Gamma-glutamyltransferase-associated glycoprotein patterns in human seminal plasma of normozoospermic men: a new aspect of biomarker heterogeneity.

BACKGROUND: Gamma-glutamyltransferase (GGT) is a well-known laboratory biomarker. In spite of high concentration and the possible biomedical importance of estimating GGT in human seminal plasma (hSP), it has not been widely explored in reproductive physiology. This study aimed to complement existing data on its diversity, previously obtained on seminal extracellular vesicles, by analyzing matched soluble fraction of hSP. The GGT-associated patterns of selected glycoproteins were analyzed in order to establish an adjunct referent parameter for differentiation between known high molecular mass forms of GGT. Getting insight into distinct GGT-associated glycoprotein patterns should contribute to define them together as possible multimarkers. METHODS: GGT forms in soluble, membrane-free-fraction isolated form hSP of normozoospermic men were analyzed using gel filtration and lectin blotting using WGA (wheat germ agglutinin) and Con A (concanavalin A). RESULTS: Widely distributed GGT (with two to three partially resolved peaks), which may correspond to high molecular mass aggregates, were detected. GGT-associated patterns of selected glycoproteins (at position of big, medium, and small-GGT) all comprised high molecular mass WGA-reactive smears, but differed in the presence of Con A-reactive glycans, as well as mucin-associated antigens CA19-9 and CA125. CONCLUSIONS: GGT contributes to several molecular patterns that differ between the soluble and extracellular vesicle fractions of hSP. Their glycobiochemical heterogeneity is due to difference in the presence of distinct sialylated and mannosylated glycans. Moreover, GGT-associated glycoprotein patterns differentiate between high molecular mass forms of GGT in the soluble fraction of hSP. They hold promise as possible targets for increasing biomarker potential of GGT.

Stratum corneum levels of inflammatory mediators and natural moisturizing factor in patch test reactions to thiurams and fragrances and their possible role in discrimination between irritant and allergic reactions to hapten mixtures.

BACKGROUND: Patch test (PT) reactions to thiuram mix (TM) and fragrance mix (FM) I or II without concomitant reactions to their single constituents are potentially caused by the irritant properties of the mixes. OBJECTIVE: Comparing inflammatory profiles of PT reactions to TM, FM I, FM II, and their constituents and assessing their potential in discrimination of irritant and allergic reactions. PATIENTS AND METHODS: Levels of 14 cytokines and natural moisturizing factor (NMF) were determined in stratum corneum samples collected from PT reactions to TM, FM I or II, their constituents, and petrolatum (pet.) control sites in 36 individuals. RESULTS: Levels of interleukin (IL)-16, chemokine (CXC motif) ligand (CXCL) 8, CXCL10, chemokine (CC motif) ligand (CCL) 17, and CCL22 were significantly increased in reactions (+, ++) to thiurams and fragrances compared to their petrolatum. controls, except for PT reactions to FM I/II with negative breakdown testing in which, however, decreased levels of NMF were observed. In doubtful reactions to FM I/II with negative breakdown testing, NMF was significantly lower than in petrolatum controls. CONCLUSIONS: PT reactions to thiurams and fragrances indicate a Th2-skewed inflammation. The inflammatory profiles suggest that weak or doubtful FM I/II reactions without accompanying reaction to a constituent were irritant. IL-16 might be suitable to distinguish irritant from allergic reaction.

Membrane-associated gamma-glutamyl transferase and alkaline phosphatase in the context of concanavalin A- and wheat germ agglutinin-reactive glycans mark seminal prostasome populations from normozoospermic and oligozoospermic men.

Background: Human seminal prostasomes are intrinsically heterogeneous extracellular vesicles (EVs) whose composition is, additionally, influenced by different physiological conditions. Aiming at the molecular properties of the prostasomal surface exemplified by glycan compositions as a possible distinction factor, we applied lectin-affinity chromatography (LAC) as a new tool for their separation. Since glycans, generally, exhibit various biological activities, introduction of glyco-parameters as reference could upgrade standardization of EVs isolated by different methods and intended for use in biomedicine.Methods: Preparations of seminal prostasomes from normozoospermic (sPro-N) and oligozoospermic (sPro-O) men were subjected to LAC on concanavalin A (Con A) and wheat germ agglutinin (WGA) columns. Prostasomes recovered in LAC-separated fractions were characterized according to the distribution of selected markers: gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), tetraspanin CD63, and total protein/glycoprotein composition.Results: Two CD63-immunoreactive populations exhibiting prostasome signature bands but differing in GGT activity and surface glycans were separated on the WGA column. Additional populations having distinct profiles of total glycoproteins and which can be tracked down by ALP activity were enriched on the Con A column. WGA-separated populations were similar in sPro-N and sPro-O, whereas Con A-separated ones were strikingly different.Conclusions: Membrane-associated gamma-glutamyl transferase and alkaline phosphatase in the context of Con A- and WGA-reactive glycans mark seminal prostasomes populations from normozoospermic and oligozoospermic men.

