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We proposed here a sequence-based approach predicting some microorganisms as possible sources of autoantigen-related molecular mimicry concerning Idiopathic Pulmonary Arterial Hypertension (IPAH) and related hypertension mostly accompanying autoimmune diseases and AIDS (APAH). This approach (SPECIES_VALENCE) processes the database occurrences of linear autoepitope-related short Dense Quasi-Pattern Sequences (DQPA) generated based on identities of important autoantigenic sequences. The corresponding enumeration comprises two types of statistical evaluations performed in each of eight proposed models. Based on this enumeration, we selected nine microorganisms, whereas revaluation of the obtained scoring values restricted Pseudomonas aeruginosa, Aspergillus fumigatus and the two co-infecting herpes viruses (Epstein Barr virus and cytomegalovirus) as most favourable. The results are discussed in terms of (a) the validity of increased DQPA occurrence in functionally correlated sequences, (b) the possible mechanisms leading to autoantibody response, (c) selected additional pathogenic effects of predicted microorganisms and (d) possible effects of cross-reactivities and immune tolerance.

This review summarizes available data concerning intradomain structures (IS) such as functionally important amino acid residues, short linear motifs, conserved or disordered regions, peptide repeats, broadly occurring secondary structures or folds, etc. IS form structural features (units or elements) necessary for interactions with proteins or non-peptidic ligands, enzyme reactions and some structural properties of proteins. These features have often been related to a single structural level (e.g. primary structure) mostly requiring certain structural context of other levels (e.g. secondary structures or supersecondary folds) as follows also from some examples reported or demonstrated here. In addition, we deal with some functionally important dynamic properties of IS (e.g. flexibility and different forms of accessibility), and more special dynamic changes of IS during enzyme reactions and allosteric regulation. Selected notes concern also some experimental methods, still more necessary tools of bioinformatic processing and clinically interesting relationships., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Virtual interactomics represents a rapidly developing scientific area on the boundary line of bioinformatics and interactomics. Protein-related virtual interactomics then comprises instrumental tools for prediction, simulation, and networking of the majority of interactions important for structural and individual reproduction, differentiation, recognition, signaling, regulation, and metabolic pathways of cells and organisms. Here, we describe the main areas of virtual protein interactomics, that is, structurally based comparative analysis and prediction of functionally important interacting sites, mimotope-assisted and combined epitope prediction, molecular (protein) docking studies, and investigation of protein interaction networks. Detailed information about some interesting methodological approaches and online accessible programs or databases is displayed in our tables. Considerable part of the text deals with the searches for common conserved or functionally convergent protein regions and subgraphs of conserved interaction networks, new outstanding trends and clinically interesting results. In agreement with the presented data and relationships, virtual interactomic tools improve our scientific knowledge, help us to formulate working hypotheses, and they frequently also mediate variously important in silico simulations.

Prolonged cultivation of separated rat lung mast cells (LMC) in vitro is necessary to better investigate a possible role of LMC in different stages of tissue remodeling induced by hypoxia. Rat lung mast cells (LMC) were separated using a protocol including an improved proteolytic extraction and two subsequent density gradient separations on Ficoll-Paque PLUS and a new generation of Percoll, i.e. Percoll PLUS. Instead of usual isotonic stock Percoll solution, an alternative "asymptotically isotonic" stock solution was more successful in our density separation of LMC on Percoll PLUS. Separated cells were cultivated for six days in media including stem cell factor, interleukins IL-3 and IL-6, and one of two alternative mixtures of antibiotics. These cultivations were performed without any contamination and with only rare changes in cell size and morphology. Model co-cultivation of two allogenic fractions of LMC often caused considerable rapid changes in cell morphology and size. In contrast to these observations no or rare morphological changes were found after cultivation under hypoxic conditions. In conclusions, we modified separation on Percoll PLUS to be widely used, altered LMC separation with respect to purposes of long-lasting cultivation and observed some model morphological changes of LMC.

