Bojana Milutinović

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.

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.

Glycome complexity of human seminal plasma high molecular mass components: Evaluation of the contribution of acid-soluble glycoproteins/mucins and extracellular vesicles.

This study was aimed at evaluation of the contribution of acid-soluble glycoproteins (ASG)/mucins and extracellular vesicles (EVs), yet unexplored components of human seminal plasma (hSP) to the complexity of its glycome. Gaining insight into the native presentation and distribution of glycans across hSP could help establish molecular environments supporting specific biological activities based on unique ligand capacities. Soluble and particulate fractions of hSP from healthy subjects were analyzed by gel filtration, electrophoresis, ion-exchange chromatography and a solid phase assay with immobilized charge-resolved glycospecies to test their reactivity with plant lectins, carbohydrate-binding antibodies and selected human lectins. Common O- and N-glycosylated species were detected on mixed or overlapped underlying protein scaffolds in both soluble and particulate fractions of hSP. Siaα2,6Gal and N-glycans were concentrated on EVs, whereas Siaα2,3Gal, T and Tn antigens were selectively associated with distinct glycospecies of ASG/mucins. Accessible ligands for the lectins, DC-SIGN and Siglec-9, were detected in all hSP components, but they preferentially bound to EVs glycospecies. Insight into the complexity of hSP glycans as recognition signals under normal physiological conditions could be of interest for regulation and possible modulation of its biological activity, as well as for biomarker potential related to male health.

Identification of pregnancy-associated CA125-reactive protein as a carbohydrate-binding immunoglobulin G

Cancer antigen 125 (CA125), also referred to as mucin 16, is expressed under both normal and pathological conditions and the complexity of its structure indicates multifunctionality, i.e. both the protein and carbohydrate parts may be involved in diverse interactions at different levels of cell and tissue organization. Its biological role is not understood, but involvement in immune response modulation and influence on cell adhesion have been speculated. This study aimed at isolation and characterization of endogenous ligands for CA125 as an initial step in gaining insight into its activity. A CA125-reactive fraction was separated from human placental extract by affinity chromatography. The isolated preparation was characterized by SDS-PAGE, immunoblotting, peptide mass fingerprinting and binding assay. The CA125-reactive fraction from placental extract was identified as carbohydrate-binding IgG. The glycan composition of inhibitors of carbohydrate-binding pointed to sialic acid as one determinant for recognition but indicated that sialylation was not alone and that glycotopes containing galactose, N-acetylgalactosamine and N-acetylglucosamine were also important. CA125-reactive IgG could be selectively enriched using fetuin as the ligand and represents a distinct IgG subfraction differing from abundant natural carbohydrate-binding antibodies. Taking advantage of the particular properties of ligands for CA125 may have biomedical potential for use as biological modifiers or delivery agents and have an impact beyond pregnancy, since many immunoregulatory molecular pathways are common to embryonic development and malignant transformation.

Short-term fasting promotes insulin expression in rat hypothalamus

In the hypothalamus, insulin takes on many roles involved in energy homoeostasis. Therefore, the aim of this study was to examine hypothalamic insulin expression during the initial phase of the metabolic response to fasting. Hypothalamic insulin content was assessed by both radioimmunoassay and Western blot. The relative expression of insulin mRNA was examined by qPCR. Immunofluorescence and immunohistochemistry were used to determine the distribution of insulin immunopositivity in the hypothalamus. After 6‐h fasting, both glucose and insulin levels were decreased in serum but not in the cerebrospinal fluid. Our study showed for the first time that, while the concentration of circulating glucose and insulin decreased, both insulin mRNA expression and insulin content in the hypothalamic parenchyma were increased after short‐term fasting. Increased insulin immunopositivity was detected specifically in the neurons of the hypothalamic periventricular nucleus and in the ependymal cells of fasting animals. These novel findings point to the complexity of mechanisms regulating insulin expression in the CNS in general and in the hypothalamus in particular.

