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. western blot analysis revealed that tPA and fibrin significantly increased proinflammatory and procoagulation proteins of intercellular adhesion molecule 1, high mobility group box 1, tumor necrosis factor α and phosphorylated nuclear factor kappa B-p65, and significantly reduced tight junction proteins zonular 1, occludin and claudin. However, Cerebrolysin significantly diminished and reversed tPA- and fibrin-impaired endothelial cell permeability, which was associated with significant reductions of tPA- and fibrin-augmented proinflammatory and procoagulation proteins and significant elevations of tPA- and fibrin-decreased tight junction proteins. The beneficial effect of Cerebrolysin appears specific because cerebroprotein hydrolysate, with a distinct peptide composition, failed to show the reduction of tPA- and fibrin-impaired permeability. These data indicate that cererbrolysin has a therapeutic effect on tPA- and fibrin-impaired cerebral endothelial cell permeability by reducing proinflammatory and procoagulation proteins and by elevating tight junction proteins.

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. Frequency of citrullinated peptides and their degree of citrullination could be determined for four known RA autoantigens, as well as a novel in vivo autocitrullination site of peptidylarginine deiminase 4. From the analysis of total and depleted synovial fluid after enrichment we could estimate the numbers of citrullinated peptides to be approximately 3600 and 2100, respectively. However, identification of these biotinylated peptides by MS/MS turned out to be very difficult due to fragmentation of the biotin moiety. By direct MS analysis of the total and depleted synovial fluid without enrichment, 119 and 157 citrullinated peptides were identified, respectively. This indicates that direct analysis allows identification of only a fraction of the citrullinated proteins present in synovial fluid and that specific enrichment is still needed for a comprehensive in-depth elucidation of the citrullinome.

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. The technique is based on highly specific in-solution biotinylation of citrulline residues followed by selective enrichment of modified peptides using streptavidin beads. We demonstrate that a synthetic citrulline-containing peptide can be selectively enriched when less than 0.5 pmol is spiked into a highly heterogeneous peptide mixture. After enrichment, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of an aliquot of the streptavidin eluate corresponding to theoretically 50 fmol of the spiked-in peptide showed a prominent signal. We further demonstrate the sensitivity of our technique by enrichment of citrullinated peptides from enzymatically deiminated myelin basic protein (MBP), when 10 pmol was spiked into a heterogeneous biological digest. In MALDI-TOF MS analysis, six MBP-derived citrullinated peptides were observed, showing the efficiency of this enrichment strategy. The high sensitivity combined with the remarkable specificity of the described technique makes it a valuable tool for elucidating citrullination in various biological processes.

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. Here we report purification of AQP4 to three silver-stained proteins on SDS-PAGE. This was achieved by organelle separation, alkaline stripping of cellular membranes, detergent solubilization and multiple chromatographic steps. The three proteins that co-purified were identified as AQP4 by mass spectrometry. These results represent the first purification of AQP4 from a native source and demonstrate by mass spectrometry the presence of a third AQP4 isoform of 36 kDa in the rat brain. Immunoblots revealed that the same isoform is present in the mouse, pig, and human brain.

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. For 118 RA patients, serum anti-hPAD4 IgG levels were stable over 10 years. Seven patients who were negative for anti-PAD4 IgG at baseline had become positive after 10 years. Further, total IgG from selected RA patients and controls were purified, and a fraction was depleted for anti-hPAD4 antibodies. Kinetic deimination assays were performed with total IgG and depleted fractions. The k ( cat ) and K ( m ) values of hPAD4-mediated deimination of N-α-benzoyl-L-arginine ethyl ester were not affected by the depletion of the anti-hPAD4 antibodies from the total IgG pool. In conclusion, RA patients remain positive for anti-hPAD4 antibodies over time and some patients who are initially anti-hPAD4 negative become positive later in the disease course. The anti-hPAD4 antibodies did not affect the enzymatic activity of hPAD4 when the small substrate N-α-benzoyl-L-arginine ethyl ester was used. However, this finding may not exclude an effect of these autoantibodies on citrullination of protein substrates in RA.

