Dissection of HLA class II haplotypes in HLA-DR4 homozygous individuals

In order to identify better markers for HLA-DR4-associated autoimmune disorders, we have studied the complexity of the HLA class II region in DR4-positive cells at the DNA level and compared the DNA polymorphism with that defined by serology, mixed lymphocyte culture (MLC) reactivity, and protein chemistry. At the DNA level, HLA-DR4 can be characterized by a homogeneous pattern of bands hybridizing to HLA class II cDNA probes. Besides, subtypes can be defined within DR4 using HLA-DR , -DQ , and -DQ cDNA probes in Southern blot analysis. Three subtypes are found using the DR cDNA probe. One of these subtypes correlates with the cellularly defined Dw15 specificity, another with the serologically defined LB4 and LB14 specificities. None of the restriction fragment length polymorphism (RFLP) patterns coincide with the MLC-defined DR4 subtypes Dw4, DW10, Dw13, and Dw14 separately. Variation of two fragments hybridizing to the DQ cDNA probe obtained after either Pvu II or Taq I digestion yields three subtypes. Pvu II- and Eco RI-digested DR4 DNA give rise to three DQ detectable subtypes. Correlation between these subtypes, isoelectric point variation of DQ molecules, and the DQ-related allelic system TA10/2B3 are demonstrated. Some of the patterns obtained with DQ and DQ cDNA probes display heterozygosity in the DQ region, as demonstrated by family segregation. No correlation was observed between DQ and the cellularly defined Dw determinants. A new polymorphism has been obtained with the DQ probe, probably due to DX polymorphism. DR RFLP divides the LB 14 supertypic specificity into two new subtypes. A combination of the four different techniques applied to a panel of 16 DR4 homozygous cell lines reveals at least nine different haplotypes in DR4. These newly defined haplotypes may be of help in further studies concerning the relationship of micropolymorphism with several diseases.     

Polymorphisms within the HLA-DRw6 haplotype

We have analyzed the HLA class II gene products from HLA-DRw6 homozygous cells. Epstein-Barr virus-transformed B-cell lines were internally labeled with [³⁵S]-methionine. An NP-40 lysate of the cells was subjected to immunoprecipitation, first with a DRw52-like-specific monoclonal antibody and subsequently with a DR-specific framework antibody. The DR region-encoded gene products were analyzed by one-dimensional gel isoelectric focusing and two-dimensional gel electrophoresis. It is shown that DRw6 homozygous cell lines contain at least two nonallelic DR chains, one carrying a DRw52 determinant and one DRw52-negative population. Both chains appear to be polymorphic between the cellularly defined subtypes of DRw6. The determinant responsible for the differential mixed lymphocyte culture reactivity of Dw18 and Dw19 cells resides on the DRw52-positive population, whereas the Dw6-Dw9 differences are attributed to determinants on both populations of DR light chains. The Dw16-derived DRw52⁺ chain much resembles the Dw18 DRw52⁺ light chain whereas there is a clear-cut difference between these two subtypes in the DRw52^– population. We conclude that, for DRw6 homozygous cells, the cellularly recognized D determinants are probably located on DR-encoded molecules, both DRw52⁺ and DRw52^–, and that charge shift of these chains is at least partly responsible for differential recognition of these cells in mixed lymphocyte cultures.Abbreviations used in this paper: MLC mixed lymphocyte culture - 1D-IEF one-dimensional isoelectric focusing - 2D two-dimensional - moab monoclonal antibody     

