The epitopes for natural polyreactive antibodies are rich in proline

Abstract

"Natural" polyreactive antibodies, which bind in a nonspecific manner to a range of biological molecules both of self- and nonself- origin, are normal constituents of serum and are a significant part of the immune repertoire in many species, including humans. Autoantibodies to sTNF-R (the 55-kDa extracellular domain of the human receptor to tumor necrosis factor alpha) were affinity purified from normal human sera using immobilized sTNF-R. The isolated anti-sTNF-R IgG bound both native and denatured forms of the receptor with low affinity. These antibodies also bound to different proteins and therefore are considered to be polyreactive. We used the anti-sTNF-R antibodies and purified polyreactive antibodies to mannose-specific lectin from garlic (Allium sativum) for screening a peptide library displayed on filamentous M13 phage. After the biopanning procedure, we failed to find epitopes with a consensus sequence; however, we found that proline is the most frequent amino acid in the selected phagotopes. Proline is commonly present at solvent-exposed sites in proteins, such as loops, turns, N-terminal first turn of helix, and random coils. Thus, structures containing proline can serve as conformation-dependent common "public" epitopes for polyreactive natural antibodies. Our findings may be important for understanding polyreactivity in general and for the significance of polyreactive natural antibodies in immunological homeostasis.

Figure 1

Dose-dependent binding of Igs to an sTNF-R-coated plate. Normal (▪, □) or IgG-depleted (▴, ▵) human serum was used for the binding experiment, before (▪, ▴) and after (□, ▵) preliminary acidification. Acidification was accomplished using 0.1 M glycine buffer (pH 3), and the acidified serum was immediately neutralized using 1 M Tris⋅HCl (pH 8). The level of bound antibodies was determined by ELISA.

Figure 2

Dependence of bound anti-sTNF-R antibodies on the total IgG concentration. The total IgG fraction was purified from normal human serum by protein A-Sepharose affinity chromatography. Solutions containing different concentrations of total human IgG in BSA–Tween buffer were added to sTNF-R-coated immunoassay plates. The level of bound Igs was monitored using enzyme-conjugated second (anti-human) antibodies.

Figure 3

Antigen-induced inhibition of anti-sTNF-R binding activity. Protein A-purified total human IgG was incubated for 14 hr at 4°C with varying concentrations of free sTNF-R, after which the samples were introduced into sTNF-R-coated wells. Detection of bound antibodies was accomplished by ELISA.

Figure 4

Identification of sTNF-R by Western blotting using affinity-purified human anti-sTNF-R antibodies. Following SDS/PAGE, the sTNF-R was transferred to nitrocellulose paper and incubated for 2 hr with affinity-purified antibodies (10 μg/ml), followed by horseradish peroxidase-conjugated second anti-human IgG.

Figure 5

Specificity of human antibodies eluted from ASA and sTNF-R affinity columns. Affinity-purified human anti-ASA (open columns) and human anti-sTNF-R (solid columns) antibodies (10 μg/ml) were added to wells of a microtiter plate coated with different proteins. Detection of bound antibodies was accomplished by ELISA. RBP, riboflavin-binding protein.

Figure 6

The frequency of amino acids in the peptides selected from the phage-display library. The frequency was calculated using the sequences deduced from the 36 phagotopes selected with the human anti-sTNF-R antibodies (solid columns) and from the 20 phagotopes selected with the human anti-ASA antibodies (open columns).