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. The peptide libraries can be designed so that the sequence length, the alignment of binding registers, the numbers and composition of random positions are controlled, and also modified amino acids can be included. Selected library peptides bound to HLA are then isolated by size exclusion chromatography and sequenced by tandem mass spectrometry online coupled to liquid chromatography. The MS/MS data are subsequently searched against a library defined database using a search engine such as Mascot, followed by manual inspection of the results. We used two dodecamer and two decamer peptide libraries and HLA-DQ2.5 to test possibilities and limits of this method. The selected sequences which we identified in the fraction eluted from HLA-DQ2.5 showed a higher average of their predicted binding affinity values compared to the original peptide library. The eluted sequences fit very well with the previously described HLA-DQ2.5 peptide binding motif. This novel method, limited by library complexity and sensitivity of mass spectrometry, allows the analysis of several thousand synthetic sequences concomitantly in a simple water soluble format.

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. This study aimed to identify and characterize neisserial DNA binding proteins in order to further elucidate the multi-factorial transformation machinery. The meningococcus inner membrane and soluble cell fractions were searched for DNA binding components by employing 1D and 2D gel electrophoresis approaches in combination with a solid-phase overlay assay with DNA substrates. Proteins that bound DNA were identified by MS analysis. In the membrane fraction, multiple components bound DNA, including the neisserial competence lipoprotein ComL. In the soluble fraction, the meningococcus orthologue of the single-stranded DNA binding protein SSB was predominant. The DNA binding activity of the recombinant ComL and SSB proteins purified to homogeneity was verified by electromobility shift assay, and the ComL-DNA interaction was shown to be Mg²+-dependent. In 3D models of the meningococcus ComL and SSB predicted structures, potential DNA binding sites were suggested. ComL was found to co-purify with the outer membrane, directly interacting with the secretin PilQ. The combined use of 1D/2D solid-phase overlay assays with MS analysis was a useful strategy for identifying DNA binding components. The ComL DNA binding properties and outer membrane localization suggest that this lipoprotein plays a direct role in neisserial transformation, while neisserial SSB is a DNA binding protein that contributes to the terminal part of the transformation process.

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). In this study we aimed to identify the preferred peptide substrates of TG2 in a heterogeneous proteolytic digest of whole wheat gluten. METHODS: A method was established to enrich for preferred TG2 substrates in a complex gluten peptide mixture by tagging with 5-biotinamido-pentylamine. Tagged peptides were isolated and then identified by nano-liquid chromatography online-coupled to tandem mass spectrometry, database searching and final manual data validation. RESULTS: We identified 31 different peptides as preferred substrates of TG2. Strikingly, the majority of these peptides were harboring known gluten T-cell epitopes. Five TG2 peptide substrates that were predicted to bind to HLA-DQ2.5 did not contain previously characterized sequences of T-cell epitopes. Two of these peptides elicited T-cell responses when tested for recognition by intestinal T-cell lines of celiac disease patients, and thus they contain novel candidate T-cell epitopes. We also found that the intact 9mer core sequences of the respective epitopes were not present in all peptide substrates. Interestingly, those epitopes that were represented by intact forms were frequently recognized by T cells in celiac disease patients, whereas those that were present in truncated versions were infrequently recognized. CONCLUSION: TG2 as well as gastrointestinal proteolysis play important roles in the selection of gluten T-cell epitopes in celiac disease.

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. Using mass spectrometry, we have identified a redox-sensitive cysteine triad consisting of Cys(230), Cys(370), and Cys(371) that is involved in oxidative inactivation of TG2. Within this triad, Cys(370) was found to participate in disulfide bonds with both Cys(230) and its neighbor, Cys(371). Notably, Ca(2+) was found to protect against formation of these disulfide bonds. To investigate the role of each cysteine residue, we created alanine mutants and found that Cys(230) appears to promote oxidation and inactivation of TG2 by facilitating formation of Cys(370)-Cys(371) through formation of the Cys(230)-Cys(370) disulfide bond. Although vicinal disulfide pairs are found in several transglutaminase isoforms, Cys(230) is unique for TG2, suggesting that this residue acts as an isoform-specific redox sensor. Our findings suggest that oxidation is likely to influence the amount of active TG2 present in the extracellular environment.