Analysis of the functional epitopes on different HLA-A2 molecules

Recent studies show that the serologically defined HLA-A2 molecule can be subdivided according to functional and biochemical characteristics. By the use of various HLA-A2-specific cytotoxic T lymphocytes (CTLs) and isoelectric focusing, the serologically homogeneous HLA-A2 molecule can be divided into four subtypes. The polymorphism of the serologically defined HLA-A2 molecule has also been demonstrated by the use of HLA-A2-restricted CTLs. This study was designed to analyze the functional epitopes on different HLA-A2 molecules with special regard to the recognition patterns of different types of HLA-A2-restricted CTLs directed against minor histocompatibility (minor H) antigens. Fifteen so-called HLA-A2 variants belonging to distinct HLA-A2 subtypes were tested as target cells in the cell-mediated lympholysis (CML) assay against (1) HLA-A2-restricted antiminor H-Y CTLs, (2) HLA-A2 and -B7-restricted antiminor H-Y CTLs, and (3) HLA-A2, -Bw62 and -B27-restricted antiminor HA CTLs. We found that those three CTLs recognized only one of those HLA-A2 variants. Furthermore, positive reactions by the antiminor H CTLs were only observed on those variant cells which carried, in addition to the HLA-A2 variant, either another normal HLA-A2 molecule or another required restricting class I molecule necessary for associative recognition. These results indicate that the absence of HLA-A2 normal allotypic target determinant(s) leads to the loss of epitope(s) necessary for recognition of minor H-Y and minor HA transplantation antigens by HLA-restricted CTLs. We can conclude from the present study that HLA-A2-restricted antiminor H CTLs use, in general, the same epitope (or cluster of epitopes) for cellular recognition as alloimmune HLA-A2-specific CTLs.     

Two-dimensional1H NMR study of recombinant insect defensin A in water: Resonance assignments,secondary structure and global folding

Summary A 500 MHz 2D¹H NMR study of recombinant insect defensin A is reported. This defense protein of 40 residues contains 3 disulfide bridges, is positively charged and exhibits antibacterial properties. 2D NMR maps of recombinant defensin A were fully assigned and secondary structure elements were localized. The set of NOE connectivities,³J_NH-H coupling constants as well as¹H/²H exchange rates and /T temperature coefficients of NH protons strongly support the existence of an -helix (residues 14–24) and of an antiparallel -sheet (residues 27–40). Models of the backbone folding were generated by using the DISMAN program and energy refined by using the AMBER program. This was done on the basis of: (i) 133 selected NOEs, (ii) 21 dihedral restraints from³J_NH-H coupling constants, (iii) 12 hydrogen bonds mostly deduced from¹H/²H exchange rates or temperature coefficients, in addition to 9 initial disulfide bridge covalent constraints. The two secondary structure elements and the two bends connecting them involve approximately 70% of the total number of residues, which impose some stability in the C-terminal part of the molecule. The remaining N-terminal fragment forms a less well defined loop. This spatial organization, in which a -sheet is linked to an -helix by two disulfide bridges and to a large loop by a third disulfide bridge, is rather similar to that found in scorpion charybdotoxin and seems to be partly present in several invertebrate toxins.Abbreviations SCUBA Stimulated Cross peaks Under Bleached Alphas - MCD analysis Main Chain Directed analysis - CSH motif Cysteine Stabilized -Helix motif     

Production, isolation and characterization of [Leu4]- and [Ile4]surfactins from Bacillus subtilis

Summary Bacillus subtilis coproduces several surfactin variants that are powerful biosurfactants and have potential applications in biology and industry. A single amino acid substitution in the heptapeptide moiety of surfactins strongly modifies their properties. To better establish structure-activity relationships and to search new variants with enhanced properties, Bacillus subtilis was grown into two modified culture media. Two new variants were isolated by chromatographic methods and studied by NMR spectroscopy. As planned, modifications consisted in the substitution of the l-valine residue at the fourth position by a more hydrophobic residue, i.e., leucine or isoleucine. These [Leu⁴]- and [Ile⁴]surfactins have a higher affinity for hydrophobic solvents and a twice improved surfactant power. Structure-property correlations were confirmed by analysis of the hydrophobic residue distribution in the three-dimensional model of the structure of surfactin in solution.Abbreviations DMSO dimethylsulfoxide - 1D, 2D, 3D one-, two-, three-dimensional - TLC thin layer chromatography - CMC critical micellar concentration - NMR nuclear magnetic resonance     

