Preeclampsia transforms membrane N-glycome in human placenta.

Posttranslational modifications (PTM) which accompany pathological conditions affect protein structure, characteristics and modulate its activity. Glycosylation is one of the most frequent PTM influencing protein folding, localisation and function. Hypertension is a common gestational complication, which can lead to foetal growth restriction (IUGR) and even to foetal or maternal death. In this work we focused on the impact of preeclampsia complicated with IUGR on placental membrane N-glycome. Results have shown that preeclampsia reduced fucosylation of placental glycans, increased the appearance of paucimannosidic and mannosidic structures with lower number of mannose residues and decreased the amount of glycans with more mannose residues. Since preeclampsia is tightly connected to IUGR, glycosylation changes were investigated also on the functional membrane receptors responsible for growth: insulin receptor and the type 1 insulin-like growth factor receptor (IR and IGF1R). It was found that IR present in the IUGR placenta contained significantly less α2,6-Sia. Therefore, glycans on placental membranes alter due to preeclampsia, but changes seen at the level of the entire N-glycome may be different from the changes detected at the level of a specific glycoprotein. The difference recorded due to pathology in one membrane molecule (IR) was not found in another homologous molecule (IGF1R). Thus, besides studying the glycosylation pattern of the entire placental membrane due to preeclampsia, it is inevitable to study directly glycoprotein of interest, as no general assumptions or extrapolations can be made.

Disturbed Plasma Lipidomic Profiles in Females with Diffuse Large B-Cell Lymphoma: A Pilot Study.

Lipidome dysregulation is a hallmark of cancer and inflammation. The global plasma lipidome and sub-lipidome of inflammatory pathways have not been reported in diffuse large B-cell lymphoma (DLBCL). In a pilot study of plasma lipid variation in female DLBCL patients and BMI-matched disease-free controls, we performed targeted lipidomics using LC-MRM to quantify lipid mediators of inflammation and immunity, and those known or hypothesised to be involved in cancer progression: sphingolipids, resolvin D1, arachidonic acid (AA)-derived oxylipins, such as hydroxyeicosatetraenoic acids (HETEs) and dihydroxyeicosatrienoic acids, along with their membrane structural precursors. We report on the role of the eicosanoids in the separation of DLBCL from controls, along with lysophosphatidylinositol LPI 20:4, implying notable changes in lipid metabolic and/or signalling pathways, particularly pertaining to AA lipoxygenase pathway and glycerophospholipid remodelling in the cell membrane. We suggest here the set of S1P, SM 36:1, SM 34:1 and PI 34:1 as DLBCL lipid signatures which could serve as a basis for the prospective validation in larger DLBCL cohorts. Additionally, untargeted lipidomics indicates a substantial change in the overall lipid metabolism in DLBCL. The plasma lipid profiling of DLBCL patients helps to better understand the specific lipid dysregulations and pathways in this cancer.

Sensitive glycoprofiling of insulin-like growth factor receptors isolated from colon tissue of patients with colorectal carcinoma using lectin-based protein microarray.

Glycosylation of cell receptors influences their function and development of tumour induces changes in glycosylation. Cell growth depends on the activation of receptors which bind growth factors and the insulin-like growth factor (IGF) receptors are among the most important ones. Usually, only small quantities of isolated receptors are available thus there is a need of suitable assay to study receptors glycosylation. Therefore, we developed a lectin-based reverse-phase protein microarray method for screening the glycosylation pattern of receptors in picomolar (pM) concentrations. The method was applied to glycoprofile IGF1 and IGF2 receptors and the solubilised membrane proteins isolated from tumour and non-tumour colon tissue of patients with colorectal cancer. We found that common to both receptors was partial overlapping of the major glycan structures with those present in the entire glycome of membrane proteins. In contrast, receptors possess higher level of α2,3 sialic acid residues and lower level of tri-/tetra-antennary complex type N-glycans and terminal mannose in high-mannose structures. Increased levels of fucosylation and branched mannose structures were observed in both receptors derived from tumour tissue compared to non-tumour tissue. The described method enabling glycan analysis of receptors has a big application potential in e.g. biomarker research, biology and diagnostics.

