Abstract
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.
Highlights
Prostasomes, extracellular vesicles (EVs) originating from the prostate, exhibit distinct heterogeneity regarding molecular composition, size, and internal morphology
In this study we aimed to explore molecular properties of the prostasomal surface exemplified by its glycan composition leading to progress in separation and purification of various populations
In relation to this, checking surface glycans is the initial step preceding affinity chromatography that is necessary for determining which lectins to use
Summary
Prostasomes, extracellular vesicles (EVs) originating from the prostate, exhibit distinct heterogeneity regarding molecular composition, size, and internal morphology. In this study we aimed to explore molecular properties of the prostasomal surface exemplified by its glycan composition leading to progress in separation and purification of various populations. To achieve this goal, a lectin-affinity chromatography (LAC) format was evaluated as a tool using seminal prostasomes from normozoospermic and oligozoospermic men. 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. 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. 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
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