In another set of experiments, T7DGA-K strongly, yet non-reciprocally, competed with T7DGA-R (= 001), and neither of these clones significantly competed with T7DGA-N (< 005; Fig

In another set of experiments, T7DGA-K strongly, yet non-reciprocally, competed with T7DGA-R (= 001), and neither of these clones significantly competed with T7DGA-N (< 005; Fig. IgM mostly comprises polyspecific natural antibodies. However, it is possible that polyspecific IgM is not Vav1 adequately registered by our functional phage-inactivation assays. In this study, we resolve the issue of C-IgM specificity by directly characterizing the binding reactivity of normal serum IgM with phage-displayed C-terminal peptides. Keywords: IgM, natural antibodies, phage display, T7 phage Introduction Natural immunoglobulin M (IgM) antibodies are produced in mice, and apparently in humans, mostly by B1 cells, and constitute an important part of the innate immune system.1,2 Their Mitoquinone mesylate major functions include the Mitoquinone mesylate interception of bacterial and viral pathogens3,4 and the promotion of primary immune responses.5,6 An intriguing aspect of the physiology of natural IgM antibodies is their reactivity with endogenous antigens. Previous studies have demonstrated that antigen-free mice express almost normal levels of serum IgM and that the binding patterns produced by this IgM in multiantigen panels are similar to those observed for mice reared on a conventional diet.7,8 Therefore, it was proposed that the production of natural IgM was specified by as-yet-unidentified endogenous antigens.7C9 This notion was later supported by the observations that an endogenous antigen specifically stimulated the B1 cells expressing a cognate antigen receptor in a transgenic murine model.9 Recent studies have demonstrated that mice deficient in secreted IgM develop high levels of autoreactive immunoglobulin G (IgG).10,11 One plausible explanation for this phenomenon is that natural IgM reacts with degraded endogenous structures and, by activating complement, mediates their non-inflammatory clearance by the cells expressing complement receptors.12 The failure to eliminate degraded structures from the circulation seems to be a strong prerequisite for the development Mitoquinone mesylate of autoimmunity. Thus, the accumulation of apoptotic cells in the lymphoid organs of mice deficient in the protein kinase is accompanied by development of a lupus-like disease.13 Other proteins, whose deficiency in mice leads to a similar manifestation of autoimmunity, include C1q that reacts with apoptotic blebs,14 DNAse I that is apparently required for the degradation of released chromatin15 and serum amyloid P that binds to chromatin and probably prevents its recognition by autoreactive B cells.16 A selective recognition by natural IgM of degraded antigens has been observed in a number of experimental systems, including apoptotic T cells12 and protease-treated17 or disease-transformed red blood cells.18 Lysolipid phosphorylcholine and structurally altered band 3 protein have been identified as IgM-reactive antigens in degraded T cells and red blood cells, respectively.12,18 We have recently identified a new group of well-defined antigenic determinants that react with natural IgM and in complement-grade serum and are probably expressed at elevated levels in degraded supramolecular structures. These are peptide sequences, approximately three amino acids (aa) long, located at protein C-termini immediately. By injecting phage-displayed peptide libraries into rats, we discovered that virtually all arbitrarily produced C-terminal peptides shown within the T7 phage layer proteins 10B (415 copies per phage particle) reacted with organic IgM.19 These IgMCpeptide reactions resulted in phage inactivation by complement and had been functionally discovered by phage plaque assays. The phage clones demonstrating a higher rate of success in rats either acquired truncated 10B proteins or shown peptides using a C-terminal K or R residue. The C-terminal K and R residues covered phage against organic IgM via binding constitutively portrayed rat C-reactive proteins through its phosphorylcholine-binding sites.19 A dazzling feature from the IgMCpeptide reactions was that different phage-displayed peptides specifically reacted with different C-terminal IgM (C-IgM) antibodies. No polyspecific C-IgM, displaying a considerable amount of cross-reactivity with nonhomologous peptide, was discovered.20 C-IgM antibodies, responding with different shown peptides, had been taken off highly purified polyclonal IgM by selectively.