Anti-adhesive action of novel ruthenium(II) chlorophenyl terpyridine complexes with a high affinity for double-stranded DNA: in vitro and in silico.

Interactions of three Ru(II) chlorophenyl terpyridine complexes: [Ru(Cl-Ph-tpy)(en)Cl]Cl (1), [Ru(Cl-Ph-tpy)(dach)Cl]Cl (2) and [Ru(Cl-Ph-tpy)(bpy)Cl]Cl (3) (Cl-Ph-tpy = 4'-(4-chlorophenyl)-2,2':6',2''-terpyridine, en = 1,2-diaminoethane, dach = 1,2-diaminocyclohexane, bpy = 2,2'-bipyridine) with human serum albumin (HSA), calf thymus DNA and a double-helical oligonucleotide d(CGCGAATTCGCG)2 (1BNA) were examined. Fluorescence emission studies were used to assess the interactions of complexes with HSA, which were of moderate strength for 1 and 2. Molecular docking allowed us to predict mostly π-π stacking and van der Waals interactions between the complexes and the protein. We suggest that the complexes bind to a novel site on HSA, which is different from its druggable sites I, II or III. We suggest a partial intercalation of complexes through the minor groove as a possible mode of interaction with double-helical DNA. Finally, when applied to normal extravillous cell line HTR8/SVneo and JAr choriocarcinoma cell line, complexes 1 and 2 exerted anti-adhesive properties at very low doses, whereas complex 3 had a negligible effect. The obtained results are completion of our studies of Ru(II) terpyridyl complexes that carry N-N ancillary ligands. We suggest a new research direction towards studying the cellular effects of Ru(II) polypyridyl compounds.

Glycoanalysis of the placental membrane glycoproteins throughout placental development.

Structural changes of glycans are observed in different (patho)physiological conditions. Human placental membrane (glyco)proteins were isolated from the first and third trimester placentas of mothers at different ages. By using lectin microarray, we demonstrated that the placental membrane N-glycome contains several N-glycan groups: high mannose, asialylated and sialylated biantennary moieties, bisected, core fucosylated, fucosylated at other positions (bearing terminal and/or antennary Fuc), α2-6 and α2-3 sialylated structures. Employing MALDI-TOF MS enabled identification of over sixty different N-glycan structures in all samples, with 17 moieties exceeding the relative abundance of 2%. The major MS peaks originated from: 1) biantennary complex type N-glycan with a bisecting GlcNAc residue and 2) a core Fuc paucimannosidic and high mannose type structures M3-M9. Age of mothers and the stage of placental development affected N-glycome. The work presented in this article is the first comprehensive mass spectrometric study of the N-glycome of human placental membrane proteins. Our results may be seen as the baseline which can serve for future MALDI MS profiling of the placental membrane N-glycome in different pathophysiological conditions.

Affinity modulation of human placental insulin and insulin-like growth factor receptors by lectins.

The ability of plant lectins to modify the interactions of the insulin receptor (IR) and insulin-like growth factor (IGF) receptors (IGFRs) with their ligands was investigated. The lectins profoundly affected the competition-binding curves for (125)I-labelled IGF-I and insulin, causing an increase in the affinity of placental IGF1R and IR towards their ligands. This increment was of such a magnitude that it could affect the receptors' specificity towards these ligands. The lower the ligand concentration, the greater was the lectin-induced affinity shift, which suggests potential physiological significance of the effect. The affinity modulation occurred in a lectin-specific and dose-dependent manner. In contrast to IGF1R and IR, the binding of (125)I-labelled IGF-II to its receptors resisted lectin modulation. Here we provide evidence of the possibility of external modulation of the affinity of placental IGF1R and IR via interactions of the receptors' carbohydrate moieties with lectins. The existence of modulators that would selectively inhibit or enhance the binding of IGFs or insulin to their corresponding receptors may have important implications for placental cell responses to these molecules.

The change in the circulating insulin-like growth factor binding protein 1 isoform pattern during the course of oral glucose tolerance test.

