Yuriy Kit

Macrophages Discriminate Glycosylation Patterns of Apoptotic Cell-derived Microparticles

Background: Apoptotic cells release vesicles, which expose “eat-me” signals. Results: Vesicles originated from endoplasmic reticulum expose immature glycoepitopes and are preferentially phagocytosed by macrophages. Conclusion: Immature surface glycoepitopes serve as “eat-me” signals for the clearance of apoptotic vesicles originated from endoplasmic reticulum. Significance: Understanding the distinction by macrophages of apoptotic blebs may provide new insights into clearance-related diseases. Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.

In vivo expression and characteristics of novel α-d-mannose-rich glycoprotein markers of apoptotic cells

We recently established that an increased expression of α‐d‐mannose (Man)‐ and β‐d‐galactose‐rich plasma membrane glycoproteins (GPs) is characteristic for apoptotic cells in vitro [Bilyy, R.O., Stoika, R.S., 2003. Lectinocytochemical detection of apoptotic murine leukemia L1210 cells. Cytometry 56A, 89–95]. It was independent of cell line or apoptosis‐inducing agent, and can therefore be considered as a selective marker for identification and isolation of apoptotic cells [Bilyy, R.O., Antonyuk, V.O., Stoika, R.S., 2004. Cytochemical study of role of alpha‐d‐mannose‐ and beta‐d‐galactose‐containing glycoproteins in apoptosis. J. Mol. Histol. 35, 829–838]. The main goals of the present study were: (1) to determine whether an increased expression of specific GPs also takes place after apoptosis induction in vivo; and (2) to identify additional characteristics of the membrane GP markers of the apoptotic cells. To reach these goals, we studied the expression of α‐Man‐rich membrane GPs in murine leukemia L1210 cells inoculated into abdominal cavities of mice which were then subjected to the action of apoptosis inducer doxorubicin. Another experimental model used in the present work was splenocytes obtained from mice treated with dexamethasone. Lectin‐affinity chromatography and PAGE electrophoresis, or PAGE electrophoresis and lectinoblot analysis were applied for isolation of plasma membrane GPs (34 kDa, and high MW of ∼600 and 800 kDa) whose expressions were increased during apoptosis. Triton X‐114 treatment of cell membrane samples showed that the apoptotic cell‐specific GPs were localized in the peripheral and integral compartments of plasma membrane. Apoptosis in vitro and in vivo was accompanied by an increased expression of the same GP, identified by MALDI‐TOF MS analysis as the microtubule‐actin cross‐linking factor 1. Other GPs, whose expressions were also increased at apoptosis, were similarly identified as G‐protein β‐subunit like (Acc# BAA06185.1) and dystonin isoform β.

Antibody-mediated sialidase activity in blood serum of patients with multiple myeloma.

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.

Detection and characterization of IgG-and sIgA-abzymes capable of hydrolyzing histone H1

Immunoglobulins IgG and sIgA actively hydrolyzing histone H1 have been detected on analyzing proteolytic activity of antibodies isolated by chromatography on Protein A-agarose from blood serum of patients with multiple sclerosis and from colostrum of healthy mothers. These antibodies hydrolyze other histones less actively and virtually failed to cleave lysozyme of chicken egg. By gel filtration at acidic pH and subsequent analysis of protease activity of chromatographic fractions, it was shown that IgG and sIgA molecules were responsible for hydrolysis of histone H1. Anti-histone H1 antibodies of IgG and sIgA classes were purified by affinity chromatography on histone H1-Sepharose from catalytically active antibody preparations. The protease activity of anti-histone H1 IgG antibodies was inhibited by serine proteinase inhibitors, whereas anti-histone H1 sIgA antibodies were insensitive to inhibitors of serine, asparagine, and cysteine proteases.

Desialylation of dying cells with catalytically active antibodies possessing sialidase activity facilitate their clearance by human macrophages.

Recently 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.

