Goran Miljuš

Interaction of Insulin-Like Growth Factor-Binding Protein 2 with α2-Macroglobulin in the Circulation

Insulin-like growth factors (IGFs) play active role in mitogenic and metabolic processes. In the peripheral circulation, they are mostly bound to specific IGF-binding proteins (IGFBPs). Proteolysis of IGFBPs releases free, active IGFs. IGFBP-2 is the second most abundant of the six binding proteins and its concentration increases in catabolic states. The possible interaction between IGFBP-2 and other proteins in the circulation was investigated in this study. Our results showed that IGFBP-2 associates with α2-macroglobulin (α2M), a protease inhibitor. Formation of IGFBP-2/α2M complexes most likely contributes to the regulation of IGFBP-2 proteolysis and, thus, the activity of IGFs.

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.

IGFBP-3/transferrin/transferrin receptor 1 complexes as principal mediators of IGFBP-3 delivery to colon cells in non-cancer and cancer tissues

PURPOSE The aim of this work was to study the involvement of IGFBP-3/Tf complexes in the pathology of colorectal carcinoma (CRC), quantify them, investigate their relation to iron concentration and binding to transferrin receptor (TfR) in colon tissue (non-cancer and cancer), and to assess the priority of this pathway for internalization of IGFBP-3. METHODS The presence of IGFBP-3/Tf complexes was analyzed in sera from healthy persons and patients with CRC, and in colon tissue by immunoblotting. Complexes were immunoprecipitated, quantified by immunoassay and structurally characterized by immunoblotting, lectin blotting and mass spectrometry. Complexes which interacted with colon cells were immunoprecipitated with anti-TfR1 antibody and studied. Colon tissue slides were subjected to immunohistochemical analysis. RESULTS The concentration of IGFBP-3/Tf complexes was three times lower in patients with CRC. They were increasingly carbonylated, sialylated, contained more Galβ4GlcNAc units, expressed altered charge density and increased affinity for metal ions. Immunoprecipitation experiments revealed more TfR1 on membranes than in cytosol of colon cells, also more in cancer than non-cancer tissue. TfR1 on membranes were less occupied with IGFBP-3/Tf complexes than in cytosol. Immunofluorescent staining indicated a remarkable degree of co-localization of IGFBP-3 and TfR1, evenly distributed in non-cancer tissue and both evenly and cell surface concentrated in cancer tissue. CONCLUSIONS Increased expression of TfR1 on colon cell membranes in patients with CRC compensates for the reduced extracellular availability of IGFBP-3/Tf and TfR1 is the principal binding partner of extracellular IGFBP-3. IGFBP-3/Tf complexes in patients with CRC exhibit increased affinity for iron ions.

Analysis of four circulating complexes of insulin-like growth factor binding proteins in human blood during aging

The primary role of insulin-like growth factor binding proteins (IGFBPs) is to regulate availability of IGFs for interacting with receptors, but IGFBPs perform IGF-independent actions as well. The availability and activity of IGFBPs in the circulation is influenced primarily by their concentration and structural modifications, but possibly also by interaction with major plasma proteins such as transferrin, alpha-2-macroglobulin (α2M), and fibrinogen. Four types of circulating IGFBP complexes were examined in this study by immuno- and ligand-binding assays in adults of different age. The amounts of IGFBP-3/transferrin and IGFBP-1/fibrinogen complexes were similar in middle- and old-aged persons, whereas the amounts of IGFBP-1 (or -2)/α2M monomer complexes were lower in the old-aged group and negatively correlated with total IGFBP-1 (or -2) amounts in blood. In contrast to IGFBP-1, IGFBP-2 was present in significantly greater quantities in complexes with α2M dimer than α2M monomer in older individuals. IGFBP complexes did not bind 125I-labeled IGF-I in amounts detectable by ligand blotting. According to the results of this study, the quantities of IGFBP-1 and IGFBP-2, which interact with α2M, are age-dependent and, in the case of complexes with α2M monomer, they are negatively correlated with the total circulating levels of these two IGFBPs.

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.

Preparatory training attenuates drastic response of the insulin-like growth factor binding protein 1 at the point of maximal oxygen consumption in handball players

Background Intensive exercise changes physiological need for glucose and several biochemical pathways responsible for its metabolism response. Among them are those which involve insulin, insulin-like growth factor (IGF-1), and IGF-binding proteins (IGFBPs). Different types and degrees of exercise, as well as an athletes fitness, may induce a range of responses regarding concentrations and time needed for the alteration. The idea of the work was to find out whether and how insulin/IGF axis responds to additional physical activity in the already trained subjects and if so, is the adaptation potentially beneficial from the aspect of metabolic control. Methods The effect of 4-week intensive training on campus (preparatory training) on the levels of insulin, IGF-1, and IGFBPs during maximal progressive exercise test (MPET) on a treadmill was compared to the results obtained during MPET conducted after a regular training season of a female elite handball team (n = 17, age: 17 ± 1 years, height: 171 ± 8 cm, weight: 65 ± 8 kg, body mass index: 22 ± 1 kg/m2 at the beginning of the study; there were no significant changes at the end). Serum samples were obtained from players immediately before the test (basal), at the end of the test after reaching the point of maximal oxygen consumption (VO2max), and after recovery. Results The concentration of insulin decreased at VO2max, but remained higher in players after preparatory training (12.2 ± 2.5 mU/L vs. 8.9 ± 4.4 mU/L, p = 0.049). The level of IGFBP-1 decreased in players at VO2max in either case of training, but it remained much higher in tests performed after the preparatory regime than before (p = 0.029). Concentrations of IGF-1, IGFBP-2, -3, and -4 did not change significantly. Conclusion The inverse relation between insulin and IGFBP-1 was lost during MPET, as these 2 molecules changed in the same direction. The results obtained suggest less severe stress-induced depression of insulin and IGFBP-1 after preparatory training. But another metabolic mechanism cannot be excluded, and that is potentially impaired insulin sensitivity resulting in higher level of IGFBP-1.