There is a tight connection between insulin-like growth factor binding protein 1 (IGFBP-1) and nutrient/energy supply, suggesting modulation of the short-term insulin-like activity and glucose homeostasis by IGFBP-1. Differential phosphorylation of IGFBP-1 alters its affinity for insulin-like growth factors (IGFs) and its capacity to modulate cellular response. The object of this study was to define the time course of changes in the IGFBP-1 isoform pattern during an oral glucose tolerance test. Besides changing in counterdirections, the alterations in glucose/insulin/C-peptide and IGF-I/IGFBP-1 concentrations were phase-shifted. Denaturing electrophoresis revealed that the IGFBP-1 proteolytic activity was not increased after glucose ingestion. In native electrophoresis, the isoform that moved most anodically, with the greatest phosphate content, was markedly reduced during the course of oral glucose tolerance test; and it disappeared after 3 hours. Our data show that both a change in the total amount of IGFBP-1 and an alteration in the relative amount (ratio) of the specific phosphoforms of IGFBP-1 are part of the mechanism involved in modulation of the insulin-like activity.

Effects of peroral insulin and glucose on circulating insulin-like growth factor-1, its binding proteins and thyroid hormones in neonatal calves.

There is disagreement in the literature about the ability of neonatal calves to absorb perorally administered insulin. This study evaluated the absorption of a bolus of insulin administered alone or with an energy souce and its effects on the circulating insulin-like growth factor system and thyroid hormones in newborn Holstein-Friesian calves. Within 1 h of dosing, mean serum insulin and triiodothyronine (T3) concentrations had increased considerably, whether the insulin was applied alone (n = 4) or together with glucose (n = 4), accompanied by marked hypoglycemia. No significant changes were observed in control calves (n = 4) given the vehicle solution. Increased serum glucose and T3 concentrations with no change in insulinemia occurred in a 4th group of calves given glucose alone. At 32 h of age and after 3 meals of colostrum there were no differences in glycemia, insulinemia, or proteinemia among the 4 groups of calves examined. Mean serum insulin-like growth factor-I (IGF-I) tended to decrease over this period in the control group. The decrease was more pronounced in the insulin-treated group but absent in both groups that received glucose. These differences were associated with equivalent differences in abundance of the 40-45K IGF-binding protein-3 (IGFBP-3); however, lower molecular mass IGFBPs were not affected. The results show that a pharmacological peroral dose of insulin can lead to rapid systemic alterations in the IGF/IGFBP system in neonatal calves that can be modified by simultaneous administration of a small energy supply in the form of glucose.

New gold pincer-type complexes: synthesis, characterization, DNA binding studies and cytotoxicity.

With the aim of assessing whether Au(iii) compounds with pincer type ligands might be utilized as potential antitumor agents, three new monofunctional Au(iii) complexes of the general formula [Au(N-N'-N)Cl]Cl2, where N-N'-N = 2,6-bis(5-tert-butyl-1H-pyrazol-3-yl)pyridine (H2LtBu, 1), 2,6-bis(5-tert-butyl-1-methyl-1H-pyrazol-3-yl)pyridine (Me2LtBu, 2) or 2,6-bis((4S,7R)-1,7,8,8-tetramethyl-4,5,6,7-tetrahydro-1H-4,7-methanoindazol-3-yl)pyridine (Me2*L, 3) were synthesized. All complexes were characterized by elemental analysis, spectroscopic techniques (IR, UV-Vis, 1D and 2D NMR) and mass spectrometry (MALDI TOF MS). The chemical behavior of the complexes under physiological conditions was studied by UV-Vis spectroscopy, which showed that all compounds were remarkably stable and that the gold center remained in the 3+ oxidation state. The kinetics and the mechanism of the reaction of complexes 1-3 with guanine derivatives (i.e. guanosine (Guo) and guanosine-5'-monophosphate (5'-GMP)) and calf thymus DNA (CT DNA) were studied by stopped-flow spectroscopy. The three complexes displayed moderately different rate constants in their reactions with Guo, 5'-GMP and CT DNA, which can be explained by the steric hindrance and σ-donicity of the methyl substituent on the bis-pyrazolylpyridine fragment in complexes 2 and 3. The measured enthalpies and entropies of activation (ΔH≠ > 0, ΔS≠ < 0) supported an associative mechanism for the substitution process. The interaction of the newly synthesized complexes 1-3 with CT DNA was investigated by UV-Vis and fluorescence spectroscopy, and also by viscosity measurements, which all indicated that complexes 1-3 bound to CT DNA with moderate binding affinity (Kb = 1.6-5.7 × 103 M-1) and stabilized the duplex of CT DNA. Molecular docking indicated that complexes 1-3 interacted with DNA via intercalation. Complex 1 reduced the cell survival of all the investigated cell lines (A549, A375, and LS-174) with IC50 values being up to 20 µM. We have shown that 1 induced perturbations of the cell cycle and led to apoptosis in human melanoma A375 cells. Complex 1 also affected the level of reactive oxygen species (ROS) in the same cells. However, pre-treatment of A375 cells with NAC (ROS scavenger) reversed the effect of 1 on their survival.

