The icosahedral T7 phage (diameter approximately 65 nm) displaying random peptides at the carboxy-terminus of the phage coat proteins was used as a model for drug and gene delivery vehicles containing peptide ligands. We found that displayed peptides were recognized by natural antibodies and induced complement activation. Strikingly, the phage inactivation by complement was peptide-specific that implied the existence of numerous natural antibodies with different peptide specificity. Selection of phage that avoided inactivation by complement allowed the identification of peptides that protected the phage by binding to serum proteins. In rat blood, peptides with carboxy-terminal lysine or arginine residues protected the phage against complement-mediated inactivation by binding C-reactive protein. In human serum, a number of protective peptides with tyrosine residues were selected. The recognition of displayed peptides by natural antibodies appears to represent a universal mechanism for activation of complement at sites that contain identical or homologous proteins with exposed carboxy-termini.

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Our previous study indicated that normal serum contains complement-fixing natural IgM antibodies reacting with a large variety of randomly generated protein carboxy-termini. Here we show that the "carboxy-terminal" IgM (C-IgM) antibodies specifically react with short peptide sequences located immediately at the protein carboxy-terminus. The specificity of C-IgM-peptide interactions is tentatively defined by three to four amino acid residues. All carboxy-terminal peptides in a large peptide library apparently react with C-IgM antibodies. Immobilized synthetic peptides also react with C-IgM antibodies. No interaction of C-IgM antibodies with internal peptide sequences has been observed. C-IgM antibodies are present in germ-free and in athymic adult rats and are absent in newborn rats. The natural ubiquity of protein carboxy-termini in biological structures suggests that C-IgM could play an important role in antigen clearance and presentation to the immune system. From a practical viewpoint, the recognition of carboxy-terminal peptides by complement-fixing C-IgM antibodies has profound implications for the use of peptide- and protein-derivatized delivery vehicles and artificial materials.

SUMMARYBoth the timely clearance of degraded endogenous structures and the presence of secreted natural immunoglobulin M (IgM) are needed to avoid autoimmunity. These requirements may be causally related provided that natural IgM preferentially reacts with degraded antigens and, by activating complement, mediates their non-inflammatory clearance through complement receptors. We have previously shown that normal serum IgM reacts in vivo and in vitro with virtually all randomly generated C-terminal peptides displayed on T7 phage. The resultant multivalent IgM-peptide complexes activate complement and are detected by a loss of phage infectivity. A striking feature of these reactions is that different C-terminal peptides (» 3-4 amino acids) specifically react with different 'C-terminal' IgM (C-IgM) antibodies. This suggests that degraded supramolecular structures, expressing elevated levels of identical C-termini as a result of proteolysis, denaturation and abnormal exposure of repetitive protein constituents, may be preferential targets of C-IgM-mediated complement activation in the physiological environment. The specificity of C-IgM-peptide reactions is much higher than one would expect, assuming that normal serum IgM mostly comprises polyspecific natural antibodies. However, it is possible that polyspecific IgM is not 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.

Natural IgM antibodies (Abs) play an important role in clearing pathogens, enhancing immune responses, and preventing autoimmunity. However, the molecular mechanisms that mediate the functions of natural IgM Abs are understood only to a limited degree. This shortcoming is largely due to the fact that isolated natural IgM Abs are commonly polyspecific and recognize a variety of antigens (Ags) with no apparent structural homology. It is generally believed that polyspecificity is an inherent property of natural Abs. However, there is increasing evidence that polyspecificity may be induced by mild denaturing conditions. In this study, we compared the specificity of three polyspecific IgM Abs in conventional buffers and undiluted sera deficient in immunoglobulins. All three Abs lost their polyspecificity in serum. They no longer reacted with conventional screening Ags, including hapten-BSA conjugates, ssDNA, thyroglobulin and myosin, but fully retained their reactivity with cognate peptide Ags selected from a T7 phage library. The acquisition of narrow specificity by polyspecific IgM in serum was also observed with muscle tissue sections used as a source of endogenous Ags. The loss of polyspecificity by different Abs was apparently dependent on the presence of different serum constituents. The results of this study suggest that the seemingly inherent polyspecificity of many natural IgM Abs may be largely an in vitro phenomenon related to the lack of normal serum components in the medium. Potential mechanisms underlying the loss of polyreactivity are discussed.