Miroslava Janković

Glycoforms of CA125 antigen as a possible cancer marker

CA125, a coelomic epithelium-related antigen, is expressed in both normal and pathological conditions. In this study, we compared the glycosylation of CA125 antigen from amniotic fluid and the ovarian carcinoma cell line OVCAR-3, in order to detect possible differences as a specific marker of their origin. Antigens from both sources were radiolabelled and subsequently subjected to the affinity chromatography, using plant lectins differing in carbohydrate specificity as ligands. A common chromatographic scheme was applied to all columns, i.e. they were eluted with: a) washing buffer to wash out non-bound and low-affinity bound fractions, b) a solution of inhibitory sugar and c) a low pH buffer, to release the high affinity bound fractions. CA125 antigen from each source was found to be heterogeneous in respect to the existence of multiple glycoforms, with O-linked glycan chains predominating. However, the binding patterns of both N- and O-linked glycan-reactive lectins indicated distinct differences in carbohydrate composition between CA125 antigen isolated from amniotic fluid and OVCAR-3 cell line. The observed specificites of CA125-oligosaccharide chains might be of special importance from the biomedical aspect, in terms of their possible use for clinical evaluation of gynecological functions in health and disease.

Isolation of urinary extracellular vesicles from Tamm- Horsfall protein–depleted urine and their application in the development of a lectin-exosome-binding assay

Urine is a readily available source of relatively large quantities of extracellular vesicles (EVs). However, the isolation of urinary EVs (uEVs) is complicated by the presence of Tamm-Horsfall protein (THP), which polymerizes and co-precipitates as a contaminant. This may make glycan analysis of uEVs difficult since THP is heavily glycosylated. To facilitate glycosylation analysis and address the need for elimination of non-uEV glycans, we present a modification of the uEV isolation procedure and use the isolated uEVs in the development of a lectin-exosome binding assay. Salt precipitation was employed to remove THP under conditions originally described for its separation from urine, followed by differential centrifugation. The quality of the isolated uEVs was examined by electron microscopy, SDS-PAGE, and immunoblotting. The uEVs were subsequently immobilized on solid phase and probed with labeled plant lectins using the lectin-exosome binding assay. Our results indicate that the isolated uEVs had preserved structural integrity and reacted with labeled plant lectins in a selective, carbohydrate-dependent manner. The basic lectin binding pattern of uEVs obtained by our method can be used as a reference for assessing the composition of their surface glycans in different physiological and pathological conditions.

Fibronectin Pattern in Benign Hyperplasia and Cancer of the Prostate

Fibronectin (FN) is a multifunctional glycoprotein involved in cell-matrix interactions. It exhibits a complex pattern of forms differing in respect to aminoacid and oligosaccharide composition. In this study we examined glycobiochemical and functional properties of the FN in benign prostatic hyperplasia (BPH) and prostatic cancer (PCa), attempting to resolve disease-related differences. Two BPH sera pools and three PCa sera pools were used as the FN source. The affinity-purified molecule was characterized by SDS-PAGE, immuno- and lectin blot, lectin-affinity chromatography and adhesion assay. BPH FN existed as intact molecule, giving the main immunoreactive band at 220 kDa. In contrast, PCa FN comprised three main immunoreactive fragments of 140, 110 and 90 kDa. As for glycosylation the ratio of altogether lectin-reactive PCa FN was different from that of BPH FN manifested as a decrease of Con A- and an increase of LCA-reactive moieties. Fibroblasts adhered to both FN preparations in a concentration dependent manner, but with a significantly lower efficiency to PCa FN. The results obtained showing distinct structural characteristics of PCa FN compared to BPH FN could be important for modulation of its ligand and recognition properties expressed as gain or loss of functions or as specific markers of its origin.