A microscale protocol for the isolation of transferrin directly from serum

A microscale procedure for the isolation of transferrin directly from human serum (hTf) is described in this study. The protocol is based on three precipitation steps without application of chromatography. It lasts 90min with the initial sample volume of 250μL. The yield of the isolated hTf is 58%, which is considerable in biochemical terms. The purity of the isolated hTf is 97%, as assessed by three methods: electrophoresis followed by protein staining, immunoblotting and HPLC. Immunoblotting with antibodies against other major serum proteins indicated that isolated hTf does not contain albumin, immunoglobulin G or alpha-2-macroglobulin. Lectin dot-blot demonstrated that isolated hTf preserved its glycan moieties. Fluorescent emission spectroscopy of the isolated hTf has shown no changes in tertiary structure. Isolated hTf was approximately 26% saturated with iron ion, which is comparable to physiological value (although a degree of saturation decreases to some extent during isolation procedure). Finally, co-immunoprecipitation experiment confirmed that isolated hTf retained its ligand characteristics crucial for the ligand-receptor type of interaction with the hTf receptor. To conclude, the procedure described in this work, is time and cost-effective, allows multiple sample handling and provides high-purity hTf isolate with preserved structural and functional properties.

Regulation of Insulin and Insulin-Like Activity in Malnourished Patients with Carcinoma Ventriculi Subjected to Total Gastrectomy and Personalized Nutritional Support/ Kontrola insulinske i aktivnosti slične insulinu kod neuhranjenih pacijenata sa tumorom želuca podvrgnutih totalnoj gastrektomiji i lično adaptiranoj ishrani

Summary Background: Insulin and insulin-like growth factor (IGF) activities are disturbed during critical illness. Time-course changes in the concentrations of insulin, IGF-I and IGFbinding proteins (IGFBPs) were monitored in this study and their correlation with interleukin (IL)-6 was assessed in patients subjected to total gastrectomy and specific nutritional regime. Methods: Patients were fed post-operatively according to the following scheme: parenteral nutrition on day 1, enteral nutrition combined with parenteral form from day 2 to 7, peroral nutrition from day 8 and full oral nutrition from day 14. Blood samples were taken periodically and the levels of IL-6, insulin, IGF-I and IGFBP-1 to -4 were determined. Results: On day 1 post-operatively, the concentration of IL- 6 reached its maximum and decreased afterwards. The concentration of insulin increased until day 3 and then started to fall. The concentration of IGF-I, already low preoperatively, continued to decrease. The concentration of IGFBP-1 peaked on day 1 post-operatively, whereas the concentration of IGFBP-3 decreased on that day. The concentration of IL-6 correlated positively with the concentration of IGFBP-1 and negatively with IGFBP-3. On day 14, the concentrations of IL-6, insulin and IGFBP-1 returned to or were close to their basal levels, whereas the concentrations of IGF-I and IGFBP-3 remained reduced. Conclusions: 14-day post-operative recovery, which included specific nutritional support, was suitable to restore insulin concentration and re-establish IGFBP-1 regulation primarily by nutrition. Very low IGF-I level on day 14 after surgery and IGFBP-3 concentration still lower than before surgery indicated that the catabolic condition was not compensated. Kratak sadržaj Uvod: Aktivnost insulina i faktora rasta sličnih insulinu (IGF) izmenjena je u kritičnom stanju. U ovoj studiji je praćena promena koncentracije insulina, IGF-I, IGF vezujudh proteina (IGFBP) i utvrdivana njihova korelacija sa interleukinom (IL)-6 kod pacijenata sa tumorom želuca, koji su pod- vrgnuti totalnoj gastrektomiji i specifičnom režimu ishrane. Metode: Pacijenti su postoperativno hranjeni prema sledećem režimu: parenteralna ishrana prvog dana, enteralna ishrana u kombinaciji sa parenteralnom od drugog do sedmog dana, peroralna ishrana osmog dana i redovna ishrana četrnaestog dana. Periodično su uzimani uzorci krvi i određivane koncentracije IL-6, insulina, IGF-I i IGFBP-1 do -4. Rezultati: Koncentracija IL-6 je dostigla maksimum prvog dana nakon operacije i dalje je opadala. Koncentracija insulina je rasla do trećeg dana, a zatim je počela da pada. Koncentracija IGF-I, koja je bila niska pre operacije, na- stavila je da opada. Prvog dana posle operacije, koncentracija IGFBP-1 je porasla, dok se koncentracija IGFBP-3 smanjila. Promene IGFBP-1 i IGFBP-3 su bile izraženije sa vedm skokom IL-6. Četrnaestog dana su se koncentracije IL-6, insulina i IGFBP-1 vratile ka referentnim vrednostima, dok su koncentracije IGF-I i IGFBP-3 ostale smanjene. Zaključak: Četrnaestodnevni postoperativni oporavak, koji je obuhvatao specifičan režim ishrane, pokazao se odgovarajućim za uspostavljanje kontrole koncentracije insulina i IGFBP-1. Sa druge strane, vrlo niska koncentracija IGF-I, kao i koncentracija IGFBP-3 ispod preoperativne vrednosti, ukazuju na to da kataboli~ki stres kod ovih pacijenata i dalje nije kompenzovan.