be a ubiquitous component of Salmonidae fish eggs are a novel type of glycoprotein. PSGP from rainbow trout has a molecular weight of 200 X 10(3), a low protein content (about 15% w/w), and a high sialic acid (N-glycolylneuraminic acid (NeuGc] content (about 60%, w/w). In any evaluation of the biological functions of PSGP, information about the complete structure of this unique macromolecular component is relevant. We have now completed the determination of the overall structural organization of the 200-kDa PSGP, and this is the first report of the complete structural analysis of this novel class of glycoprotein: (Asp)0-2-Ala-Thr*-Ser*-Glu-(Ala-Ala-Thr*-Gly-Pro-Ser-Gly-Asp-Asp-Ala-Thr *-Ser*- Glu)n-Ala-Ala-Thr*-Gly-Pro-Ser-Gly where * indicates the amino acid residues to which oligo- and/or polysialylglycan units are attached and n = 25. Thus the most outstanding structural features of PSGP isolated from the unfertilized eggs of rainbow trout are now the occurrence of (a) tandem repeats of a tridecapeptide and (b) an alpha-2----8-linked oligo(poly)sialyl group on each of the core oligosaccharide chains, i.e. GalNAc- beta 1----4(NeuGc alpha 2----3)GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Fuc alpha 1----3GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n ----6)GalNAc alpha 1----Ser (or Thr), GalNAc beta 1----3Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), Gal beta 1----4Gal beta 1----3[----8NeuGc alpha 2)n----6)GalNAc alpha 1----Ser (or Thr), and Gal beta 1----3[----8NeuGc alpha 2)n----6) GalNAc alpha 1----Ser (or Thr).