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. This study presents a simple technique for a highly specific enrichment of citrullinated peptides that is based on the specific reaction of glyoxal derivatives with the citrulline ureido group under acidic conditions. Beads were functionalized with 4-hydroxyphenylglyoxal attached via a base-labile linker. Incubation of these "citrulline reactive beads" with peptide mixtures at low pH resulted in selective immobilization of citrullinated peptides. Unbound noncitrullinated peptides were removed by extensive washing. Finally, citrullinated peptides carrying a modified ureido group were cleaved off at high pH and were analyzed by mass spectrometry. The procedure was validated by enrichment of synthetic citrulline-containing peptides from a tryptic digest of bovine serum albumin and from an endoproteinase LysC digest of a cytosolic fraction of a cell line. The technique was further applied to enrich citrullinated peptides from a digest of deiminated myelin basic protein.

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. In dermatitis herpetiformis (DH), TG3 appears to be the target autoantigen whereas in gluten ataxia (GA) autoantibodies reactive with TG6 are present. A functional role for TG3 and TG6 in these diseases has yet to be described. It is also not known whether these enzymes can use gluten peptides implicated in the pathology as substrates. We here report that similar to TG2, TG3 and TG6 can specifically deamidate gluten T cell epitopes. However, the fine specificities of the enzymes were found to differ. TG2 can form covalent complexes with gluten by iso-peptide and thioester bonds. We found that both TG3 and TG6 were able to complex with gluten peptides through thioester linkage although less efficiently than TG2, whereas TG6 but not TG3 was able to form iso-peptide linked complexes. Our findings lend credence to the notion that TG3 and TG6 are involved in the gluten-induced autoimmune responses of DH and GA.

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. We have used a positional scanning nonapeptide library to determine the optimal amino acids for each position of the HLA-DQ2 binding frame. By combining the optimal residues in each position, we were able to design high affinity binders to HLA-DQ2. Interestingly, the decapeptide with highest affinity was composed of the most favorable residues in each position. This sequence bound 50-fold better than the immunodominant gluten epitope DQ2-alpha-I-gliadin, which makes it an interesting lead compound for the development of blockers. For some natural HLA-DQ2 ligands, the correlation between measured and predicted affinities was poorer, but notably these peptides did not have optimal amino acids at all positions. Our approach represents a straightforward strategy for developing high-affinity binders to HLA class II molecules.

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. We have recently reported on the details of this reaction. Here, we show that this chemical modification can be utilized to specifically detect and identify citrullinated peptides and their citrullination sites by liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Using model compounds, we demonstrate that in collision-induced dissociation (CID) a specific, modification-derived fragment ion appears as the dominating signal at m/z 201.1 in the MS/MS spectra. When applying electron transfer dissociation (ETD), however, the chemical modification of citrulline remained intact and extensive sequence coverage allowed identification of peptides and their citrullination sites. Therefore, LC/MS/MS analysis with alternating CID and ETD has been performed, using CID for specific, signature ion-based detection of derivatized citrullinated peptides and ETD for sequence determination. The usefulness of this targeted analysis was demonstrated by identifying citrullination sites in myelin basic protein deiminated in vitro. Combining antibody-based enrichment of chemically modified citrulline-containing peptides with specific mass spectrometric detection will increase the potential of such a targeted analysis of protein citrullination in the future.

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. Several T-cell epitopes of gluten exist, and most of these epitopes derive from the alcohol-soluble gliadin fraction. For some of these epitopes, specific T cells can be isolated from intestinal biopsies from nearly all patients, whereas for others, T-cell reactivity could be demonstrated in only a few patients. One reason for this observation could be that the rate of transglutaminase 2 (TG2)-mediated deamidation significantly differs between these peptides, resulting in different amounts of epitopes generated in vivo. In this study, we established a quantitative, mass spectrometry-based approach to measure the kinetics of TG2-mediated deamidation of gliadin-derived, DQ2-restricted epitopes. Our results demonstrate large variations in the degree of deamidation between different peptides and also between individual glutamine residues within each peptide. In general, alpha-gliadin derived epitopes that are frequently recognized by patient T cells showed a significant higher level of deamidation compared to the majority of epitopes from gamma-gliadin that are less frequently recognized. The degree of deamidation of individual residues within a peptide also seems to influence whether some epitopes are better recognized in context of DO2 or DQ8. Thus, the rate of deamidation by TG2 appears to be a factor of importance for the T-cell response to gluten in celiac disease.