Plasma phospholipid changes are associated with response to chemotherapy in non-Hodgkin lymphoma patients.

Limited studies have been performed to associate abnormal phospholipid (PL) profile and disease activity in hematological malignancies, including non-Hodgkin lymphoma (NHL). The aim of his study was to evaluate the levels of plasma PL fractions in NHL patients, in response to chemotherapy. Forty non-treated patients with NHL and 25 healthy individuals were recruited. Blood samples from patients were taken before chemotherapy, after 3 cycles and after the end of the treatment, and PL fractions were resolved by one-dimensional thin-layer chromatography. To assess potential relationship between plasma PL profile and response to therapy, patients were divided according to clinical outcome in 3 groups: complete remission (CR), stable disease (SD) and progression (PG). Despite significant differences between NHL patients and healthy controls, no differences were found at baseline among patients divided according to clinical outcome. During and after chemotherapy important alterations in PL profile were observed. Levels of total PLs and all PL fractions decreased in patients with PG while in patients who responded to therapy (CR, SD) PLs significantly increased. Results of our study suggest that changes of total PLs and PL fractions during the therapy are associated with the effects of therapy and clinical outcome in patients with NHL.

Characterisation of insulin-like growth factor receptors and insulin receptors in the human placenta using lectin affinity methods.

Insulin and insulin-like growth factor receptors (IR, IGF-IR, IGF-IIR) from human placental cell membranes were solubilised and their glycoprotein properties were studied in terms of their interaction with five lectins: wheat germ agglutinin (WGA), banana lectin (BanLec), phytohaemagglutinin (PHA), concanavalin A (Con A), and Sambucus nigra agglutinin (SNA). The pattern of binding to the immobilised lectins indicated that the glycosylation of the IGF-IR, IGF-IIR and IR differed. We found several populations of receptors in placental cell membranes, differing with respect to their oligosaccharide moieties. IGF-IIR populations bore highly branched complex type N-glycans with a very high content of oligosaccharides terminating with Sia, high-mannose type N-glycans and hybrid type N-glycans. All these glycans seemed to be attached to the same IGF-II receptor molecules. Two major glycoforms of IR were detected, one having multiple highly branched N-glycans with a low content of terminal Sia and the other, having high-mannose type glycans attached to multiple N-glycosylation sites. As for the IGF-IR, multiple glycoforms were detected, bearing complex type N-glycans with various content of Sia-terminating branches, hybrid type N-glycans or high-mannose type N-glycans. The specific binding of (125)I-IGF-II to its receptor increased in the presence of immobilised WGA and SNA, which might imply the existence of a mammalian lectin counterpart whose potential physiological significance may lie in different targeting to various membrane compartments, thereby potentially modifying their cell signalling pathways.

Gestation-associated changes in the glycosylation of placental insulin and insulin-like growth factor receptors.

