Therapeutic effect of Cerebrolysin on reducing impaired cerebral endothelial cell permeability.

Cerebrolysin has been shown to promote neurovascular protection and repair in preclinical models of stroke and neural injury and is demonstrating promise for stroke and neural injury therapeutic application in the clinic. The effect of Cerebrolysin on the human cerebral endothelial cell function has not been investigated. Using an in-vitro cerebral endothelial cell permeability assay and western blot analyses of tight junction and proinflammatory and procoagulant proteins, the present study showed that tissue plasminogen activator (tPA) and fibrin substantially impaired human cerebral endothelial cell barrier function and increased permeability, which persisted for at least 24 h.

Assessing the citrullinome in rheumatoid arthritis synovial fluid with and without enrichment of citrullinated peptides.

Protein citrullination is a posttranslational modification that has attracted increased attention, especially for its involvement in rheumatoid arthritis (RA). Here, we assess the citrullinome in RA synovial fluid by direct LC-MS/MS analysis and by the use of an enrichment strategy based on citrulline specific biotinylation. RA synovial fluid was depleted for abundant proteins, and total and depleted fractions were analyzed.

Specific biotinylation and sensitive enrichment of citrullinated peptides.

Protein citrullination is a posttranslational modification where peptidylarginine is enzymatically deiminated to form peptidylcitrulline. Although the role of protein citrullination in both health and disease is being increasingly recognised, techniques available to identify citrullinated proteins and to map their citrullination site(s) are rare and often show poor sensitivity. Here, we present a sensitive technique for specific modification and selective enrichment of citrullinated peptides from complex biological samples.

Small-scale purification and mass spectrometry analysis reveal a third aquaporin-4 protein isoform of 36 kDa in rat brain.

Aquaporin-4 (AQP4) is known to have two main isoforms M1 and M23 in the brain. Immunoblot analyses have provided evidence of additional AQP4 immunopositive bands, suggesting that the repertoire of AQP4 isoforms is broader than previously assumed. As isoforms beyond M1 and M23 are not observed in recombinant systems, investigation of novel isoforms requires the use of a native source.

Assessing high affinity binding to HLA-DQ2.5 by a novel peptide library based approach.

Here we report on a novel peptide library based method for HLA class II binding motif identification. The approach is based on water soluble HLA class II molecules and soluble dedicated peptide libraries. A high number of different synthetic peptides are competing to interact with a limited amount of HLA molecules, giving a selective force in the binding.

Structure-function relationships of the competence lipoprotein ComL and SSB in meningococcal transformation.

Neisseria meningitidis, the meningococcus, is naturally competent for transformation throughout its growth cycle. The uptake of exogenous DNA into the meningococcus cell during transformation is a multi-step process. Beyond the requirement for type IV pilus expression for efficient transformation, little is known about the neisserial proteins involved in DNA binding, uptake and genome integration.

Anti-PAD4 autoantibodies in rheumatoid arthritis: levels in serum over time and impact on PAD4 activity as measured with a small synthetic substrate.

Isoform 4 of the human peptidylarginine deiminase (hPAD4) enzyme may be responsible for the citrullination of antigens in rheumatoid arthritis (RA) and has been shown to be itself the target of disease-specific autoantibodies. Here, we have tested whether the level of serum anti-hPAD4 antibodies in RA patients is stable over a period of 10 years and whether the antibodies influence hPAD4-mediated deimination of the small substrate N-α-benzoyl-L-arginine ethyl ester. RA sera (n = 128) obtained at baseline and after 10 years were assessed for anti-hPAD4 antibodies by a specific immunoassay.

The preferred substrates for transglutaminase 2 in a complex wheat gluten digest are Peptide fragments harboring celiac disease T-cell epitopes.

Background: Celiac disease is a T-cell mediated chronic inflammatory disorder of the gut that is induced by dietary exposure to gluten proteins. CD4+ T cells of the intestinal lesion recognize gluten peptides in the context of HLA-DQ2.5 or HLA-DQ8 and the gluten derived peptides become better T-cell antigens after deamidation catalyzed by the enzyme transglutaminase 2 (TG2).

