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.

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.

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.

Glycosylation of urinary prostate-specific antigen in benign hyperplasia and cancer: assessment by lectin-binding patterns.

OBJECTIVES: In the present study, we examined the glycosylation of urinary prostate-specific antigen (PSA) from benign prostatic hyperplasia (BPH) and prostate cancer (PCa) subjects, specifically looking at alterations in its oligosaccharide chain as a potential biomarker of these pathophysiological conditions. DESIGN AND METHODS: First morning urine voids were collected from subjects with PCa and BPH before initiation of any treatment. Urinary PSA was characterized by ion-exchange chromatography, followed by lectin affinity chromatography on the columns using immobilized plant lectins. RESULTS: Four isoforms of urinary PSA from both BPH and PCa samples were separated by ion-exchange chromatography. The elution profiles from lectin-affinity columns reflected molecular heterogeneity of PSA isoforms and the main differences observed were in the reactivity to Ulex europaeus agglutinin, Aleuria aurantia agglutinin, Phaseolus vulgaris erythroagglutinin and Phaseolus vulgaris leukoagglutinin. CONCLUSIONS: The observed differences in the lectin reactivities between BPH PSA and PCa PSA may be of clinical importance in the evaluation of prostate health.