INTRODUCTION: Insulin receptor (IR) and type 1 and type 2 insulin-like growth factor receptors (IGF1R and IGF2R) play important roles in regulation of placental and foetal growth. All three receptors are abundantly glycosylated. N-glycosylation significantly affects protein conformation and may alter its function. We have recently found that the N-glycome of placental membrane proteins alters during gestation. The aim of the study presented herein was to investigate whether there were gestation-related changes in N-glycan profiles of placental IR and IGFRs. METHODS: Placentas from healthy women at first (FTP) and third trimester (TTP) of pregnancy were collected, membrane proteins isolated, solubilised and used as the source of IR and IGFRs. Reactivity of glycoforms of IR and IGFRs with lectins was monitored by measuring radioactivity of (125)I-ligands-receptors complexes. RESULTS: Significant differences in the binding pattern of all three receptors to the lectins were observed between FTP and TTP, which suggest gestational changes in N-glycans bound to receptors. These changes include decrease in total fucosylated, core-fucosylated biantennary N-glycan (NA2F) and α2,6-sialo-N-glycans (for IR); decrease in total fucosylated and α2,6-sialo-N-glycans and an increase in NA2F N-glycans (for IGF1R) and an increase in the total fucosylation and NA2F N-glycans (for IGF2R). DISCUSSION: The gestational alterations in N-glycans attached to IR and IGFRs may represent a mechanism by which these receptors acquire new/additional roles as gestation progresses.

Elucidation of the binding sites of two novel Ru(II) complexes on bovine serum albumin.

Hyphenated mass spectrometry (MS) techniques have attained an important position in analysis of covalent and non-covalent interactions of metal complexes with peptides and proteins. The aim of the present study was to qualitatively and quantitatively determine ruthenium binding sites on a protein using tandem mass spectrometry and allied techniques, i.e. liquid chromatography (LC) and inductively coupled plasma optical emission spectrometry (ICP-OES). For that purpose, two newly synthesized Ru(II) complexes of a meridional geometry, namely mer-[Ru(4' Cl-tpy)(en)Cl](+) (1) and mer-[Ru(4' Cl-tpy)(dach)Cl](+) (2) (where 4' Cl-tpy=4'-chloro-2,2':6',2″-terpyridine, en=1,2-diaminoethane and dach=1,2-diaminocyclohexane), and bovine serum albumin were used. The binding of the complexes to the protein was investigated by means of size exclusion- and reversed phase-LC, ICP OES, matrix-assisted laser desorption ionization MS and MS/MS. Ruthenated peptide sequence and a binding target amino acid were revealed through accurate elucidation of MS/MS spectra. The results obtained in this study suggest a high binding capacity of the protein towards both complexes, with up to 5.77±0.14 and 6.95±0.43mol of 1 and 2 bound per mol of protein, respectively. The proposed binding mechanism for the selected complexes includes the release of Cl ligand, its replacement with water molecule and further coordination to electron donor histidine residue.

The N-glycan profile of placental membrane glycoproteins alters during gestation and aging.

Alterations in the glycosylation of few membrane proteins from human placenta during gestation have been documented, but data on N-glycome of placental membrane proteins are still missing. The primary goal of this study was to obtain N-glycan profiles of human placental membrane proteins using a reliable, simple and high-throughput method. The second goal was to examine whether the N-glycan profile alters during gestation. Placental membrane proteins were isolated from women of different ages after first and third trimesters of pregnancy. The N-glycan fingerprint of membrane proteins was obtained using DNA sequencer-assisted fluorophore-assisted carbohydrate electrophoresis (DSA-FACE). Lectin blotting was used to confirm DSA-FACE results. Observed gestation-related alterations were: greater abundance of core-fucosylated and multiantennary N-glycans, but lower amounts of bisected biantennary N-glycans together with a decrease in α2,3-sialylation. Age-related alterations were: more core Fuc and more α2,3-Sia in first trimester placentas from older women than in those from younger women; also less core Fuc and less α2,6-Sia in third trimester placentas from older women compared to those from younger women. This study represents the first N-glycan profiling of placental cell membrane proteins. These data represent a basis for future research on the N-glycome of placental proteins in different (patho)physiological conditions.

Posttranslational modifications of the insulin-like growth factor-binding protein 3 in patients with type 2 diabetes mellitus assessed by affinity chromatography.