400 MHz 1H n.m.r. study of the conformation and self-association of peptidolipin NA

Peptidolipin NA is a lipocyclopeptide extracted from Nocardia Asteroides having the formula:

Its conformation and self-association properties and those of its l-Val(6) analogue have been investigated in three solvents of different polarities: chloroform, pyridine and dimethylsulphoxide, using 400 MHz ¹H n.m.r. A model of the conformation of peptidolipin NA in pyridine has been proposed in which the peptide backbone is folded into a γ-turn around the l-Pro residue. Conformational changes are caused by l-Ala(6) → l-Val(6) replacement and by changing the solvent polarity. Nevertheless, the general shape of the peptide ring seems to be maintained. In chloroform, self-association occurs involving the (2) and, to a lesser extent, the (6) residues. Peptidolipin NA could be representative of natural amphiphiles in which a long hydrophobic tail is bound to a polar peptide moiety.     

1H and 13C nmr studies of cyclic and linear dipeptides containing threonyl,seryl, aspartyl,and histidyl residues

The side-chain conformations of D - orL - Thr, D - or L -Ser, L -Asp, and L - His residues in cyclic and linear dipeptides in D₂O or in DMSO-d₆ are deduced from vicinal (¹H,¹H) and (¹³C, ¹H) coupling constants. Vicinal (¹³C, ¹³C) coupling constants strongly depend on substituents and cannot be used without a more sound analysis. In cyclic dipeptides, the Thr and Ser side chains are folded above the DKP ring, with χ¹ near 60°. The L -Asp side chain interacts more specifically with peptide bonds (χ¹ near 300°). The L - His side chain is more flexible and its conformation depends on the proximity of a second side chain and on solute-solvent interactions. In all cases, this side chain is not completely folded. In linear dipeptides, the conformation of a C-terminal L -His residue is mainly influenced by the end carboxylic group. On the other hand, a N-terminal L -His residue interacts more easily with a neighboring L -Asp residue. In aqueous solution, the imidazole pK_a depends on the proximity of terminal and lateral charged groups but does not reveal any specific interaction in cyclic dipeptides. A comparison between the conformations of cyclic peptides observed in solution, in the crystalline state and calculated by empirical methods, allows one to point out the discriminating role of the packing in crystals, and of solute-solvent interactions in solution.     

Species divergence under competition and shared predation

Species competing for resources also commonly share predators. While competition often drives divergence between species, the effects of shared predation are less understood. Theoretically, competing prey species could either diverge or evolve in the same direction under shared predation depending on the strength and symmetry of their interactions. We took an empirical approach to this question, comparing antipredator and trophic phenotypes between sympatric and allopatric populations of threespine stickleback and prickly sculpin fish that all live in the presence of a trout predator. We found divergence in antipredator traits between the species: in sympatry, antipredator adaptations were relatively increased in stickleback but decreased in sculpin. Shifts in feeding morphology, diet and habitat use were also divergent but driven primarily by stickleback evolution. Our results suggest that asymmetric ecological character displacement indirectly made stickleback more and sculpin less vulnerable to shared predation, driving divergence of antipredator traits between sympatric species.     

The nucleotide sequence of the bovine MHC class II alpha genes:DRA,DQA, andDYA

The nucleotide sequence of the exons 2, 3, and 4, and parts of the intervening sequences of aBoLA-DRA and-DQA gene and one other class IIBoLA-A gene have been determined. The structure of theBoLA-DRA and-DQA gene was found to be very similar to that of the corresponding human HLA class II genes. An analysis of the structure of the other class IIBoLA-A gene showed that thisA gene was clearly very different from both the humanA genes and the bovineDRA andDQA genes. The results indicate that this other type of class IIA gene probably represents the class II gene that has already been identified in restriction fragment length polymorphism (RFLP) studies asBoLA-DYA. Since no clear homologue of this presumedBoLA-DYA gene was found among the human HLA class II genes, these results indicate that, at least as far as theA genes are concerned, a distinct class II gene is present in cattle.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30117–M30120. Address correspondence and offprint requests to: J. van der Poel.