CA-125 of fetal origin can act as a ligand for dendritic cell-specific ICAM-3-grabbing non-integrin

CA-125 (coelomic epithelium-related antigen) forms the extracellular portion of transmembrane mucin 16 (MUC16). It is shed after proteolytic degradation. Due to structural heterogeneity, CA-125 ligand capacity and biological roles are not yet understood. In this study, we assessed CA-125 as a ligand for dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), which is a C-type lectin showing specificity for mannosylated and fucosylated structures. It plays a role as a pattern recognition molecule for viral and bacterial glycans or as an adhesion receptor. We probed a human DC-SIGN-Fc chimera with CA-125 of fetal or cancer origin using solid- or fluid-phase binding and inhibition assays. The results showed that DC-SIGN binds to CA-125 of fetal origin and that this interaction is carbohydrate-dependent. By contrast, cancerderived CA-125 displayed negligible binding. Inhibition assays indicated differences in the potency of CA-125 to interfere with DC-SIGN binding to pathogen-related glycoconjugates, such as mannan and Helicobacter pylori antigens. The differences in ligand properties between CA-125 of fetal and cancer origin may be due to specificities of glycosylation. This might influence various functions of dendritic cells based on their subset diversity and maturation-related functional capacity.

Nano-sized CA125 antigen glycocamouflage: Mucin - Extracellular vesicles alliance to watch?

Mucin 16 (MUC16) is a transmembrane type mucin and its released extracellular portion is designated as CA125 antigen. It is considered to be part of a supramolecular glycoprotein complex having a complicated epitope map and extreme structural heterogeneity. Starting from the initial transmembrane localization of MUC16/CA125 antigen and its alternative routes of release by shedding or putative secretion, CA125 antigen from human amniotic fluid soluble and extracellular vesicles (EVs)-containing fractions were characterized aiming at the possible glycosylation-associated mode of distribution as a factor contributing to the reported conflicting structural data. Ultracentrifugation, sucrose density gradient centrifugation, ion-exchange chromatography and TEM were used for analysis. The results indicated that the smeared abundantly glycosylated high molecular mass CA125-immunoreactive species, which follow the wheat germ agglutinin-binding pattern, were shared across amniotic fluid soluble and particulate fractions. A lower molecular mass glycoprotein-like CA125-immunoreactive species which follows the peanut agglutinin-binding pattern and was specifically associated with the EVs-enriched fraction was observed. CA125 presentation in the particulate amniotic fluid fraction was found to be shaped by a complex interactome partially involving lactose-sensitive galectin-3 binding. The MUC16 - EVs alliance as well as heterogeneous mucin/macromolecular complexes, at membranes or extracellularly, may represent cryptic pools of distinct CA125 species.

Analysis of CA125 antigen in normal human seminal plasma highlightsthe molecular heterogeneity of underlying glycosylated species

Cancer antigen CA125 represents an extracellular part of transmembrane mucin MUC16 and may be expressed in tissues of the male reproductive tract. It reaches human seminal plasma (hSP) after undergoing the main processes involved in protein speciation: synthesis, posttranslational modifications, compartmentalization, and auto/proteolytic degradation. This study was aimed at profiling CA125-immunoreactive species in hSP from healthy subjects as a specific and unexplored source of this tumor-associated antigen expressed under normal physiological conditions. High molecular mass components from total hSP and corresponding acid-soluble hSP preparations were analyzed. The results indicated that antibodies recognizing two distinct immunogenic areas of CA125 antigen exhibited a common pattern of immunoreactive bands affected by low pH and reducing agents. They comprise a large, heavily glycosylated moiety and a mixture of low glycosylated species ranging from 100 to 150 kDa. High molecular mass CA125-immunoreactive smears overlapped core 2 O-glycans and the Lex glycotope, the latter also being abundant on distinct lower molecular mass immunoreactive bands. Within the bulk of normal hSP mucins, the CA125 antigen is present in low amounts and resides on heterogeneously glycosylated species that may be proteolysis-derived and sensitive to redox environments. Both factors influence the composition of hSP and therefore might affect speciation of mucin16 under normal and pathological conditions.