Tag Archives: glycosylation
Lectinocytochemical study of rat stomach mucosa under the conditions of cyclooxygenase-1/-2 blockage and pretreatment witH H-Glu-Asp-Gly-OH
C. M. Nasadyuk1*, E. A. Sogomonyan2, A. M. Yashchenko2, A. Y. Sklyarov1
1Department of Biochemistry, Danylo Halytsky Lviv National Medical University, Ukraine;
2Department of Histology, Danylo Halytsky Lviv National Medical University, Ukraine;
*e-mail: nasadyukch@gmail.com
Received: 22 December 2019; Accepted: 27 March 2020
Assessment of glycoconjugate expression on cell membranes using the lectin histochemistry technique may be a feasible approach for evaluating the functional state of the cell. The aim of this study was to evaluate carbohydrate determinants of rat stomach mucosa cell membranes under the conditions of COX-1/-2 blockage with indomethacin and pretreatment with the tripeptide H-Glu-Asp-Gly-OH. Male Wistar rats were divided into 3 groups (n = 6 per group): 1st group (control) received vehicle; 2nd – indomethacin (35 mg/kg); 3rd – H-Glu-Asp-Gly-OH (10 µg) 30 min before indomethacin. Rats were sacrificed 24 hours later. Gastric mucosa (GM) carbohydrate determinants were studied by lectin-peroxidase technique. The lectins panel included α-fucose- (LABA), syalo- (WGA, SNA), mannose- (Con A, LCA) and galactose-specific (HPA, PNA, SBA) lectins. Intensity of lectin-receptor reaction was scored: 0 – no reaction; 1 – weak; 2 – mild; and 3 – strong reaction. COX-1/2 blockage caused GM lesions, attenuated by H-Glu-Asp-Gly-OH. WGA and SNA showed the highest affinity to GM. Indomethacin decreased SNA-labeling of epitheliocytes and mucocytes and LABA-labeling of chief cells. H-Glu-Asp-Gly-OH reversed the glycosylation changes, caused by COX-1/COX-2 blockage only in regards to labeling of chief cells with LABA, epitheliocytes and mucocytes with LCA, mucocytes with SNA. Predominantly H-Glu-Asp-Gly-OH under COX-1/COX-2 blockage had an effect opposite to indomethacin alone but glycosylation changes under these conditions differed significantly also from the control. COX-1/COX-2 blockage causes alteration of glycosylation processes in rat GM, mainly reduction of NeuNAc(α2-6)DGal and α-Fuc content. H-Glu-Asp-Gly-OH under the conditions of COX-1/COX-2 blockage leads to more profound changes in GM lectin-binding pattern compared to the independent effect of indomethacin and to control.
Freezing influences, the exposure of IgG glycans in sera from multiple sclerosis patients
M. Bozhenko1, M. Boichuk1, G. Bila2, T. Nehrych1*, R. Bilyy2*
1Department of Neurology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine;
2Department of Histology, Cytology and Embryology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine;
*e-mail: r.bilyy@gmail.com; tnehrych@gmail.com
Received: 08 January 2020; Accepted: 27 March 2020
N-glycan residues attached to Asn297 of the immunoglobulin IgG molecule are responsible for changing its structural conformation and are used as markers of many inflammatory diseases. Freezing stabilizes protein structure, while recent solution NMR data showed greatly altered IgG glycan mobility at different temperatures. The aim of the current work was to investigate whether freezing sera samples from multiple sclerosis (MS) patients and normal healthy donors (NHD) influences exposure of IgG glycans. The developed lectin immunosorbent assay was used to evaluate exposure of native IgG glycans with fucose-binding AAL lectin and sialic acid-binding SNA lectin. Sera samples were divided and either immediately frozen at -20 °C or stored at 4 °C. Lectin exposure was compared between 5 MS patient groups (n = 75) vs NHD (n = 23) and in paired samples with and without freezing. A significant increase in the exposure of fucose residues on IgG glycans in MS patients, compared to NHD, was observed. This increase was only observed if sera were frozen before analysis. The exposure of sialic acid was decreased in MS vs NHD samples after freezing sera samples. The exposure of core fucose residues and terminal sialic residues differed significantly in paired sera samples after freezing. Combined parameters of fucose and sialic acid exposure on native IgG glycans using frozen sera samples serve as a discriminative marker between MS and NHD. For AAL exposure, the discrimination of MS was characterized by AUROC of 0.906, sensitivity of 76.7% and specificity of 59.0% (P < 0.0001).
Stability of native and modified α-galactosidase of Cladosporium cladosporioides
N. V. Borzova, L. D. Varbanets
Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
e-mail: nv_borzova@bigmir.net
By modifying carbohydrate component of glycoproteins it is possible to elucidate its role in manifestation of structural and functional properties of the enzyme. The comparison of activity and stability of the native and modified by oxidation with sodium periodate α-galactosidase of Cladosporium cladosporioides was carried out. To determine α-galactosidase activity the authors used n-nitrophenyl synthetic substrate, as well as melibiose, raffinose and stachyose. Modification of the carbohydrate component had a significant effect on catalytic properties of the enzyme. Both the reduction of Vmax and enzyme affinity for natural and synthetic substrates were observed. The native enzyme retained more than 50% of the maximum activity in the range of 20-60 °C, while for the modified enzyme under the same conditions that temperature range was 30-50 °C. The modified α-galactosidase demonstrated a higher thermal stability under neutral pH conditions. The residual activity of the modified α-galactosidase was about 30% when treated with 70% (v/v) methanol, ethanol and propanol. About 50% of initial activity was observed when 40% ethanol and propanol, and 50% methanol were used. It was shown that the modification of C. cladosporioides α-galactosidase by sodium periodate is accompanied by a significant decrease in enzyme activity and stability, probably caused by topological changes in the tertiary and quaternary structure of the protein molecule.