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. Inner membrane fractions from a panel of neisserial strains were subjected to a solid-phase overlay assay with DNA substrates, and MS was subsequently employed to identify proteins that bind DNA. A number of DNA binding components were detected, including the pilus biogenesis component PilG, the competence protein ComL, and the cell division ATP-binding protein FtsE, as well as two hypothetical proteins. The DNA binding activity of these components was not dependent on the presence of the neisserial DNA uptake sequence. Null mutants, corresponding to each of the proteins identified, were constructed to assess their phenotypes. Only mutants defective in pilus biogenesis were non-competent and non-piliated. The DNA binding activity of the pilus biogenesis components PilQ and PilG and the phenotypes of their respective null mutants suggest that these proteins are directly involved as players in natural transformation, and not only indirectly, through pilus biogenesis.

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 11.5. After affinity purification, the sDQ2/DCB-45 complex exists in substantial quantities next to a small amount of free heterodimeric sDQ2 and large amounts of aggregated sDQ2 free of DCB-45. Motivated by the stable complex formation and our interest in the development of reagents which inhibit HLA-DQ2 peptide binding, we have further characterized the sDQ2/DCB-45 interaction. Several lines of evidence indicate that an N-terminal fragment of DCB-45 is involved in the interaction with the peptide binding groove of sDQ2. Further mapping of this fragment of 54 residues identified a pentadecapeptide with high affinity for sDQ2 which may serve as a lead compound for the design of HLA-DQ2 blockers.

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. Using two peptide substrates, residues in positions -2, -1, +1, and +2 relative to the central arginine targeted by PAD-4 were systematically replaced by all natural L-amino acids except cysteine. Each peptide was treated with recombinant human PAD-4 and deimination was analyzed by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. In all four flanking positions, amino acids which positively or negatively influenced deimination were identified. We next designed peptides with expected high or low deimination rates and determined their Km and kcat values. These peptides showed PAD-4 substrate behavior as predicted, demonstrating that residues flanking the targeted arginine are important for deimination. Further truncation of peptide substrates suggested additional effects on deimination by residues outside the -2 to +2 region. Finally, we observed that a methylated lysine residue flanking the targeted arginine influences PAD-4-mediated deimination, also suggesting that posttranslational modifications can affect substrate efficiency.

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. We hypothesized that DQ2-CLIP1/2 might be poor substrates for DM. We measured DM-mediated exchange of CLIP and other peptides for high-affinity indicator peptides and found it is inefficient for DQ2. DM-DQ-binding and DM chaperone effects on conformation and levels of DQ are also reduced for DQ2, compared with DQ1. We suggest that the unusual interaction of DQ2 with Ii and DM may provide a basis for the known disease associations of DQ2.

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. Here we have measured peptide deamidation and transamidation in the same reaction to reveal factors that affect the relative propensity for the two possible products. We report that the propensity for deamidation by TG2 is both substrate dependent and influenced by the reaction conditions. Direct deamidation is favored for poor substrates and at low concentrations of active TG2, while indirect deamidation (i.e. hydrolysis of transamidated product) can significantly contribute to the deamidation of good peptide substrates at higher enzyme concentrations. Further, we report for the first time that TG2 can hydrolyze iso-peptide bonds between two peptide substrates. This was observed also for gliadin peptides introducing a novel route for the generation of deamidated T cell epitopes in celiac disease.

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. To study FcRn in vitro and to select and characterize FcRn binders, large quantities of soluble human FcRn are needed. In this report, we explored the impact of two free cysteine residues (C48 and C251) of the FcRn heavy chain on the overall structure and function of soluble human FcRn and described an improved bacterial production strategy based on removal of these residues, yielding approximately 70 mg.L(-1) of fermentation of refolded soluble human FcRn. The structural and functional integrity was proved by CD, surface plasmon resonance and MALDI-TOF peptide mapping analyses. The strategy may generally be translated to the large-scale production of other major histocompatibility complex class I-related molecules with nonfunctional unpaired cysteine residues. Furthermore, the anti-FcRn response in goats immunized with the FcRn heavy chain alone was analyzed following affinity purification on heavy chain-coupled Sepharose. Importantly, purified antibodies blocked the binding of both ligands to soluble human FcRn and were thus directed to both binding sites. This implies that the FcRn heavy chain, without prior assembly with human beta2-microglobulin, contains the relevant epitopes found in soluble human FcRn, and is therefore sufficient to obtain binders to either ligand-binding site. This finding will greatly facilitate the selection and characterization of such binders.