The motif DGYW/WRCH (Mh) and its frequently discussed simplified derivative GYW/WRC (Mhs) are involved in immunoglobulin (Ig) hypermutation. Both these motifs appear to be markedly shorter than the corresponding conventionally predicted minima of valid sequence lengths (MVSL). The same conclusion concerning both Mh and Mhs can also be obtained in the combined case including a less strict semi-empirically defined w-value and one nucleotide length tolerance related to MVSL. Such disagreement indicates considerably low information content in Mh and Mhs when evaluating these motifs as alphabetical structures (words). This fact raises a question of actually recognized structures (presumably longer than Mh and Mhs). Interestingly, both Mh and Mhs dimers or pairs of closely located Mh or Mhs achieve confirmation of length validity in the case of w=0.05, suggesting thus double-motif recognition as one of statistically consistent explanations. This possibility is also in agreement with the results of our model sequence study of mRNA derived from variable Ig gene sequences (rIgV) with respect to the most frequently occurring structures formed by motif overlaps in all model sequence sets. On the other hand, additional superior occurrence of motif pairs at a structurally important distance of a single DNA thread was found in the conserved domain (cd00099) related sequences of Elasmobranchii origin and less markedly in the corresponding human rIgV, but not in a randomly selected human subset of rIgV. The data are discussed with respect to statistical evaluation and structural properties of hypermutation motifs or the competent enzyme, i.e. activation-induced cytidine deaminase.

Many structures and molecules closely related to those involved in the specific process of immunoglobulin (Ig) hypermutation existed before the appearance of primordial Ig genes. Consequently, these structures can be found even in animals and organisms distinct from vertebrates; likewise, homologues of hypermutation enzymes are present in a broad range of species, from bacteria to mammals. Our analysis, based predominantly on primary structure, demonstrates the existence of molecules similar to Ig domains, variable Ig domains (IGv), and antigen receptors (AR) in unicellular organisms, nonvertebrate metazoans, and nonvertebrate Coelomata, respectively. In addition, we deal here with some important structural properties of CDR1-like segments of the selected sponge adhesion molecule GCSAMS exhibiting chimerical Ig domain similarities, and demonstrate the occurrence of conserved regions corresponding to Ohno's modern intact primordial building block in the C-terminal part of IGv-related segments of nonvertebrate origin. The results of our analysis are also discussed with respect to the possible phylogeny of molecules preceding the hypothetical common antigen receptor ancestor.

Sequences of immunoglobulin (Ig) domains of adhesive molecule GSAMS from the living fossil sponge Geodia cydonium were compared with the important motif of peptide protein kinase substrates and inhibitors (PKSI), detail PKSI sequences, and a common template sequence, derived from structures determined previously. We found the site-restricted sequence similarities to these peptide sequences predominantly in the GSAM Ig1 domain of GSAMS in the domain region related to corresponding Ig similarities detected earlier. Additional sequence block-related analysis revealed the presence of CDR1-like segments within PKSI-related regions and resulted in the detection of increased numbers of hypermutation motifs just in the CDR1-like segment of GSAM Ig1 (GSAM(cdrl.1)). In the following database searches with PKSI-related regions and GSAM(cdr1.1) we looked for: (i) peptide similarities present in the context of Ig domains or related structures in a large range of species from Archaea to Vertebrata, and (ii) some special nucleotide similarities.

