Metal dependent hydrolysis of β-casein by sIgA antibodies from human milk

We present the first evidence that electrophoretically and immunologically homogeneous sIgAs purified from milk of healthy human mothers by chromatography on Protein A-Sepharose and FPLC gel filtration contain intrinsically bound metal ions (Ca > Mg ≥ Al > Fe ≈ Zn ≥ Ni ≥ Cu ≥ Mn), the removal of which by a dialysis against ethylenediamine tetraacetic acid (EDTA) leads to a significant decrease in the β-casein-hydrolyzing activity of these antibodies (Abs). An affinity chromatography of total sIgAs on benzamidine-Sepharose interacting with canonical serine proteases separates a small metalloprotease sIgA fraction (6.8 ± 2.4%) from the main part of these Abs with a serine protease-like β-casein-hydrolyzing activity. The relative activity of this metalloprotease sIgA fraction containing intrinsically bound metal ions increases ∼1.2–1.9-fold after addition of external metal ions (Mg²⁺ > Fe²⁺ > Cu²⁺ ≥ Ca²⁺ ≥ Mn²⁺) but decreases by 85 ± 7% after the removal of the intrinsically bound metals. The metalloprotease sIgA fraction free of intrinsic metal ions demonstrates a high β-casein-hydrolyzing activity in the presence of individual external metal ions (Fe²⁺ > Ca²⁺ > Co²⁺ ≥ Ni²⁺) and especially several combinations of metals: Co²⁺ + Ca²⁺ < Mg²⁺ + Ca²⁺ < Ca²⁺ + Zn²⁺ < Fe²⁺ + Zn²⁺ < Fe²⁺ + Co²⁺ < Fe²⁺ + Ca²⁺. The patterns of hydrolysis of a 22-mer oligopeptide corresponding to one of sIgA-dependent specific cleavage sites in β-casein depend significantly on the metal used. Metal-dependent sIgAs demonstrate an extreme diversity in their affinity for casein-Sepharose and chelating Sepharose, and interact with Sepharoses bearing immobilized monoclonal mouse IgGs against λ- and κ-type light chains of human Abs. Possible ways of the production of metalloprotease abzymes (Abz) by human immune system are discussed. Copyright © 2010 John Wiley & Sons, Ltd.