Identification of a 48 kDa form of unconventional myosin 1c in blood serum of patients with autoimmune diseases

We searched for protein markers present in blood serum of multiple sclerosis (MS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients in comparison to healthy human individuals. We used precipitation/extraction methods and MALDI TOF/TOF mass spectrometry, and identified a protein with Mr ~46 kDa as a fragment of human unconventional myosin IC isoform b (Myo1C). Western blotting with specific anti-human Myo1C antibodies confirmed the identity. Screening of blood serum samples from different autoimmune patients for the presence of Myo1c revealed its high level in MS and RA patients, relatively low level in SLE patients, and undetected in healthy donors. These data are suggesting that the level of p46 Myo1C in blood serum is a potential marker for testing of autoimmune diseases.

Calf thymus histone-conjugated magnetic poly(2-oxoethyl methacrylate) microspheres for affinity isolation of anti-histone IgGs from the blood serum of patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a heterogeneous, inflammatory and multisystem autoimmune disease in which antinuclear antibodies are present in blood often years before clinical symptoms occur. Isolating the antibodies is thus of crucial importance to confirm the diagnosis and prognosis of patients with some autoimmune diseases. Isolation can be performed advantageously using magnetic microspheres, which offer easy and quick manipulation with a magnet and avoid sample dilution. Here, we developed calf thymus histone-conjugated magnetic poly(2-oxoethyl methacrylate) (POEMA–His) microspheres using a multiple-stage swelling technique followed by His immobilization. Magnetic POEMA–His microspheres were characterized using scanning and transmission electron microscopy, SQUID, ATR FT-IR spectroscopy, elemental analysis and atomic absorption spectrometry. The microspheres were successfully used for rapid purification of the anti-histone immunoglobulins (IgGs) from blood serum samples of a cohort of systemic lupus erythematosus patients.

Characteristics of Potential Protein Biomarkers Extracted with 10% TCA from Blood Serum of Non-Hodgkin’s Lymphoma and Multiple Myeloma Patients

1. Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine. 2. Laboratory of Immunocytology and Genetics of Blood Tumors , Institute of Blood Pathology and Transfusion Medicine, National Academy of Medical Sciences of Ukraine, Lviv, Ukraine. 3. Biological Faculty of Ivan Franko Lviv National University, Lviv, Ukraine.

Identification of SER-PRO-CYS Peptide in Blood Serum of Multiple Sclerosis Patients

Monitoring of multiple sclerosis (MS) requires additional molecular markers. Recently, we used original TCA-precipitation/extraction approach in combination with MALDI TOF/TOF mass-spectrometry and identified earlier unknown 48 kDa form of the unconventional myosin IC isoform b (Myo1C) in blood serum of the MS patients. Further examination of TCA-extracted fraction of blood serum of these patients by means of thin-layer chromatography and HPLC gel-filtration allowed detecting 300-500 Da peptides. MALDI TOF/TOF massspectrometry of these peptides showed that they contain Ser-Pro-Cys amino acid sequence. We discussed potential mechanisms of a release of these peptides that were earlier unknown in blood serum of the MS patients.

Identification of the Unique Properties of IgGs and their Heavy chains in BloodSerum of Multiple Sclerosis Patients

Monitoring of multiple sclerosis (MS) requires of molecular markers. Recently, using TCA-precipitation/ extraction and MALDI TOF/TOF mass-spectrometry, we identified in blood serum of multiple sclerosis (MS) patients earlier unknown 46 kDa form of human unconventional myosin IC isoform b (Myo1C) (Myronovskij et al., 2015). During SDS-electrophoresis of TCA-extracted proteins of blood serum of 28 MS patients, 3 additional polypeptides with 55, 50 and 25 kDa molecular masses were detected. Western-blot analysis using monospecific anti-human IgG rabbit HRP-conjugated antibodies showed that 55 and 25 kDa polypeptides belong to heavy and light chains of IgG molecules, while 50 kDa protein corresponds to free heavy chains of IgGs. None of these polypeptides was found in fractions of TCA extracted polypeptides isolated from blood serum of healthy human donors or patients with systemic lupus erythematosis and rheumatoid arthritis.