Surface glycans contribute to differences between seminal prostasomes from normozoospermic and oligozoospermic men.

Background: Extracellular vesicles (EVs), released from the plasma membrane or intracellular compartments, have a specific composition related to their parent cells, but they can, additionally, be modified by the extracellular environment. Although glycans are known to contribute to EV composition and may have biomedical importance as biomarkers and recognition signals, they have not been extensively investigated. In this study, seminal prostasomes, i.e. EVs from seminal plasma (SP) of normo- and oligozoospermic men, were analyzed in order to detect possible changes in their surface glycans under altered physiological conditions. Methods: Prostasomes were isolated from pooled SP by differential centrifugation and gel filtration, followed by glycobiochemical characterization using lectin/immune-transmission microscopy and ion-exchange chromatography. Results: Within the frame of overall similarity in protein composition, surface glycans specifically contributed to the differences between the examined groups of prostasomes in terms of presentation of sialylated and mannosylated moieties. These changes did not affect their anti-oxidative capacity, but implied a possible influence on the accessibility of galectin-3 to its ligands on the prostasomal surface. Conclusions: Subtle differences in the presentation of surface molecules may be helpful for differentiation among vesicles sharing the same physical properties. In addition, this may point to some unexpected regulatory mechanisms of interaction of distinct populations of vesicles with their binding partners.

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.

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.

Human Serum Low Molecular Mass Prostate-specific Antigen As Biomarker.

BACKGROUND: Prostate-specific antigen (PSA) is a glycoprotein tumor marker known to exist as numerous glycospecies. Investigations on its glycobiochemical properties aimed at their use in the preparation of adjuncts in determining PSA concentration for clinical purposes have accumulated a lot of data on its structural properties. In this study, we reconsidered unexplored ubiquitously present low molecular mass species of PSA regarding to molecular mass, origin and pathophysiological source specificity in order to evaluate them as biomarkers. METHODS: Data on low molecular mass PSA-immunoreactive species from sera of subjects with prostate cancer (PCa), benign prostatic hyperplasia (BPH), breast cancer (BCa), and urine of healthy males obtained by on-chip immunoaffinity chromatography combined with mass spectrometry were analyzed. RESULTS: The results obtained indicated PSA species common to BCa, PCa, and BPH at 12-13 kDa, 17-19 kDa and 21-24 kDa. The striking difference in predominant frequencies made the profile characteristic in each examined pathophysiological condition. On the other hand, paired groups of prostatic and extraprostatic PSA contained rare species with small differences among groups concerning individual species. Low molecular mass PSA also included rare species unique for each group of samples. CONCLUSION: The results obtained revealed that uniformity of low molecular mass PSA-immunoreactive species in sera prevails over diversity related to cancer and non-cancer conditions, but at the same time some of them are molecules with biomarker potential for BPH detection.

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.

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.

On chip immuno-affinity profiling of cancer- and benign hyperplasia-associated free prostate specific antigen.

Prostate specific antigen (PSA) exhibits pronounced heterogeneity in both primary structure and glycan composition, resulting in the existence of different molecular forms. Investigation of PSA structure is a demanding task facing limitations due to inadequate sensitivity of analytical techniques and low concentrations of the different forms. This study aimed to profile free PSA (fPSA), especially lower molecular mass species lacking detailed classification, in normal seminal plasma and in sera from subjects with benign hyperplasia (BPH) or cancer of the prostate (PCa) as samples of known clinical relevance. fPSA forms were separated from complex proteomes on chips with immobilized anti-fPSA antibody followed by detection using surface-enhanced laser desorption/ionization time of flight mass spectrometry. At least 39 fPSA-immunoreactive species, ranging from 3-29 kDa were detected in seminal plasma. General fPSA profiles in seminal plasma and sera were similar, but differed in the abundance and presence of particular peaks/clusters of the lower molecular mass species. No striking difference in fPSA forms was observed between BPH and PCa samples, but some distinct peaks varied in intensity and frequency within or between groups. Obtained data verify fPSA heterogeneity that might be important for better exploration of all their molecular and marker potentials.