Characterization of molecular species of human prostate-specific antigen by on-chip immunoaffinity chromatography

Specifičan antigen prostate (prostate-specific antigen; PSA) ili kalikrein 3 (hK3), pripada grupi ekstracelularnih serin-zavisnih proteaza iz porodice kalikreina. PSA u najvećoj meri eksprimiraju epitelne ćelije acinusa i duktusa prostate i periuretralne žlezde, a u manjoj meri i neka druga tkiva. PSA je, u normalnim fiziološkim uslovima, prisutan u semenoj tečnosti u izrazito visokoj koncentraciji za razliku od seruma gde je ona veoma niska. Usled narušene strukture prostate, do koje može doći pri različitim poremećajima i oboljenjima, nivo PSA se u sistemskoj cirkulaciji znatno povećati pa se on koristi kao serumski biomarker. Brojne studije su pokazale da PSA nije, u striktnom smislu, specifičan samo za prostatu, već da ga eksprimiraju i neka hormon-zavisna tkiva žene, pre svega dojka, endometrijum i ovarijum, kako u normalnom, tako i u patološkim stanjima. Ekspresija gena za PSA je, u tkivu prostate, regulisana androgenima, dok je, u ćelijskim linijama kancera dojke ona posredovana aktivacijom receptora za progesteron, androgene, mineralokortikoide i glukokortikoide, ali ne i receptora za estrogene. PSA je glikoprotein molekulske mase 28,43 kDa, koji sadrži jedan biantenaran lanac N-acetil-laktozaminskog tipa sa sijalinskom kiselinom na krajevima obe grane. Ovi podaci se odnose na kanonsku sekvencu/strukturu enzimski aktivnog molekula PSA. On se u cirkulaciji nalazi u kompleksima sa inhibitorima proteaza kao što je α1-antihimotripsin (ACT) ili α2-makroglobulin, ali pored njega postoje i molekuli PSA koji nemaju enzimsku aktivnost ili je ona znatno smanjena. Njihova zastupljenost u kompleksima je niska i oni predstavljaju formu slobodnog PSA (fPSA). Za razliku od ispitivanja PSA formi, njegove pojedinačne molekulske vrste koje pripadaju ovim formama do sada nisu bile predmet sistematske analize. Proteinska vrsta je najmanja strukturna i funkcionalna jedinica proteoma i označava jedan individualan protein iz familije proteina koji kodira pojedinačan gen. Ispitivanje specijacije proteoma i kinetike njegovih proteinskih vrsta se nalazi u fokusu savremenih proteomskih istraživanja. Cilj ove doktorske disertacije je da se ustanovi profil molekulskih vrsta PSA u subproteomima klinički relevantnih bioloških tečnosti: serum, urin i semena plazma..

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

Kompozicija i distribucija glikana na membrani prostazoma kod muškaraca sa normospermijom i oligospermijom

Простазоми су екстраћелијске везикуле (EVs) које епителијалне ћелије простате секретују у семену течност. Они имају двослојну липидну мембрану и садрже компоненте ћелија које их излучују (протеинe, фрагментe ДНK, информациону РНK, микрo РНK, дугу некодирaјућу РНK, липидe и метаболитe). Циљ овог рада је био да се анализом композициje и обрасца дистрибуције манозилованих/сијалинизованих N-гликана и маркера EVs: тетраспанина (CD9, CD63, CD81), гама-глутамил-трансферазе (GGT) и галектина-3 (gal-3), утврди јединствени гликански састав мембране простазома као нови референтни маркер. Простазоми су изоловани из семених плазми мушкараца са нормоспермијом и олигоспермијом. Дистрибуција N-гликана, тетраспанина, GGT и gal-3 на површини мембране простазома је добијена анализом солубилизованих простазома. N-гликани су детектовани на оснoву реактивности са лектинима: Wheat germ agglutinin (WGA) и Concanavalin A (ConA), а тетраспанини и gal-3 имуно-дотблот-ом. GGT је праћенa мерењем ензимске активности. Установљена су два обрасца дистрибуције код обе испитиване групе: CD9/gal-3/WGA-реактивни гликопротеини и CD63/GGT/ConA-реактивни гликопротеини. Показано је да постоји сличност у дистрибуцији N-гликана и GGT, док су разлике уочене у дистрибуцији тетраспанина и gal-3. Разлике у композицији и дистрибуцији гликана на површини мембране простазома могле би представљати потенцијални индикатор промена у репродуктивној физиологији

Surface-Associated Glycans as a Possible Distinct Factor for Establishing the Molecular Properties of Prostasomes