Redox regulation of transglutaminase 2 activity.

Transglutaminase 2 (TG2) in the extracellular matrix is largely inactive but is transiently activated upon certain types of inflammation and cell injury. The enzymatic activity of extracellular TG2 thus appears to be tightly regulated. As TG2 is known to be sensitive to changes in the redox environment, inactivation through oxidation presents a plausible mechanism.

A technique for the specific enrichment of citrulline-containing peptides.

Protein citrullination results from enzymatic deimination of peptidylarginine and plays an important role in health and disease. Despite increasing scientific interest, the identity and function of citrullinated proteins in vivo remain widely unknown. Thorough proteomic studies could contribute to a better understanding of the role of this posttranslational modification but will require tools for enrichment of citrullinated polypeptides.

Gluten T cell epitope targeting by TG3 and TG6; implications for dermatitis herpetiformis and gluten ataxia.

Transglutaminase 2 (TG2) is well characterized as the main autoantigen of celiac disease. The ability of TG2 to deamidate and crosslink gluten peptides is essential for the gluten-dependent production of TG2 specific autoantibodies. In patients with primarily extraintestinal manifestation of gluten sensitivity the repertoire of autoantibodies may be different.

Design of new high-affinity peptide ligands for human leukocyte antigen-DQ2 using a positional scanning peptide library.

Human leukocyte antigen (HLA)-DQ2 (DQA1 x 0501/DQB1 x 0201) is associated with several immune disorders, including celiac disease, which is caused by an inappropriate T-cell response to gluten. Interference with peptide presentation by HLA-DQ2, for example, by the use of peptide blockers, is a possible treatment strategy for such HLA-associated disorders. A successful implementation of this strategy will depend on the identification of ligands that bind much better to HLA-DQ2 than the disease related epitopes.

Targeted analysis of protein citrullination using chemical modification and tandem mass spectrometry.

Protein citrullination originates from enzymatic deimination of polypeptide-bound arginine and is involved in various biological processes during health and disease. However, tools required for a detailed and targeted proteomic analysis of citrullinated proteins in situ, including their citrullination sites, are limited. A widely used technique for detection of citrullinated proteins relies on antibody staining after specific derivatization of citrulline residues by 2,3-butanedione and antipyrine.

Identification of neisserial DNA binding components.

Neisseria meningitidis, a causative agent of meningitis and septicaemia, expresses type IV pili, a feature correlating with the uptake of exogenous DNA from the environment by natural transformation. The outer membrane complex PilQ, through which pili are extruded and retracted, has previously been shown to bind DNA in its pore region. In order to further elucidate how DNA is transported across the membranes, we searched for DNA binding proteins within the meningococcal inner membrane.

A quantitative analysis of transglutaminase 2-mediated deamidation of gluten peptides: implications for the T-cell response in celiac disease.

Celiac disease develops in genetically predisposed individuals as the result of an inappropriate intestinal immune response to dietary gluten proteins. T cells present in the intestine of celiac patients recognize gluten peptides in the context of HLA-DQ2 or -DQ8 molecules. Notably, T-cell recognition is increased after these peptides have been deamidated by the enzyme transglutaminase 2.

Primary sequence, together with other factors, influence peptide deimination by peptidylarginine deiminase-4.

Enzymes of the peptidylarginine deiminase (PAD) family catalyze the posttranslational deimination of polypeptide-bound arginine residues. Here, we report the selection of peptide substrates by PAD-4, an isoform thought to be involved in the pathogenesis of rheumatoid arthritis. First, we investigated whether PAD-4-mediated deimination is influenced by the nature of amino acid residues flanking the targeted arginine.

Soluble HLA-DQ2 expressed in S2 cells copurifies with a high affinity insect cell derived protein.