Glycans as Biomarkers: Status and Perspectives

Glycans as Biomarkers: Status and Perspectives Protein glycosylation is a ubiquitous and complex co- and post-translational modification leading to glycan formation, i.e. oligosaccharide chains covalently attached to peptide backbones. The significance of changes in glycosylation for the beginning, progress and outcome of different human diseases is widely recognized. Thus, glycans are considered as unique structures to diagnose, predict susceptibility to and monitor the progression of disease. In the »omics« era, the glycome, a glycan analogue of the proteome and genome, holds considerable promise as a source of new biomarkers. In the design of a strategy for biomarker discovery, new principles and platforms for the analysis of relatively small amounts of numerous glycoproteins are needed. Emerging glycomics technologies comprising different types of mass spectrometry and affinity-based arrays are next in line to deliver new analytical procedures in the field of biomarkers. Screening different types of glycomolecules, selection of differentially expressed components, their enrichment and purification or identification are the most challenging parts of experimental and clinical glycoproteomics. This requires large-scale technologies enabling high sensitivity, proper standardization and validation of the methods to be used. Further progress in the field of applied glycoscience requires an integrated systematic approach in order to explore properly all opportunities for disease diagnosis. Glikani Kao Biomarkeri: Status i Perspektive Glikozilacija proteina je univer zalna i složena ko- i post-translaciona modifikacija koja dovodi do formiranja glikana, tj. oligosaharidnih lanaca koji su kovalentno vezani za polipeptidnu kičmu. Dobro je poznat značaj promena u glikozilaciji proteina za nastanak, razvoj i krajnji ishod različitih bolesti kod ljudi. Glikani se smatraju jedinstvenim strukturama za dijagnozu, i praćenje toka bolesti. U »omics« eri, glikom, glikanski analog proteoma i genoma, predstavlja mogući izvor novih biomarkera. Kreiranje strategije za otkriće biomarkera zahteva nove principe i platforme za analizu relativno malih količina brojnih glikoproteina. Očekuje se da glikomske tehnologije, koje su još u razvoju, a koje obuhvataju različite tipove masene spektrometrije i afinitivnih tehnika, rezultiraju novim analitičkim procedurama u oblasti odre đivanja biomarkera. Najveći izazovi za eksperimentalnu i kliničku glikoproteomiku su: pretraga različitih tipova glikomolekula, odabir potencijalnih markera i njihova selekcija ili prečišćavanje i identifikacija. Za ovo je neophodno razviti tehnologije koje će omogućiti visoku senzitivnost detekcije biomarkera kao i odgovarajuću standardizaciju i validaciju novih metoda, kako bi se one mogle primeniti u laboratorijskom radu. Dalji razvoj na polju primenjene glikonauke zahteva integrisani sistemski pristup sa ciljem da se na pravi način iskoriste sve njene mo gućnosti u dijagnostici.

Assessment of Sialic Acid Diversity in Cancer- and Non-Cancer Related CA125 Antigen Using Sialic Acid-Binding Ig-Like Lectins (Siglecs)

This study was aimed at obtaining insight into the diversity of sialic acids in cancer- and non-cancer-related CA125 antigen, tumour marker of serous ovarian cancer. Starting from available data suggesting the possible relevance of sialic acids for discriminating CA125 antigens of different origin, we have employed a new experimental approach based on the use of human sialic acid-binding Ig-like lectins, Siglecs, as tools for the investigation of sialylation. Siglec−2, belonging to the group of evolutionarily conserved Siglecs, and Siglec−3, −6, −7, −9 and −10, which are CD33-like Siglecs, were probed in solid-phase binding assays with cancer-related CA125 antigens from pleural fluid of patients with ovarian carcinoma (pfCA125), the OVCAR-3 ovarian carcinoma cell line (clCA125) and a non-cancer-related CA125 antigen, i.e. pregnancy-associated pCA125 antigen. All Siglecs used showed detectable binding to pCA125 antigen. Siglec−3, Siglec−7 and Siglec−2 exhibited moderately stronger binding to pCA125 antigen than the others. In contrast to this, Siglec−2 and Siglec−3 preferentially recognized pfCA125 with greater total binding than for pCA125, whereas Siglec−9 and Siglec−10 were highly selective for clCA125. Siglecs promise to be powerful tools for discriminating CA125 of different origin and could propagate further research on other molecular markers of biomedical and diagnostic importance.

