Mapping of B cell epitopes on desmoglein 3 in pemphigus vulgaris patients by the use of overlapping peptides.

BACKGROUND: Pemphigus vulgaris (PV) is a severe autoimmune blistering disease associated with autoantibodies to desmoglein 3 (Dsg 3), a transmembrane glycoprotein of the cadherin family. Previous studies mainly focused on the mapping of conformational epitopes of Dsg 3 using recombinant fragments of Dsg 3 and competition ELISA. OBJECTIVE: Here, we performed a mapping of linear B cell epitopes on Dsg 3 in PV patients by the use of overlapping synthetic peptides. METHODS: A set of 254 overlapping synthetic peptides of 14 amino acids length covering the entire Dsg 3 extracellular domain was generated. Sera of patients with active PV (n=10) and healthy volunteers (n=10) were tested for IgG reactivity with the 254 peptides by ELISA. Testing each peptide separately, 7 major antigenic sites were identified. In order to validate these reactivities, 7 corresponding peptides of 13-33 amino acids in length were generated and employed by ELISA. Additional sera of active PV patients (n=17) and healthy volunteers (n=20) were tested and the most reactive peptide was used to specifically purify anti-Dsg 3 antibodies from PV sera (n=3). RESULTS: The major autoantibody reactivity in PV sera was mapped to amino acids 333-356 within the EC3 domain. Purifying patients IgG using the identified peptide, however, failed to induce acantholysis in keratinocyte dissociation assay. CONCLUSION: We conclude that linear epitopes do not play a major pathogenic role in human PV.

Intracellular degradation of somatostatin-14 following somatostatin-receptor3-mediated endocytosis in rat insulinoma cells.

Somatostatin receptor (SSTR) endocytosis influences cellular responsiveness to agonist stimulation and somatostatin receptor scintigraphy, a common diagnostic imaging technique. Recently, we have shown that SSTR1 is differentially regulated in the endocytic and recycling pathway of pancreatic cells after agonist stimulation. Additionally, SSTR1 accumulates and releases internalized somatostatin-14 (SST-14) as an intact and biologically active ligand. We also demonstrated that SSTR2A was sequestered into early endosomes, whereas internalized SST-14 was degraded by endosomal peptidases and not routed into lysosomal degradation. Here, we examined the fate of peptide agonists in rat insulinoma cells expressing SSTR3 by biochemical methods and confocal laser scanning microscopy. We found that [(125)I]Tyr11-SST-14 rapidly accumulated in intracellular vesicles, where it was degraded in an ammonium chloride-sensitive manner. In contrast, [(125)I]Tyr1-octreotide accumulated and was released as an intact peptide. Rhodamine-B-labeled SST-14, however, was rapidly internalized into endosome-like vesicles, and fluorescence signals colocalized with the lysosomal marker protein cathepsinD. Our data show that SST-14 was cointernalized with SSTR3, was uncoupled from the receptor, and was sorted into an endocytic degradation pathway, whereas octreotide was recycled as an intact peptide. Chronic stimulation of SSTR3 also induced time-dependent downregulation of the receptor. Thus, the intracellular processing of internalized SST-14 and the regulation of SSTR3 markedly differ from the events mediated by the other SSTR subtypes.

Agonist-induced endocytosis of rat somatostatin receptor 1.

Somatostatin-receptor 1 (sst1) is an autoreceptor in the central nervous system that regulates the release of somatostatin. Sst1 is present intracellularly and at the cell surface. To investigate sst1 trafficking, rat sst1 tagged with epitope was expressed in rat insulinoma cells 1046-38 (RIN-1046-38) and tracked by antibody labeling. Confocal microscopic analysis revealed colocalization of intracellularly localized rat sst1-human simplex virus (HSV) with Rab5a-green fluorescent protein and Rab11a-green fluorescent protein, indicating the distribution of the receptor in endocytotic and recycling organelles. Somatostatin-14 induced internalization of cell surface receptors and reduction of binding sites on the cell surface. It also stimulated recruitment of intracellular sst1-HSV to the plasma membrane. Confocal analysis of sst1-HSV revealed that the receptor was initially transported within superficial vesicles. Prolonged stimulation of the cells with the peptide agonist induced intracellular accumulation of somatostatin-14. Because the number of cell surface binding sites did not change during prolonged stimulation, somatostatin-14 was internalized through a dynamic process of continuous endocytosis, recycling, and recruitment of intracellularly present sst1-HSV. Accumulated somatostatin-14 bypassed degradation via the endosomal-lysosomal route and was instead rapidly released as intact and biologically active somatostatin-14. Our results show for the first time that sst1 mediates a dynamic process of endocytosis, recycling, and re-endocytosis of its cognate ligand.

Development of peptide microarrays for epitope mapping of antibodies against the human TSH receptor.

Accurate characterization of the antigen binding region of antibodies is of great value in many fields of research, assay development and clinical diagnostics. Up to now, there is an unmet clinical need to use antibodies as diagnostic markers for the prediction of both prognosis and therapeutic response. To this end, comprehensive but differentiated immunoassays need to be generated. We have developed a peptide microarray for the diagnosis and epitope mapping of anti-thyrotropin receptor antibodies. The primary sequence of the human thyrotropin receptor (hTSHR) was represented by a library of 251 synthetic peptides. The peptides were site-specifically immobilized in a two-step procedure first by coupling of biotinylated peptides to hydrazide-modified streptavidin and then utilizing a subsequent chemoselective reaction between the hydrazide linkers of the streptavidin and an aldehyde coated glass surface. The technology was used to map the epitopes of seven commercially available murine monoclonal antibodies specific for the human TSH receptor (mTSHRAb). A previously unknown epitope recognized by mTSHRAb 4C1 was identified at amino acids (AA) 379 through 384 and the epitope recognized by mTSHRAb A9 was also localized (AA 214-222). Previously identified epitopes recognized by mTSHRAbs 2C11 (AA 349-360), 28 (AA 34-39), 49 (AA 289-297), A7 (AA 406-411) and A10 (AA 34-39) were confirmed. The peptide microarray exhibited excellent performance in single and multiplex antibody analysis and high specificity. This technology may have potential as a multi-determinate in vitro diagnostic assay for the differential analysis of a heterogeneity of antibodies involved in the pathogenesis of autoimmune diseases.

Functional peptide microarrays for specific and sensitive antibody diagnostics.

Peptide microarrays displaying biologically active small synthetic peptides in a high-density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site-specific solution-phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide-NA-complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence-labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA-mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low-picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.

Peptide microarrays with site-specifically immobilized synthetic peptides for antibody diagnostics.

Peptide microarrays bear the potential to discover molecular recognition events on protein level, particularly in the field of molecular immunology, in a manner and with an efficiency comparable to the performance of DNA microarrays. We developed a novel peptide microarray platform for the detection of antibodies in liquid samples. The system comprises site-specific solution phase coupling of biotinylated peptides to NeutrAvidin, localized microdispensing of peptide-NeutrAvidin conjugates onto activated glass slides and a fluorescence immuno sandwich assay format for antibody capture and detection. Our work includes synthetic peptides deduced from amino acid sequences of immunodominant linear epitopes, such as the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c-myc protein and three domains of the Human coronavirus 229E polymerase polyprotein. We demonstrate that our method produces peptide arrays with excellent spot morphology which are capable of specific and sensitive detection of monoclonal antibodies from fluid samples.