Dragana Robajac

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.

Lectin-based protein microarray analysis of differences in serum alpha-2-macroglobulin glycosylation between patients with colorectal cancer and persons without cancer

Glycosylation is co‐ and posttranslational modifications affecting proteins. The glycopattern changes are associated with changes in biological function and are involved in many diseases including cancer. We present the lectin‐based protein microarray method enabling determination of differences in protein glycosylation. The method involves isolation of targeted protein from samples by immunoprecipitation, spotting of protein from multiple samples into arrays on a microarray slide, incubation with set of biotinylated lectins, the reaction with fluorescent conjugate of streptavidin, and detection of fluorescent intensities by microarray scanner. Lectin‐based protein microarray was applied in investigation of differences in alpha‐2‐macroglobulin (α2M) glycosylation isolated from sera samples of healthy persons and patients with colorectal cancer (CC). From 14 lectins used in analysis, statistically significant differences (Students t‐test, P < 0.05) between two groups of samples (persons without cancer and CC patients) were found for 5 of them. α2M molecules isolated from sera of CC patients have higher content of α2,6 sialic acid, N‐acetylglucosamine and mannose residues, and tri‐/tetraantennary complex type high‐mannose N‐glycans. A novel lectin‐based protein microarray developed and described can serve as a suitable analytical technique for sensitive, simple, fast, and high‐throughput determination of differences in protein glycosylation isolated from serum or other samples.

Detection and identification of oxidized insulin-like growth factor-binding proteins and receptors in patients with colorectal carcinoma

Colorectal cancer (CRC) is one of the most prevalent cancers worldwide and also the one with the highest mortality rate. Tumor growth is assisted by various growth factors, and insulin-like growth factors (IGFs) are among the most important. A majority of the IGFs are bound to IGF-binding proteins (IGFBPs) and their release is dependent on the rate of IGFBP proteolysis. The action of free IGFs is exerted and controlled by binding to cell membrane receptors (IGF-Rs). The objective of this work was to connect two determinants of the CRC pathology: oxidation as a process that underlies tumor development and the members of the IGF system that control it. Carbonyl groups (CO) on IGFBP-2, IGFBP-3, IGF-1R, and IGF-2R were determined in samples obtained from patients with CRC, and IGF-binding properties of these proteins were analyzed. According to our results, IGFBP-2 and IGFBP-3 in serum had increased content of CO groups due to CRC. Oxidation of IGFBP-2 increased its affinity for IGF molecules, whereas oxidation of IGFBP-3 reduced it. As for receptors, only intact CO-IGF-2R was detected on solubilized colon membranes, whereas CO-IGF-1R was degraded into fragments. Oxidative changes in the IGF axis may be regarded as part of the mechanism of its action. IGFs bound to IGFBP-3 remain in the circulation, whereas those bound to IGFBP-2 freely reach target tissues. Therefore, oxidation supports IGF distribution toward tissues and, consequently, promotes tumor growth.

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.

Oxidation of placental insulin and insulin-like growth factor receptors in mothers with diabetes mellitus or preeclampsia complicated with intrauterine growth restriction

Abstract Placental insulin receptor (IR) and insulin-like growth factor receptors (IGFRs) are essential for fetal growth. We investigated structural changes of these receptors exposed to increased oxidative stress in mothers diagnosed with diabetes mellitus (DM) or preeclampsia (PE) complicated with intrauterine growth restriction. Increased amount of IR and decreased amounts of IGF1R and IGF2R were found in both pathologies, accompanied by significant elevation in protein carbonyls. When isolated receptors were examined, increased carbonylation of IR and IGF1R in PE placentas was detected, whereas the amounts of carbonylated IR and IGF1R were similar in DM and healthy placentas. Carbonylation status of IGF2R did not change due to pathology, confirming the detrimental role of primary structure and conformation in oxidative susceptibility. Ligand binding was similar in all three groups of samples and did not seem to be affected by receptor oxidation. Since babies delivered by mothers with PE were smaller than the referent population, increased carbonylation of receptors might have affected downstream receptor signaling post-ligand binding.

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.

Analysis of changes in the glycan composition of serum, cytosol and membrane glycoprotein biomarkers of colorectal cancer using a lectin-based protein microarray

The altered glycosylation of proteins is a cancer-related marker and the monitoring of glycosylation status can significantly increase the informative value of protein biomarkers. Herein is presented an analytical method for the investigation of the glycan composition of colorectal cancer (CRC) protein biomarkers applied to different sample types using a lectin-based microarray platform. The samples included sera (from healthy persons and patients with CRC), cytosol/membrane protein fractions (from non-tumor and tumor colon tissue), and insulin-like growth factor-binding protein 3 (IGFBP-3) isolated from sera and cytosol/membrane fractions. All samples were spotted into arrays on a microarray slide, and were then incubated with a panel of 14 biotinylated lectins and a fluorescent conjugate of streptavidin. The signal intensities were detected using a microarray scanner. Statistically significant differences (Mann–Whitney test, P < 0.05) in signal intensities were found between the two groups of serum samples (with stronger signal intensities from Sambucus nigra lectin for the samples from patients with CRC than in samples from healthy individuals) and between the two groups of samples containing IGFBP-3 isolated from serum (with stronger signals from Maackia amurensis lectin-II and weaker signals from Aleuria aurantia lectin for the samples from patients with CRC). Weaker signal intensities from Aleuria aurantia lectin were observed also for samples of IGFBP-3 isolated from tumor cell membranes than for IGFBP-3 samples isolated from non-tumor tissue. The described method is applicable to the fast and high-throughput glyco-recognition analysis of differences in glycosylation patterns in various types of samples containing glycoprotein biomarkers.