Expression of Galectin-1 and Galectin-3 in Human Fetal Thyroid Gland

High levels of expression of galectin-1 and galectin-3, the β-galactoside-binding proteins, have been recently described in malignant thyroid tumors but not in adenomas nor in normal thyroid tissue. However, there are no data about the expression of these galectins during fetal thyroid development. In this study we analyzed immunohistochemically the presence of galectin-1 and galectin-3 in human fetal thyroid glands (16–37 weeks of gestation). Weak to moderate cytoplasmic staining for galectin-1 was observed in follicular cells of all fetal thyroids. Galectin-3 could not be detected in thyroid follicular cells of any fetal thyroid investigated. Both galectins were detected in stromal tissue, but staining for galectin-1 was more intense. The absence of galectin-3 in thyroid cells during fetal development suggests that galectin-3 is expressed de novo during malignant transformation of thyroid epithelium, and that galectin-1 could be considered an oncofetal antigen. The results obtained indicated potential roles for galectin-1 and galectin-3 during the investigated period of human fetal thyroid gland development. Both galectins might participate in developmental processes regarding stromal fetal thyroid tissue organization, whereas galectin-1 might have a function in thyroid epithelium maturation.

Molecular heterogeneity of gelatin-binding proteins from human seminal plasma.

Defining the molecular characteristics of seminal plasma proteins is essential for understanding their function in physiological and pathological conditions. Starting from the predicted importance of human seminal plasma gelatin-binding proteins, comprising fibronectin (FN) and FN-related molecules, for male fertility, this study aims at gaining insight into their immuno-glycobiochemical properties. Human seminal plasma from subjects with normal semen parameters were separated on a gelatin-Sepharose column and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting using antibodies against distinct FN forms. Heterogeneity of the isolated molecular species was examined by protein chip arrays combined with surface-enhanced laser desorption/ionization time of flight mass spectrometry, on normal, metal and hydrophobic surfaces. Carbohydrate composition was investigated using mannose-, fucose- and sialic acid-specific plant lectins and galectin-1. The results obtained indicated a pattern of isolated proteins corresponding to that of known FN fragments, as confirmed by immunoreactivity. Among them heparin-binding ability was preferentially associated with low molecular mass species. As for posttranslational modifications, phosphorylation and glycosylation of distinct fragments were revealed. Lectin binding to fragments containing the gelatin-binding domain, particularly with Ricinus communis agglutinin I, was stronger than to fragments containing the cell-binding site of FN. A low level of sialylation and distinctive concanavalin A- and Lens culinaris agglutinin-reactive species were also observed. Galectin-1 did not interact with the isolated preparation. Resolving the molecular heterogeneity of normal human seminal plasma FN and gaining initial insight into possible similarities/differences with known FN molecular species may be considered a prerequisite step preceding challenging the clinical usefulness of these molecular properties.

