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

Abstract

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 approximately 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 (Mg(2+) > Fe(2+) > Cu(2+) ≥ Ca(2+) ≥ Mn(2+)) 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 (Fe(2+) > Ca(2+) > Co(2+) ≥ Ni(2+)) and especially several combinations of metals: Co(2+) + Ca(2+) < Mg(2+) + Ca(2+) < Ca(2+) + Zn(2+) < Fe(2+) + Zn(2+) < Fe(2+) + Co(2+) < Fe(2+) + Ca(2+). 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.

Catalytic Antibodies in Norm and Systemic Lupus Erythematosus

“…Protease IgGs from the sera of ~95-100% of patients with different autoimmune pathologies [9,66,116], human milk Abs hydrolyzing casein [96,117], Abs from AIDS patients hydrolyzing HSA, casein, and HIV reverse transcriptase [95] are serine-like proteases, whose activity is strongly decreased after their preincubation with serine protease-specific inhibitors PMSF. In addition, a high metal-dependent MBP-hydrolyzing activity for MS IgGs [86] and caseinhydrolyzing of human milk sIgAs [96] were recently revealed.…”

“…In addition, a high metal-dependent MBP-hydrolyzing activity for MS IgGs [86] and caseinhydrolyzing of human milk sIgAs [96] were recently revealed. It was shown that antiintegrase IgGs and IgMs of HIV-infected patients can contain abzymes hydrolyzing viral integrase of four types, resembling serine, thiol, metal-dependent, and acidic proteases, the ratio of which may be individual for every AIDS patient [97,98].…”

Natural Catalytic Antibodies in Norm and in HIV-Infected Patients

“…We have analyzed the type of proteolytic activity of AIDS pIgGs and pIgMs in the hydrolysis of IN and -casein. It was shown, that in contrast to milk sIgAs 2011) and similarly to several other proteolytic Abzs, only specific inhibitor of serine protease AEBSF significantly suppress AIDS pIgG-dependent hydrolysis of -casein, while EDTA has no significant effect. We have analyzed the type of IN-hydrolyzing activity of ten AIDS pIgGs; several typical examples are given in Fig.…”

“…Proteolytic IgGs from the sera of patients with asthma hydrolyzing VIP (Paul et al, 1989), Hashimoto thyroiditis and rheumatoid arthritis hydrolyzing thyroglobulin (Li eta l., 1995;Kalaga et al, 1995) are serine-like proteases, and their activity is most strongly reduced after incubation with specific serine protease inhibitors PMSF or AEBSF. It was shown that casein-hydrolyzing sIgAs from human milk 2011) and hMBP-hydrolyzing IgGs and IgMs from the sera of patients with MS (Polosukhina et al, 2004(Polosukhina et al, , 2005(Polosukhina et al, , 2006 contain not only Ab subfractions with serine-like, but also specific subfractions with metal-dependent activity. We have analyzed the type of proteolytic activity of AIDS pIgGs and pIgMs in the hydrolysis of IN and -casein.…”

“…Proteolytic activity of five of ten AIDS IgGs was inhibited by 40-96 % after incubating the IgGs with EDTA. An incubation of IgGs with iodoacetamide (a specific inhibitor of thiol proteases) usually has no remarkable effect on their proteolytic activity (Paul et al, 1989;Kalaga et al, 1995;2011;Polosukhina et al, 2004Polosukhina et al, , 2005Polosukhina et al, , 2006. Surprisingly, however, IN-hydrolyzing activity of all 10 IgGs was inhibited by iodoacetamide by 12-98 % (Baranova et al, 2009).…”