Screening for the best detergent for the isolation of placental membrane proteins

Although membrane proteins (MPs) play crucial roles in physiological processes, information on them are insufficient, mostly due to their peculiar nature and surrounding which demand specific procedures for their extraction (using detergents) and analysis. A pallet of ten detergents and β-cyclodextrin was employed to investigate their efficiency in extracting total placental MPs, glycoproteins and insulin-like growth factor receptors (IR/IGF1R/IGF2R). Regardless of detergent used, the identity of major extracted proteins was the same. Glycoproteins extracted with Triton X-100 contained the greatest variety and quantity of glycans recognised by fifteen lectins, pointing to this detergent as universal medium for the extraction of membrane glycoproteins. Glycoproteins extracted using Brij 35 exhibited weak interaction with only seven lectins and were differently recognised by lectins of the similar glycan specificity. Brij 35, Tween 20, saponin and digitonin selectively extracted IGF2R compared to other two receptors. Pilot experiments should be conducted in order to choose adequate detergent for the extraction of specific MP. To obtain preparations enriched in specific receptor of the insulin/IGF system sequential solubilisation of placental MPs can be proposed: to use Brij 35 to extract IGF2R and subject the insoluble remaining suspension to Triton X-114 in order to extract most of IGF1R with small amounts of IR.

N-Glycosylation Pattern of Human Placental Insulin-Like Growth Factor and Insulin Receptors in Well-Controlled Pregestational Diabetes Mellitus

N-Glycosylation Pattern of Human Placental Insulin-Like Growth Factor and Insulin Receptors in Well-Controlled Pregestational Diabetes Mellitus Diabetes mellitus is a complex disease that leads to alterations in the glycosylation of proteins. Insulin-like growth factor and insulin receptors are involved in the regulation of fetal and placental growth and development. In this work the N-glycans of these receptors, originating from placentas obtained from pregnancies complicated by pregestational insulin dependent diabetes mellitus, were studied. Diabetic mothers were under regular insulin therapy. Solubilised membrane samples from healthy and diabetic placentas were analysed using lectin-affinity chromatography. N-glycans bound to insulin-like growth factor and insulin receptors were studied in terms of their interaction with eleven agarose-immobilised lectins: wheat germ agglutinin, succinylated wheat germ agglutinin, Ricinus communis agglutinin I, Sambucus nigra agglutinin, Erythrina cristagalli lectin, Ulex europaeus agglutinin, Lens culinaris agglutinin, Canavalia ensiformis lectin, Phaseolus vulgaris erythro- and leukoagglutinin and Maackia amurensis agglutinin. A very similar type of N-glycans and content of the terminal saccharide residues were found in both groups of placentas. The results of this work suggest that the tight glycemic control may prevent alterations in the glycosylation of insulin-like growth factor and insulin receptors, thus maintaining physiological homeostasis during pregnancy and fetal growth. N-Glikozilovanje Receptora za Insulin i Insulinu Slične Faktore Rasta iz Humane Placente Kod Majki sa Dobro Kontrolisanim Pregestacijskim Dijabetesom Dijabetes melitus je kompleksno oboljenje koje uzrokuje promene u načinu glikozilovanja proteina. Receptori za insulin i insulinu slične faktore rasta učestvuju u regulaciji rasta i razvitka fetusa i placente. U ovom radu ispitivani su N-glikani vezani kovalentnom vezom za pomenute receptore. Receptori za insulin i insulinu slične faktore rasta dobijeni su iz placenti majki sa pregestacijskim insulin-zavisnim dijabetesom. Dijabetične majke bile su na redovnoj terapiji insulinom. Uzorci ćelijskih membrana, izolovani i solubilizovani iz placenti zdravih majki i majki sa dijabetesom, analizirani su primenom lektinske afinitetne hromatografije. Ispitivana je interakcija N-glikana vezanih za receptore sa jedanaest različitih lektina, imobilisanih na agaroznom gelu: lektin iz pšeničnih klica, sukcinilovani lektin iz pšeničnih klica, Ricinus communis lektin I, lektin iz kore zove, lektin iz koralnog drveta, lektin iz Ulex europaeus-a, lektin iz sočiva, konkanavalin A, dva lektina iz pasulja i lektin iz Maackia amurensis-a. Rezultati ovog rada pokazali su da receptori za insulin i insulinu slične faktore rasta poreklom iz humane placente zdravih majki i onih sa pregestacijskim dijabetesom imaju veoma sličan tip N-glikana kao i sastav terminalnih ostataka saharida, što ukazuje na to da se dobrom kontrolom glikemije kod trudnica sa dijabetesom mogu sprečiti promene u načinu glikozilovanja ovih receptora, čime bi se pomoglo održavanju fiziološke homeostaze za vreme rasta fetusa.