Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263–275) epitope: an MHC anchor variant peptide for immune modulation

We sought to identify an altered peptide ligand (APL) based on the endogenously expressed synovial auto-epitope of human cartilage glycoprotein-39 (HC gp-39) for modulation of cognate, HLA-DR4-restricted T cells. For this purpose we employed a panel of well-characterized T cell hybridomas generated from HC gp-39-immunized HLA-DR4 transgenic mice. The hybridomas all respond to the HC gp-39(263–275) epitope when bound to HLA-DR4(B1*0401) but differ in their fine specificities. First, the major histocompatibility complex (MHC) and T-cell receptor (TCR) contact residues were identified by analysis of single site substituted analogue peptides for HLA-DR4 binding and cognate T cell recognition using both T hybridomas and polyclonal T cells from peptide-immunized HLA-DR4 transgenic mice. Analysis of single site substituted APL by cognate T cells led to identification of Phe265 as the dominant MHC anchor. The amino acids Ala268, Ser269, Glu271 and Thr272 constituted the major TCR contact residues, as substitution at these positions did not affect HLA-DR4(B1*0401) binding but abrogated T cell responses. A structural model for visualisation of TCR recognition was derived. Second, a set of non-classical APLs, modified at the MHC key anchor position but with unaltered TCR contacts, was developed. When these APLs were analysed, a partial TCR agonist was identified and found to modulate the HC gp-39(263–275)-specific, pro-inflammatory response in HLA-DR4 transgenic mice. We identified a non-classical APL by modification of the p1 MHC anchor in a synovial auto-epitope. This APL may qualify for rheumatoid arthritis immunotherapy

Optimizing heterologous expression in Dictyostelium: importance of 5′ codon adaptation

Expression of heterologous proteins in Dictyostelium discoideum presents unique research opportunities, such as the functional analysis of complex human glycoproteins after random mutagenesis. In one study, human chorionic gonadotropin (hCG) and human follicle stimulating hormone were expressed in Dictyostelium. During the course of these experiments, we also investigated the role of codon usage and of the DNA sequence upstream of the ATG start codon. The Dictyostelium genome has a higher AT content than the human, resulting in a different codon preference. The hCG-β gene contains three clusters with infrequently used codons that were changed to codons that are preferred by Dictyostelium. The results reported here show that optimizing the first 5–17 codons of the hCG gene contributes to 4- to 5-fold increased expression levels, but that further optimization has no significant effect. These observations suggest that optimal codon usage contributes to ribosome stabilization, but does not play an important role during the elongation phase of translation. Furthermore, adapting the 5′-sequence of the hCG gene to the Dictyostelium ‘Kozak’-like sequence increased expression levels ~1.5-fold. Thus, using both codon optimization and ‘Kozak’ adaptation, a 6- to 8-fold increase in expression levels could be obtained for hCG

Human cartilage gp-39+, CD16+monocytes in peripheral blood and synovium : correlation with joint destruction in rheumatoid arthritis

Objective. To investigate the expression of human cartilage (HC) gp-39, a possible autoantigen in rheumatoid arthritis (RA), in peripheral brood and synovium, to characterize its cellular source, and to analyze correlations with clinical features, Methods. The expression of HC gp-39 in synovium and peripheral blood mononuclear cells (PBMC) was assessed by immunohistochemistry and flow cytometry, Synthesis and secretion were investigated by both reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Results. PBRIC expressing HC gp-39 were increased in RA patients compared with spondylarthropathy patients (P = 0.0029) and with healthy control subjects (P = 0.0013), HC gp-39+ cells were also slightly overrepresented in RA synovium (P = 0.01). In both blood and synovium, HC gp-39+ cells were identified as CDT4dim, CD16+ monocytes, a phenotype which can differentiate from classic CD14++ monocytes by maturation in vitro. HC gp-39 messenger RNA was detected in RA synovium and PBMC, and PBMC secreted HC gp-39 in vitro. The number of HC gp-39+ PBMC correlated with serum levels of C-reactive protein (r = 0.39, P = 0.003) and HC gp-39 (r = 0.52, P = 0.014). HC gp-39 expression in Ri synovial lining correlated with joint destruction (r = 0.77, P < 0.001), Conclusion, CD16+ monocytes, a cellular source of HC gp-39 in vivo, are overrepresented in both RA peripheral blood and synovial tissue. The presence of HC gp-39+ cells in RA synovium is correlated with the degree of joint destruction. These data support a role of these cells in the local autoimmune response that leads to chronic inflammation and joint destruction

A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA

Proteomic

Structures of the HC gp-39(263–275) sequence (compound 1) and of a series of modified peptides at anchor position 1 (P1, Phe265; compounds 24 to 35) and associated bioactivities

<p><b>Copyright information:</b></p><p>Taken from "Identification of an altered peptide ligand based on the endogenously presented, rheumatoid arthritis-associated, human cartilage glycoprotein-39(263–275) epitope: an MHC anchor variant peptide for immune modulation"</p><p>http://arthritis-research.com/content/9/4/R71</p><p>Arthritis Research & Therapy 2007;9(4):R71-R71.</p><p>Published online 23 Jul 2007</p><p>PMCID:PMC2206373.</p><p></p> Binding of MHC anchor variant peptides to HLA-DR4(B1*0401) was determined in a competition binding assay. IC= 50% inhibitory concentration. All peptides were found to bind HLA-DR4 with high relative affinity (ICranged between 0.001 and 0.38 μM). The hybridoma response (IL-2 production) to wild-type (WT) peptide and MHC anchor variant peptides presented by HLA-DRB1*0401 expressing B lymphoblastoid cells as source of antigen-presenting cells is expressed as stimulation index (SI). The SI values are based on mean fluorescence counts derived from duplicate or triplicate measurements and calculated as the ratio of mean fluorescence counts of antigen stimulated cultures and control cultures. Background (no peptide added) values for hybridoma 5G11, 8B12 and 14G11 were 29,203, 16,288 and 7,152 mean fluorescence units/counts, respectively. The standard deviation of measurements did not exceed 15%. Values greater or less than 30% (2 × the standard deviation) of the positive control (response to WT peptide) are defined as super agonists (+30%) and partial agonists (-30%) respectively and are indicated in bold