Forty original sequences of peptide substrates and inhibitors of protein kinases and phosphatases were aligned in a chain matrix without artificial gaps. Fifteen protein kinase peptide substrates and inhibitors (PKSI peptides) contained a common dipeptide ArgArg and also additional important tetra-, tri- and dipeptide homologies. Three further peptide substrates were significantly similar to these peptides but lacked the ArgArg dipeptide. Sequence comparison of individual PKSI peptides revealed probabilistically restricted consensus sequence--PKSI motif--comprising 8 homologous and 13 non-randomly distributed amino acids without considering mutation analysis. This template motif was compared with the consensus sequences of 12 different immunoglobulin domains. In 11 of 12 these domains, the starts of homologous segments were found at nearly the same domain related sites, beginning with serine. A single-triplet mutation of any of the first two triplet bases that encode equally localized amino acids in each of the two sequence sets (PKSI and Ig) revealed additional homologies with the other set. A primary derived motif version composed of 9 homologous and seven non-randomly distributed amino acids was consequently established by its feedback projection into the original sequence sets. This procedure yielded a second preliminary motif version (revised motif) formed by a sequence of 9 homologous amino acids and two non-randomly distributed amino acids. In addition, three shorter oligopeptide motifs called important stereotypes were derived, based on repeated homology between Ig chains and the revised motif. The most extensive similarities in terms of these stereotypes occurred in the CH2 and CH4 domains of Ig peptides, and inhibitors of cAMP dependent protein kinase and protein kinase A. Further comparisons based on a reference sequence set arranged with the aid of feedback projection revealed a lower similarity between variable Ig chains reflected in a decreased number of homologous amino acids. Two final motif versions, FMC and FMV, were found in two different subsets of constant and variable Ig chains, respectively. FMC was composed of seven homologous and one non-randomly distributed amino acids forming the dispersed structure STLR(C)LVSD, whereas 6 homologous and one questionable amino acid constituted FMV. Only CH4 and CH1 domain segments contained all five high-incidence amino acids, which represented a higher level of similarity than homologous amino acids of all preliminary and final motifs. Four such amino acids were present also in three PKSI peptides. All similarities described here occur in domain segments positionally overlapping with the CDR1 region of variable chains. The results are discussed in terms of immunoglobulin evolution, the position of Fc receptor binding sites and degeneration or mutability of the triplets of motif-constituting amino acids.

This review summarizes some important data, principles, opinions, commentaries, and modern methodology concerning the receptor structure, interactions, signaling and receptor-mediated cell functions. Three sections give a brief overview of the signaling synergy, multivariant signaling, and some reactions in phosphorylation networks. A concise report about the cytotoxic reaction of NK cells represents an example of multistage recognition reaction, involving differently acting receptors, changes in affinities of cell-cell interactions, and secretion of regulatory and cytotoxic molecules. Some interesting trends in receptor engineering, including antibody molecules as a special receptor phenomenon are mentioned in the final section.

Membrane lectins of mammalian large granular lymphocytes are thought to be important receptors in their non-major-histocompatibility complex-restricted activation. A triantennary desialylated oligosaccharide has been reported as the most effective triggering structure [Pospísil M., Kubrycht J., Bezouska K., Táborský O., Novák M. & Kocourek J. (1986) Immunol. Lett. 12, 83-90] while its cell surface receptor has recently been identified in pig natural killer cells as a 205-kDa membrane lectin resembling the proteins of the leukocyte common antigen family (LCA). In this study we have prepared 4-azidophenyl (photoactivatable) and 4-hydroxyphenyl (radio-iodinatable) derivatives of triantennary oligosaccharides by a new procedure which allows the natural conformation of the N-glycosidic linkage between the oligosaccharide and the respective labeling group to be retained. We used these high-affinity ligands to investigate the oligosaccharide-combining site of the 205-kDa lectin. Photoaffinity labeling of the whole cells and solubilized proteins confirmed that a 205-kDa polypeptide constitutes the major cell-surface calcium-independent receptor for triantennary oligosaccharides in pig lymphocytes. Isolation and manual sequencing of two ligand-labeled and eleven other peptides proved that the 205-kDa lectin represents a member of the LCA family expressing exons 4 and 6 during alternative splicing and that the high-affinity binding site is localized in the N-terminal 70-kDa extracellular domain. Binding studies with radiolabeled oligosaccharides and the above carbohydrate-recognition domain subjected to various chemical and enzymatic treatments indicated that the binding of oligosaccharides might be significantly modulated by sialylated O-glycosidically linked lineage-specific carbohydrate epitopes localized within this domain. Affinity chromatography of LCA isolated by conventional methods on immobilized oligosaccharides revealed that only a fraction of these cell-surface glycoproteins expressed high-affinity binding sites for the oligosaccharide ligands. Thus, N-linked oligosaccharide moieties of cell-surface glycoproteins seem to represent possible ligands of LCA that may be important in intercellular adhesion and oligosaccharide-mediated activation of lymphocytes.

