Type II collagen (CII) is a candidate cartilage‐specific autoantigen, which can become post‐translationally modified by hydroxylation and glycosylation. T cell recognition of CII is essential for the development of murine collagen‐induced arthritis (CIA) and also occurs in rheumatoid arthritis (RA). The common denominator of murine CIA and human RA is the presentation of an immunodominant CII‐derived glycosylated peptide on murine Aq and human DR4 molecules, respectively. To investigate the importance of T cell recognition of glycosylated CII in CIA development after immunization with heterologous CII, we treated neonatal mice with different heterologous CII‐peptides (non‐modified, hydroxylated and galactosylated). Treatment with the galactosylated peptide (galactoseat position 264) was superior in protecting mice from CIA. Protection was accompanied by a reduced antibody response to CII and by an impaired T cell response to the glycopeptide. To investigate the importance of glycopeptide recognition in an autologous CIA model, we treated MMC‐transgenic mice, which express the heterologous CII epitope with a glutamic acid in position 266 in cartilage, with CII‐peptides. Again, a strong vaccination potential of the glycopeptide was seen. Hence CII‐glycopeptides may be the optimal choice of vaccination target in RA, since humans share the same epitope as the MMC mouse.

Antibody affinity limits sensitivity of detection in many areas of biology and medicine. High affinity usually depends on achieving the optimal combination of the natural 20 amino acids in the antibody binding site. Here, we investigate the effect on recognition of protein targets of placing an unnatural electrophile adjacent to the target binding site. We positioned a weak electrophile, acrylamide, near the binding site between an affibody, a non-immunoglobulin binding scaffold, and its protein target. The proximity between cysteine, lysine, or histidine on the target protein drove covalent bond formation to the electrophile on the affibody. Covalent bonds did not form to a noninteracting point mutant of the target, and there was minimal cross-reactivity with serum, cell lysate, or when imaging at the cell surface. Electrophilic affibodies showed more stable protein imaging at the surface of mammalian cells, and the sensitivity of protein detection in an immunoassay improved by two orders of magnitude. Thus electrophilic affibodies combined good specificity with improved detection of protein targets.Human blood contains a panoply of proteins below the detection limit of standard immunoassays, including early markers of cancer or neurodegeneration (1, 2). Immunoassays have advanced enormously over the last 60 years, opening up whole new fields of research from the ability to detect lower and lower abundance species (3). Nearly all enzyme-linked immunosorbent assays (ELISAs) 2 stop working at low picomolar concentration of target (4), but recently, methods such as nanowires (5) or the bio-barcode assay (6) have made leaps in detection sensitivity. These advances mean that amplification is not the limitation on immunoassay sensitivity any longer; it is the antibody itself that is the limiting factor (4). Antibody binding strength correlates with protection from disease agents (7, 8) and can be a limiting factor in live cell imaging, since antibody off-rates are on the order of 30 min (9 -11) but lifetimes of cell surface proteins are often ϳ1 day.Affinity-matured antibodies generally have micromolar to nanomolar monovalent affinity for their target (9). It has proved very difficult to generate antibodies of subnanomolar monovalent affinity. This may be because of a limit to the affinity of antibodies that can be selected in an immunized animal, as a result of the kinetics of B cell stimulation (11). In vitro selection methods based on phage display (12), mRNA/ribosome display (13), or yeast cell surface display (14) have sometimes managed to obtain femtomolar affinity antibodies. There are a few examples of antibodies that form covalent bonds to artificial small molecule antigens (15-17), but these strategies, such as the antibody made by Meares' group to an electrophilic metal chelator, do not enable recognition of protein antigens composed of the regular 20 amino acids. Immunizing mice with electrophilic antigens generated an antibody that formed SDS-stable complexes with its target, but the chemistry of the i...

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Presentation of (glyco)peptides by the class II major histocompatibility complex molecule Aq to T cells plays a central role in collagen-induced arthritis, an animal model for the autoimmune disease rheumatoid arthritis. A peptide library was designed using statistical molecular design in amino acid space in which five positions in the minimal mouse collagen type II binding epitope CII260-267 were varied. A substantially reduced peptide library of 24 peptides with diverse and representative molecular characteristics was selected, synthesized, and evaluated for the binding strength to Aq. A multivariate QSAR model was established by correlating calculated descriptors, compressed to its principle properties, with the binding data using partial least-square regression. The model was successfully validated by an external test set. Interpretation of the model provided a molecular property binding motif for peptides interacting with Aq. The information may be useful in future research directed toward new treatments of rheumatoid arthritis.

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The complement serum proteins C3 and C4 and the protease inhibitor α-2 macroglobulin are all members of the C3/α-2M thioester protein family, an evolutionarily ancient and conserved family that contains an intrachain thioester bond. The chemistry of the thioester bond is a key to the function of the thioester proteins. All these proteins function by covalently linking to their target by acyl transfer of the protein via the thioester moiety. We show that the signature thioester bond can be targeted with nucleophiles linked to a bioreporter molecule, site-specifically modifying the whole, intact thioester protein. Conditions were optimised to label selectively and efficiently pull-down unprocessed thioester-containing proteins from serum. We demonstrated pull-down of full-length C3, α-2M and C4 from sera in high salt, using a biotinylated nucleophile and streptavidin-coated resin, confirmed by MALDI-TOF MS identification of the gel bands. The potential for the development of a quantitative method for measuring active C3 in serum was investigated in patient sera pre and post operation. Quantifying active C3 in clinical assays using current methods is difficult. Methods based on antibody detection (e.g. nephelometry) do not distinguish between active C3 and inactive breakdown products. C3-specific haemolytic assays can be used, but these require use of relatively unstable reagents. The current work represents a promising robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in blood, plasma or serum.

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