Glycans, complex carbohydrates bound to lipids and proteins, play important roles in biological processes. Prostasomes, extracellular vesicles from prostate epithelial cells, have a glycan composition influenced by their cellular origin and by the composition of the seminal plasma into which they are secreted. We hypothesized that the membrane-associated glycans could be used as selective targets for separation of prostasomes and as relevant parameter for distinction of their populations. Prostasomes from seminal plasma of normozoospermic and oligozoospermic men were separated by ion-exchange- and lectin-affinity chromatography using concanavalin A (ConA) lectin, specific for mannosylated structures, and wheat germ agglutinin (WGA), specific for sialic acid. The presence of tetraspanins (CD63, CD9 and CD81), galectin-3 (gal-3) and gamma-glutamyl transferase (GGT) as known vesicle markers were monitored in association with distinct glycans. Their distribution was also analysed upon treatment of prostasomes with non-ionic detergent. Membrane-associated GGT in the context of Con A- and WGA-reactive glycans mark prostasome populations from normozoospermic and oligozoospermic men. The assembly of tetraspanins, gal-3, and distinct N-glycans defines the solubilisation signature of prostasomes. WGA-reactive glycoproteins co-localize with CD9 and gal-3 in detergent-resistant domains, whereas ConA-reactive glycoproteins were distributed in detergent-sensitive domains along with CD63 and GGT. Subtle differences in the composition/presentation of examined molecules made difference among vesicles sharing the same physical properties in each group as well as between them. The results obtained suggest the potential of glyco-parameters as reference markers for EVs populations

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

Characterization of molecular species of human prostate-specific antigen by on-chip immunoaffinity chromatography

Specifičan antigen prostate (prostate-specific antigen; PSA) ili kalikrein 3 (hK3), pripada grupi ekstracelularnih serin-zavisnih proteaza iz porodice kalikreina. PSA u najvećoj meri eksprimiraju epitelne ćelije acinusa i duktusa prostate i periuretralne žlezde, a u manjoj meri i neka druga tkiva. PSA je, u normalnim fiziološkim uslovima, prisutan u semenoj tečnosti u izrazito visokoj koncentraciji za razliku od seruma gde je ona veoma niska. Usled narušene strukture prostate, do koje može doći pri različitim poremećajima i oboljenjima, nivo PSA se u sistemskoj cirkulaciji znatno povećati pa se on koristi kao serumski biomarker. Brojne studije su pokazale da PSA nije, u striktnom smislu, specifičan samo za prostatu, već da ga eksprimiraju i neka hormon-zavisna tkiva žene, pre svega dojka, endometrijum i ovarijum, kako u normalnom, tako i u patološkim stanjima. Ekspresija gena za PSA je, u tkivu prostate, regulisana androgenima, dok je, u ćelijskim linijama kancera dojke ona posredovana aktivacijom receptora za progesteron, androgene, mineralokortikoide i glukokortikoide, ali ne i receptora za estrogene. PSA je glikoprotein molekulske mase 28,43 kDa, koji sadrži jedan biantenaran lanac N-acetil-laktozaminskog tipa sa sijalinskom kiselinom na krajevima obe grane. Ovi podaci se odnose na kanonsku sekvencu/strukturu enzimski aktivnog molekula PSA. On se u cirkulaciji nalazi u kompleksima sa inhibitorima proteaza kao što je α1-antihimotripsin (ACT) ili α2-makroglobulin, ali pored njega postoje i molekuli PSA koji nemaju enzimsku aktivnost ili je ona znatno smanjena. Njihova zastupljenost u kompleksima je niska i oni predstavljaju formu slobodnog PSA (fPSA). Za razliku od ispitivanja PSA formi, njegove pojedinačne molekulske vrste koje pripadaju ovim formama do sada nisu bile predmet sistematske analize. Proteinska vrsta je najmanja strukturna i funkcionalna jedinica proteoma i označava jedan individualan protein iz familije proteina koji kodira pojedinačan gen. Ispitivanje specijacije proteoma i kinetike njegovih proteinskih vrsta se nalazi u fokusu savremenih proteomskih istraživanja. Cilj ove doktorske disertacije je da se ustanovi profil molekulskih vrsta PSA u subproteomima klinički relevantnih bioloških tečnosti: serum, urin i semena plazma..

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

Extracellular vesicles in a maze of glycomic complexity

The carbohydrate portion of proteins and lipids mediates a variety of biological processes. Revealing their underlying principles is a challenging task that could contribute to a better understanding of many patho/physiological conditions. On the other hand, the interest in extracellular vesicles (EVs) has increased in recent years due to their involvement in intercellular communication leading to an array of functional and structural changes in recipient cells. Their characterization uncovered an exceptional diversity in size, morphology, as well as in membrane and cargo content. Monitoring/analysis of surface glycosylation of EVs originating from the prostate, termed prostasomes, revealed their substantial contribution to the complexity of seminal plasma (SP) glycome. Heterogeneity of surface glycans confirm the existence of several prostasome subpopulations. Presentation of surface glycans on prostasomal membrane is strongly affected by co-localized membrane-associated glycoproteins and tetraspanins. They appear to be organized in established/regular distribution patterns on membrane domains. Surface glycans are a component of EVs membrane that affects its functionality and potentially a distinction marker of prostasome subpopulations. Further understanding of the complex composition of glycans on EVs might explain the relation of their structure with functional alterations in distinct patho/physiological conditions

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