Cell surface sialylation is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential, clearance of aged cells, while the sialylation of IgG molecules determines their anti-inflammatory properties. Four sialidases - hydrolytic enzymes responsible for cleavage of sialic residues - were described in different cellular compartments. However, sialidases activity in body fluids, and specifically in blood serum, remains poorly studied. Here, we characterize first known IgG antibodies possessing sialidase-like activity in blood serum of multiple myeloma (MM) patients. Ig fractions were precipitated with ammonium sulfate (50% of saturation) from blood serum of 12 healthy donors and 14 MM patients, and screened for the presence of sialidase activity by using 4-MUNA (2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid) as substrate. High level of sialidase activity was detected in the MM patients, but not in healthy donors. Subsequent antibody purification by protein-G affinity chromatography and HPLC size exclusion chromatography at acidic conditions demonstrated that sialidase activity was attributable to IgG molecules. Sialidase activity was also specific for (Fab)(2) fragment of IgG and blocked by sialidase inhibitor DANA. Sialidase activity of IgG molecule was also confirmed by in gel assay for cleavage of sialidase substrate. Kinetic parameters of the catalysis reaction were described by Michaelis-Menten equation with K(m) = 44.4-108 µM and k(cat) = 2.7-23.1 min(-1). The action of IgG possessing sialidase-like activity towards human red blood cells resulted in a subsequent increase in their agglutination by the peanut agglutinin, that confirms their desialylation by the studied IgG. This is the first demonstration of the intrinsic sialidase activity of IgG isolated from blood serum of MM patients.
OTHER ARTICLES PUBLISHED IN THIS SERIES Dying autologous cells as instructors of the immune system. Clinical and Experimental Immunology 2015, 179: 1-4. Anti-dsDNA antibodies as a classification criterion and a diagnostic marker for systemic lupus erythematosus: critical remarks. Clinical and Experimental Immunology 2015, 179: 5-10. The effect of cell death in the initiation of lupus nephritis. Clinical and Experimental Immunology 2015, 179: 11-16 SummaryRecently we reported the first known incidence of antibodies possessing catalytic sialidase activity (sialidase abzymes) in the serum of patients with multiple myeloma and systemic lupus erythematosus (SLE). These antibodies desialylate biomolecules, such as glycoproteins, gangliosides and red blood cells. Desialylation of dying cells was demonstrated to facilitate apoptotic cell clearance. In this study we assessed the possibility to facilitate dying cell clearance with the use of F(ab)2 fragments of sialidase abzymes. Two sources of sialidase abzymes were used: (i) those isolated from sera of patients with SLE after preliminary screening of a cohort of patients for sialidase activity; and (ii) by creating an induced sialidase abzyme through immunization of a rabbit with synthetic hapten consisting of a non-hydrolysable analogue of sialidase reaction conjugated with bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH). Antibodies were purified by ammonium sulphate precipitation, protein-G affinity chromatography and size exclusion-high performance liquid chromatography (HPLC-SEC). Effect of desialylation on efferocytosis was studied using human polymorphonuclear leucocytes (PMN), both viable and aged, as prey, and human monocyte-derived macrophages (MoMa). Treatment of apoptotic and viable prey with both disease-associated (purified from blood serum of SLE patients) and immunization-induced (obtained by immunization of rabbits) sialidase abzymes, its F(ab)2 fragment and bacterial neuraminidase (as positive control) have significantly enhanced the clearance of prey by macrophages. We conclude that sialidase abzyme can serve as a protective agent in autoimmune patients and that artificial abzymes may be of potential therapeutic value.
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