We here describe that soluble HLA-DQ2 (sDQ2) molecules, when expressed in Drosophila melanogaster S2 insect cells without a covalently tethered peptide, associate tightly with the D. melanogaster calcium binding protein DCB-45. The interaction between the proteins is stable in S2 cell culture and during affinity purification, which is done at high salt concentrations and pH 11.

Complexes of two cohorts of CLIP peptides and HLA-DQ2 of the autoimmune DR3-DQ2 haplotype are poor substrates for HLA-DM.

Atypical invariant chain (Ii) CLIP fragments (CLIP2) have been found in association with HLA-DQ2 (DQ2) purified from cell lysates. We mapped the binding register of CLIP2 (Ii 96-104) to DQ2 and found proline at the P1 position, in contrast to the canonical CLIP1 (Ii 83-101) register with methionine at P1. CLIP1/2 peptides are the predominant peptide species, even for DQ2 from HLA-DM (DM)-expressing cells.

The propensity for deamidation and transamidation of peptides by transglutaminase 2 is dependent on substrate affinity and reaction conditions.

Transglutaminase 2 (TG2) catalyzes cross-linking or deamidation of glutamine residues in peptides and proteins. The in vivo deamidation of gliadin peptides plays an important role in the immunopathogenesis of celiac disease (CD). Although deamidation is considered to be a side-reaction occurring in the absence of suitable amines or at a low pH, a recent paper reported the selective deamidation of the small heat shock protein 20 (Hsp20), suggesting that deamidation could be a substrate dependent event.

Ligand binding and antigenic properties of a human neonatal Fc receptor with mutation of two unpaired cysteine residues.

The neonatal Fc receptor (FcRn) is a major histocompatibility complex class I-related molecule that regulates the half-life of IgG and albumin. In addition, FcRn directs the transport of IgG across both mucosal epithelium and placenta and also enhances phagocytosis in neutrophils. This new knowledge gives incentives for the design of IgG and albumin-based diagnostics and therapeutics.

Modulation of Lck function through multisite docking to T cell-specific adapter protein.

T cell-specific adapter protein (TSAd), encoded by the SH2D2A gene, interacts with Lck through its C terminus and thus modulates Lck activity. Here we mapped Lck phosphorylation and interaction sites on TSAd and evaluated their functional importance. The three C-terminal TSAd tyrosines Tyr(280), Tyr(290), and Tyr(305) were phosphorylated by Lck and functioned as docking sites for the Lck Src homology 2 (SH2) domain.

A strategy for bacterial production of a soluble functional human neonatal Fc receptor.

The major histocompatibility complex (MHC) class I related receptor, the neonatal Fc receptor (FcRn), rescues immunoglobulin G (IgG) and albumin from lysosomal degradation by recycling in endothelial cells. FcRn also contributes to passive immunity by mediating transport of IgG from mother to fetus (human) or newborn (rodents), and may translocate IgG over mucosal surfaces. FcRn interacts with the Fc-region of IgG and domain III of albumin with binding at pH 6.

Cyclic and dimeric gluten peptide analogues inhibiting DQ2-mediated antigen presentation in celiac disease.

Celiac disease is an immune mediated enteropathy elicited by gluten ingestion. The disorder has a strong association with HLA-DQ2. This HLA molecule is involved in the disease pathogenesis by presenting gluten peptides to T cells.

Immunocytochemical analysis of rat vagus nerve by antibodies against glycogen phosphorylase isozymes.

Glycogen is an endogenous store of glucose equivalents for energy metabolism in many tissues. The brain contains a significant amount of glycogen the role of which as an energy reserve is currently under debate. Apparently little is known concerning a possible role of glycogen in peripheral nerves.

HLA-DQ2 and -DQ8 signatures of gluten T cell epitopes in celiac disease.

Celiac disease is associated with HLA-DQ2 and, to a lesser extent, HLA-DQ8. Type 1 diabetes is associated with the same DQ molecules in the opposite order and with possible involvement of trans-encoded DQ heterodimers. T cells that are reactive with gluten peptides deamidated by transglutaminase 2 and invariably restricted by DQ2 or DQ8 can be isolated from celiac lesions.