Carbamylation reduces the capacity of IgG for hexamerization and complement activation.

Carbamylation is a post-translational modification that can be detected on a range of proteins, including immunoglobulin (Ig)G, in several clinical conditions. Carbamylated IgG (ca-IgG) was reported to lose its capacity to trigger complement activation, but the mechanism remains unclear. Because C1q binds with high affinity to hexameric IgG, we analyzed whether carbamylation of IgG affects binding of C1q, hexamerization and complement-dependent cytotoxicity (CDC). Synovial tissues of rheumatoid arthritis (RA) patients were analyzed for the presence of ca-IgG in vivo. Synovial tissues from RA patients were analyzed for the presence of ca-IgG using mass spectrometry (MS). Monomeric or hexameric antibodies were carbamylated in vitro and quality in solution was controlled. The capacity of ca-IgG to activate complement was analyzed in enzyme-linked immunosorbent (ELISAs) and cellular CDC assays. Using MS, we identified ca-IgG to be present in the joints of RA patients. Using in vitro carbamylated antibodies, we observed that ca-IgG lost its capacity to activate complement in both solid-phase and CDC assays. Mixing ca-IgG with non-modified IgG did not result in effective inhibition of complement activation by ca-IgG. Carbamylation of both monomeric IgG and preformed hexameric IgG greatly impaired the capacity to trigger complement activation. Furthermore, upon carbamylation, the preformed hexameric IgG dissociated into monomeric IgG in solution, indicating that carbamylation influences both hexamerization and C1q binding. In conclusion, ca-IgG can be detected in vivo and has a strongly reduced capacity to activate complement which is, in part, mediated through a reduced ability to form hexamers.

Identification and characterisation of peptide binding motifs of six autoimmune disease-associated human leukocyte antigen-class I molecules including HLA-B*39:06.

Research on CD8 T cell-mediated inflammatory diseases requires a better understanding of target epitopes and the constraints placed upon these by major histocompatibility complex (MHC) class I binding restrictions, especially those that relate to predisposing alleles. We used linear trap quadrupole fourier transform (LTQ-FT) tandem mass spectrometry to identify naturally processed and presented peptides eluted from the MHC-negative myeloid leukaemia cell line K562 transfected with specific MHC class I genes. We provide information on the peptidome of HLA-B*39:06, which is associated with the autoimmune disease type 1 diabetes, and extend the analysis to include a further five human leukocyte antigen (HLA) alleles (HLA-A*02:01/-A*11:01/-A*24:02/-B*18:01/-B*38:01) studied under identical experimental conditions. We identified a total of 3095 individual peptides with a mascot score ≥40 (HLA-A*02:01 = 569 peptides, -A*11:01 = 904, A*24:02 = 257, -B*18:01 = 615, -B*38:01 = 453, -B*39:06 = 297). Peptides had a preferential length of nine amino acids and originated mainly from cytoplasmic or nuclear proteins. Eluted peptides revealed a strong binding motif with binding anchor positions at position 2 (P2) and the C-terminus (PΩ). Peptides eluted from HLA-A*02:01 showed a P2 preference for leucine (62% of total peptides have Leu at P2) and PΩ preference for valine (49%). Similar data are provided for HLA-A*11:01 (P2:Thr, 29%; PΩ:Lys, 49%), -A*24:02 (P2:Tyr, 78%; PΩ:Phe, 41%), -B*18:01 (P2:Glu, 77%; PΩ:Tyr, 32%), -B*38:01 (P2:His, 51%; PΩ:Leu, 45%) and -B*39:06 (P2:Arg/His, 24%; PΩ:Ala, 64%). This work thus gives an overview of the naturally processed and presented repertoire of several common and autoimmune disease-related HLA alleles, which may be useful in studying autoreactive CD8 T cell responses and the role of HLA in disease susceptibility.

BCR-ABL fusion regions as a source of multiple leukemia-specific CD8+ T-cell epitopes.

For immunotherapy of residual disease in patients with Philadelphia-positive leukemias, the BCR-ABL fusion regions are attractive disease-specific T-cell targets. We analyzed these regions for the prevalence of cytotoxic T lymphocyte (CTL) epitopes by an advanced reverse immunology procedure. Seventeen novel BCR-ABL fusion peptides were identified to bind efficiently to the human lymphocyte antigen (HLA)-A68, HLA-B51, HLA-B61 or HLA-Cw4 HLA class I molecules. Comprehensive enzymatic digestion analysis showed that 10 out of the 28 HLA class I binding fusion peptides were efficiently excised after their C-terminus by the proteasome, which is an essential requirement for efficient cell surface expression. Therefore, these peptides are prime vaccine candidates. The other peptides either completely lacked C-terminal liberation or were only inefficiently excised by the proteasome, rendering them inappropriate or less suitable for inclusion in a vaccine. CTL raised against the properly processed HLA-B61 epitope AEALQRPVA from the BCR-ABL e1a2 fusion region, expressed in acute lymphoblastic leukemia (ALL), specifically recognized ALL tumor cells, proving cell surface presentation of this epitope, its applicability for immunotherapy and underlining the accuracy of our epitope identification strategy. Our study provides a reliable basis for the selection of optimal peptides to be included in immunotherapeutic BCR-ABL vaccines against leukemia.

A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide.

E(rns) is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. E(rns) is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine cysteines present in the mature dimer was established by analysis of the proteolytically cleaved protein. Fragments were obtained after digestion with multiple proteolytic enzymes and subsequently analyzed by liquid chromatography-electrospray ionization mass spectrometry. The analysis demonstrates which cysteine is involved in dimerization and reveals an extremely rare vicinal disulfide bridge of unknown function. With the assistance of the disulfide arrangement, a three-dimensional model was built by homology modeling based on the alignment with members of the Rh/T2/S RNase family. Compared to these other RNase family members, E(rns) shows an N-terminal truncation, a large insertion of a cystine-rich region, and a C-terminal extension responsible for membrane translocation. The homology to mammalian RNase 6 supports a possible role of E(rns) in B-cell depletion.