Romana Masnikosa

Serum insulin-like growth factor (IGF)-II is more closely associated with liver dysfunction than is IGF-I in patients with cirrhosis

The aim of this investigation was to determine the total concentrations of the insulin-like growth factors (IGF-I and IGF-II) in the blood serum of patients with liver cirrhosis and to evaluate their association with the condition. Cirrhosis was alcohol induced (n=27), of viral origin (n=17) or due to combined or other causes (n=21) and was moderate or severe in similar numbers of cases (Child A: n=21; Child B: n=21; Child C: n=23). While serum levels of both peptides were lower in patients than in age-matched healthy subjects (n=81), there was considerable overlap into the lower normal range for IGF-I. Moreover, no correlation between disease severity (Child score) and serum IGF-I was observed. Since a total of 78% of the results for IGF-II were outside the normal range (95% confidence interval) and serum concentrations were correlated with Child score (P=0.007), it is suggested that serum IGF-II concentrations may reflect compromised hepatic function more closely than IGF-I.

Alterations of IGF-binding proteins in patients with alcoholic liver cirrhosis

The protein synthetic activity of the liver is diminished in cirrhosis. The aim of this study was to investigate possible changes in the serum IGF-IGFBP system among patients with alcoholic liver cirrhosis (ALC). The results obtained demonstrated that serum IGF-I and IGF-II concentrations were significantly lower in patients with ALC than in healthy persons (P=0.0008 for IGF-I and 0.0002 for IGF-II). The IGFBP profile was markedly altered and the 34 kDa IGFBP from patients had higher affinity towards 125I-IGF-II compared to the 34 kDa IGFBP of control individuals. Moreover, the 40-45 kDa IGFBP (in isolated complex with 125I-IGF-II) exhibited diminished interaction with concanavalin A, wheat germ, and breadfruit lectins. Modification of the glyco-component of the 40-45 kDa IGFBP seems to be an early event in ALC since change in reactivity towards lectins was noticed in patients with ALC classified as Child score A, whose serum IGF-I and IGF-II levels were within reference limits (the existence of carbohydrate microheterogeneity of this IGFBP was also assessed by lectin-affinity electrophoresis). It is possible that these biochemical alterations may affect the functional activity of the IGFs by changing the dynamics and distribution of these growth factors in the organism.

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.

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.

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.

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.

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.

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.

Receptors and Binding Proteins for Insulin and Insulin-Like Growth Factors in the Placenta of Healthy Mothers and Mothers with Insulin-Dependent Diabetes Mellitus

Receptors and Binding Proteins for Insulin and Insulin-Like Growth Factors in the Placenta of Healthy Mothers and Mothers with Insulin-Dependent Diabetes Mellitus The IGF system of human placenta consists of insulin-like growth factors (IGF)-I and -II, their receptors (IGF-1R and IGF-2R), and binding proteins (IGFBP-1 to -6). Due to many structural and metabolic similarities with insulin, the IGF system cannot be examined separately from insulin and its receptor (IR). In this study gel filtration was used to detect solubilized membrane proteins of the placenta obtained from healthy mothers and mothers with IDDM. In order to detect placental membrane proteins that bind IGF molecules (and insulin), the solubilized membranes were incubated with each of the three 125I-labelled ligands: 125I-IGF-I, 125I-IGF-II and 125I-insulin prior to gel filtration chromatography. The biochemical evidence of the presence of receptors for insulin and IGFs, as well as that of IGFBP-1 were obtained by immunoblotting. Herein we demonstrated that, considering IGF and insulin receptor content, the placental tissue obtained from mothers with IDDM was not different from that obtained from healthy mothers. However, the concentration of IGFBP-1 differed between the examined placentas. IDDM in mothers caused an increase in the amount of IGFBP-1 in their placentas and, consequently, the amount of the labelled ligand bound to it. The redistribution of IGFs between the receptors and IGFBP-1 may be involved in regulatory mechanisms in the placenta of mothers with IDDM. Receptori I Vezujući Proteini ZA Insulin I Insulinu Slične Faktore Rasta U Placenti Zdravih Majki I Majki SA Insulin-Zavisnim Dijabetes Melitusom IGF sistem humane placente sačinjavaju insulinu slični faktori rasta (IGF)-I i -II, receptori za koje se oni vezuju (IGF-1R i IGF-2R), kao i njihovi vezujući proteini (IGFBP-1 do -6). Usled strukturnih i metaboličkih sličnosti sa insulinom, IGF sistem se ne može izučavati odvojeno od insulina i njegovog receptora (IR). U ovom radu gel filtracija je korišćena kako bi se detektovali membranski proteini iz solubilizata membrana dobijenih iz placenti zdravih majki i majki sa IDDM. Pojedinačni uzorci solubilizata preinkubirani su sa tri 125I-liganda: 125I-IGF-I, 125I-IGF-II ili 1255I-insulinom, a zatim hromatografisani. Biohemijski dokazi prisustva receptora za insulin i insulinu sličnih faktora rasta, kao i IGFBP-1 dobijeni su imunoblotingom. Rezultati su pokazali da se tkiva placenti dijabetičnih majki ne razlikuju od zdravih u pogledu IGF i insulinskog receptora, ali razlike postoje na nivou IGFBP-1. IDDM izaziva povećanje količine IGFBP-1 i, shodno tome, povećanje količine liganda koji se vezuje za njega. Ova preraspodela IGF molekula između receptora i IGFBP-1 može učestvovati u regulatornim mehanizmima u placenti majki sa IDDM.