Some leukocyte effector cell-surface molecules movement toward the adjoining target cells takes place during the reaction of NK cytotoxicity (NK R). The majority of the moving molecules are usually anchored via a divalent-ion-dependent interaction (PMM-M2+). The released PMM-M2+ can interact also with the secreted tumor necrosis factor alfa (TNF-alpha). In agreement with PMM-M2+ movement, the number of TNF-alpha binding sites on the target cell surface increases during NK R. In addition, antibodies against PMM-M2+, as well as D-mannose- or N-acetyl-D-glucosamine-terminated oligosaccharides of PMM-M2+ inhibit NK R. A more detailed analysis of PMM-M2+ with monoclonal antibodies used flow cytometry and cell-surface biotinylation. Only 3 of 31 tested CD antigens (CD2, LAK-1 and CD45) were passed through this first strongly restricted experimental screening. The EDTA-released LAK-1 antigen, but not CD2 and CD45, interact with TNF-alpha and cell surface via a mannose-inhibitable interaction dependent on the presence of Ca2+ ions. The mechanism of possible participation of PMM-M2+ in cytotoxic events is discussed in relation to Ca2+ influx and subsequent cytolysin secretion.

Mannan-binding proteins found in the liver and serum of several vertebrate species are supposed to play an important role in the intracellular transport of glycoproteins, as well as in several protective reactions including complement activation and elimination of various pathogens. To study these protective functions at molecular level it is necessary to understand the fine oligosaccharide specificity and mutual relation among various forms of these soluble lectins. We have isolated mannan-binding protein as peripheral membrane proteins of porcine lymphocytes. This lectin was purified to homogeneity and shown to possess many properties in common with the well studied rat liver proteins (mol. mass, subunit composition and general organization of the molecule). Binding studies performed with three series of defined oligosaccharides (high mannose, hybrid type, and complex) on native lectin molecules as well as isolated carbohydrate-binding domains revealed distinctive features of this mannan-binding protein, including its impaired ability to bind the oligosaccharide ligand after reduction and decyclization at core N-acetyl-D-glucosamine 1.

Inhibition of pig NK cell activity by asialooligosaccharides (aOS) isolated from human serum glycoproteins was investigated. Train-tennary aOS (aOSIII) of ceruloplasmin was found to be the most potent inhibitor up to the concentration 0.1 micrograms/ml, which is in agreement with its highly specific binding to NK-activity-enriched pig lymphocytes (with a morphology similar to human large granular lymphocytes (LGL]. Only lectins with the specificity to Gal(beta 1----4)GlcNAc or Gal(beta 1----3)GalNAc structures exhibited inhibition of NK cytotoxicity. F(ab)2 fragments of rabbit antibodies against pig spleen membrane lectin cross-reacting with the pig liver membrane lectin completely inhibited NK activity when preincubated with the effectors or present in the incubation mixture during the assay. These data suggest that lectin receptors on cells of pig NK-activity-enriched fraction specific for aOSIII and antigenically related to membrane lectins isolated from pig spleen and liver, are involved in the NK recognition of several xenogeneic targets.

Oligosaccharides with four different types of branching were prepared from purified human transferrin, alpha 2-macroglobulin, caeruloplasmin and alpha 1-acid glycoprotein and labelled with NaBH3 3H. Binding of these oligosaccharides to rat liver plasma membrane, rat leucocytes, pig liver plasma membranes and pig leucocyte plasma membranes was investigated. A striking dependence of binding on oligosaccharide branching was observed. The values of apparent association constants Ka at 4 degrees C vary from 10(6) M-1 (biantennary structure) to 10(9) M-1 (tetra-antennary structure) in the liver, whereas in the leucocytes the Ka values were found to be of reversed order, from 1.8 X 10(9) M-1 for biantennary to 2.2 X 10(6) M-1 for tetra-antennary structures. The binding is completely inhibited by 150 mM-D-galactose, but 150 mM-D-mannose has almost no effect on binding. Leucocyte plasma membranes bind preferentially 125I-asialoglycoproteins with biantennary oligosaccharides, thus completing the specificity pattern of the hepatic recognition system for desialylated glycoproteins. Possible physiological roles of these two complementary recognition systems under normal and pathological conditions are discussed.