GLYCANS AS A TARGET IN THE DETECTION OF REPRODUCTIVE TRACT CANCERS

Glycans as a Target in the Detection of Reproductive Tract Cancers The significance of changes in glycosylation for the beginning, progress and outcome of different human diseases is highly recognized. In this review we summarized literature data on the alteration of glycans in cancer, especially glycoforms of tumor markers of reproductive tract cancers: prostate-specific antigen (PSA) and cancer antigen 125 (CA125). We aimed to highlight the diagnostic potential and relevance of glycan microheterogeneity and to present some novel methods for cancer detection. A computerized search of articles published up to 2007 was performed through the PubMed database. Search terms utilized included prostate/ovarian cancer glycosylation, prostate/ovarian cancer detection, PSA/CA125 glycosylation. Additional sources were identified through cross-referencing and researching in available biomedical books. The comparative studies of sugar chain structures of the PSA and CA125 indicated specific structural alterations associated with malignant transformation, in relation to glycan branching, sialylation and fucosylation. These glycan modifications should be better in distinguishing between benign and malignant conditions than the measurement of marker concentrations alone, which is widely used in practice. Cancer-associated changes in the glycosylation could yield more sensitive and discriminative diagnostic tests for reproductive tract cancer detection, i.e. for improvement of the clinical utility of known tumor markers or the discovery of new ones. Glikani Kao Mete U Detekciji Kancera Reproduktivnog Trakta Poznat je značaj promena glikozilacije za nastanak, razvoj i ishod različitih bolesti. U ovom radu sumirani su podaci iz literature o promenama glikana kod kancera, posebno glikoformi tumorskih markera kancera reproduktivnog trakta: specifičnog antigena prostate (PSA) i kancerskog antigena 125 (CA125). Cilj ovog rada je da se naglase dijagnostički potencijal i značaj mikroheterogenosti glikana kao i da se prezentuju neke nove metode za detekciju kancera. Izvršena je pretraga radova objavljenih do 2007. godine u bazi podataka PubMed, pomoću termina > prostate/ovarian cancer glycosylation<, >prostate/ovarian cancer detection<, >PSA/CA125 glycosylation<. Kao dodatni izvor podataka korišćene su i dostupne biomedicinske knjige. Uporedna studija strukture šećernih lanaca PSA i CA125 ukazala je na specifične promene povezane sa malignom transformacijom, koje se tiču grananja, sijalinizacije i fukozilacije glikana. Ovakve glikanske modifikacije tumorskih markera mogle bi imati veći potencijal u smislu diferencijalne dijagnostike benignih i malignih stanja nego samo merenje njihove koncentracije, koje se sada široko primenjuje u praksi. Promene glikozilacije, vezane za kancer, mogu biti osnov za razvoj senzitivnijih i specifičnijih dijagnostičkih testova za detekciju kancera reproduktivnog trakta, tj. poboljšati kliničku upotrebu poznatih ili doprineti pronalaženju novih tumorskih markera.

Ion-exchange chromatography purification of extracellular vesicles.

Despite numerous studies, isolating pure preparations of extracellular vesicles (EVs) has proven challenging. Here, we compared ion-exchange chromatography (IEC) to the widely used sucrose density gradient (SDG) centrifugation method for the purification of EVs. EVs in bulk were isolated from pooled normal human amniotic fluid (AF) by differential centrifugation followed by IEC or sucrose density gradient separation. The purity of the isolated EVs was evaluated by electrophoresis and lectin blotting/immuno blotting to monitor the distribution of total proteins, different EVs markers, and selected N-glycans. Our data showed efficient separation of negatively charged EVs from other differently charged molecules, while comparative profiling of EVs using SDG centrifugation confirmed anion-exchange chromatography is advantageous for EV purification. Finally, although this IEC-based method was validated using AF, the approach should be readily applicable to isolation of EVs from other sources as well.

Evaluation of Molecular Species of Prostate-Specific Antigen Complexed with Immunoglobulin M in Prostate Cancer and Benign Prostatic Hyperplasia

This study was aimed at defining molecular species of prostate-specific antigen (PSA) in immune complexes with immunoglobulin M (IgM). Having in mind the oligoreactivity of IgM and its preference for carbohydrate antigens, there is the possibility that it can selectively recognize known PSA glycoisoforms. PSA-IgM complexes and free PSA fractions were separated from the sera of subjects with prostate cancer (PCa) and benign prostatic hyperplasia (BPH) by gel filtration and subjected to on-chip immunoaffinity and ion-exchange chromatography. PSA-immunoreactive species were detected using surface-enhanced laser desorption/ionization time of flight mass spectrometry. The obtained spectra were analyzed for protein and glycan composition. The general pattern of the molecular species of PCa PSA and BPH PSA found in complexes with IgM was similar. It comprised major peaks at 17 kDa and minor peaks at 28 kDa, corresponding to the entire mature glycosylated PSA. The main difference was the presence of incompletely glycosylated 26.8 kDa species, having putative paucimannosidic structures, observed in PCa PSA-IgM, but not in BPH PSA-IgM. Characteristic PCa PSA-IgM glycoforms pose the question of the possible role of glycosylation as a framework for immune surveillance and may be of interest in light of recent data indicating mannose-containing glycans as cancer biomarker.