Structural and ligand-binding properties of the insulin-like growth factor-binding protein (IGFBP)-3 in patients with poorly controlled diabetes mellitus type 2 were investigated using boronic acid- and lectin-affinity chromatography. IGFBP-3 species separated by chromatography were analyzed by immunoblotting and surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF MS). Increased IGFBP-3 binding to boronic acid in patients was shown to be accompanied by the increased ligand-binding. Increased binding of IGFBP-3 forms to lectins from Sambucus nigra (SNA) and Canavalia ensiformis (ConA) in patients, on the other hand, was either not accompanied by altered ligand-binding (in the case of ConA) or it was reduced (in the case of SNA). Strong and opposite effects of glycation and additional sialylation on ligand binding qualify them as factors that may be involved in the regulation of the amount of free, physiologically active IGFs, and modulation of processes that accompany development and progression of diabetes. SELDI-TOF MS analysis revealed a fragment of 13.9 kDa as representative for the non-glycosylated form of IGFBP-3, whereas a fragment of 28.0 kDa profiled as typical for the glycosylated/glycated IGFBP-3 species. The same fragmentation pattern found in healthy persons and in patients indicates that the same degradation process predominantly occurs in both groups of individuals.

Characterisation of N-glycans bound to IGFBP-3 in sera from healthy adults.

Human IGFBP-3 contains three potential N-linked glycosylation sites. Published data concerning the type and saccharide composition of the N-glycans is scarce. The aim of this study was to characterise N-glycans covalently attached to IGFBP-3 from sera of healthy adults (men and women). In order to do that a panel of eight lectins covering broad saccharide specificity was used: agarose-immobilised SNA (Sambucus nigra agglutinin), Con A (lectin from Canavalia ensiformis), RCA I (Ricinus communis agglutinin I), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (P. vulgaris leukoagglutinin), succinylated WGA (wheat germ agglutinin), ECL (Erythrina cristagalli lectin) and UEA (Ulex europaeus agglutinin). IGFBP-3 interacted with SNA, Con A, RCA I, PHA-E and, to a much lesser extent, with PHA-L. These results indicate that human IGFBP-3 bears mostly biantennary complex type N-glycans with a very high content of alpha-2,6-linked Sia at their termini. Hybrid type and high-mannose type N-glycans are present, as well as a bisecting GlcNAc residue, which may be core fucosylated. N-glycosylation of IGFBP-3 follows the N-glycosylation pattern of major serum proteins. This study represents a ground for the future research of glycosylation pattern of IGFBP-3 from the circulation of men and women diagnosed with different illnesses.

Alterations of insulin-like growth factor binding protein 3 (IGFBP-3) glycosylation in patients with breast tumours.

OBJECTIVES: Insulin-like growth factor binding protein-3 (IGFBP-3) is an important modulator of development and progression of breast cancer as it regulates the amount of free, physiologically active IGF-I and IGF-II. Changes in the glycosylation pattern within IGFBP-3 may affect its interaction with ligands. The aim of this study was to investigate whether such changes occur during disease progression. DESIGN AND METHODS: IGFBP-3 in serum samples from healthy women and from women with breast tumours was characterised in terms of its concentration (IRMA), glycosylation moiety (lectin-affinity chromatography) and distribution of molecular species (immunoblotting). RESULTS: In patients with benign tumours the concentration and carbohydrate content of IGFBP-3 was unaltered compared to healthy women. In patients with malignant tumours in most cases these two parameters were unchanged, but there were women whose concentration of IGFBP-3 was reduced and its structure was altered. In non-surviving cancer patients the concentration of IGFBP-3 was significantly reduced and these molecules contained a greater amount of biantennary complex type N-glycans having more mannose, fucose, bisecting GlcNAc and terminal sialic acid residues. CONCLUSION: Our results showed that breast cancer progression causes alterations of IGFBP-3 glycosylation. The extent of changes increases with breast cancer severity.

Separation of the molecular forms of the insulin-like growth factor (IGF)-Binding proteins by affinity chromatography.

Association of IGFBP-1, IGFBP-2 and IGFBP-3 with other proteins in human serum and placental cell membranes was investigated using affinity chromatography matrix with immobilized antibodies. Circulating IGFBP-1 was found to be predominantly bound to alpha(2)-macroglobulin and not in the binary complex with its ligand, IGFBP-2 complexes and/or polymers were detected, which was not acknowledged before, and IGFBP-3 molecular forms were differentiated into those that form binary/ternary complexes and those that form stable associations with other serum proteins. As for placental membranes, both IGFBP-1 dimers and high molecular mass IGFBP-1 associations, most probably with alpha(2)-macroglobulin, were recognized and resolved.