BACKGROUND: The aim of this study was to identify the proteolytic activity which triggers the transformation of human alpha2-macroglobulin (alpha2-M) in seminal fluid and its binding to its receptor. METHODS: Measurement of the concentrations of total and transformed alpha2-M in seminal fluid was accomplished by ELISA. Zymography of seminal plasma was performed in SDS-polyacrylamide gels containing casein as proteolytic substrate. Rate electrophoresis, SDS-PAGE, and Western blotting were applied to study the complex formation of prostate-specific antigen (PSA) with alpha-M. Ligand-binding analysis of sperm cells was performed using [125I] labeled proteins. Detection of receptor on sperm cells was achieved by immunofluorescence. RESULTS: The mean concentration of total alpha2-M in a random collection of seminal plasma was 4.6 microg/ml. On average, between 33-98% of the inhibitor was found to be transformed. Zymography of seminal plasma revealed a proteolytic activity which is associated with a 33-kDa protein identified as PSA. Its proteolytic activity could be inhibited by 0.2-M. Both purified PSA and seminal plasma were capable of transforming native alpha2-M. Binding of PSA to alpha2-M triggers the exposition of receptor binding sites in the inhibitor molecule, which causes binding of the complex to alpha2-M-R/LRP identified on spermatozoa. CONCLUSIONS: PSA, the main proteinase in seminal fluid, is responsible for the transformation of alpha2-M and for its binding to alpha2-M-R/LRP present on spermatozoa. The binding of alpha2-M-PSA complexes to the spermatozoa receptor may exert an impact on normal sperm-cell functions.
BACKGROUND: The aim of this study was to identify the proteolytic activity which triggers the transformation of humanalpha2-macroglobulin (alpha2-M) in seminal fluid and its binding to its receptor. METHODS: Measurement of the concentrations of total and transformed alpha2-M in seminal fluid was accomplished by ELISA. Zymography of seminal plasma was performed in SDS-polyacrylamide gels containing casein as proteolytic substrate. Rate electrophoresis, SDS-PAGE, and Western blotting were applied to study the complex formation of prostate-specific antigen (PSA) with alpha-M. Ligand-binding analysis of sperm cells was performed using [125I] labeled proteins. Detection of receptor on sperm cells was achieved by immunofluorescence. RESULTS: The mean concentration of total alpha2-M in a random collection of seminal plasma was 4.6 microg/ml. On average, between 33-98% of the inhibitor was found to be transformed. Zymography of seminal plasma revealed a proteolytic activity which is associated with a 33-kDa protein identified as PSA. Its proteolytic activity could be inhibited by 0.2-M. Both purified PSA and seminal plasma were capable of transforming native alpha2-M. Binding of PSA to alpha2-M triggers the exposition of receptor binding sites in the inhibitor molecule, which causes binding of the complex to alpha2-M-R/LRP identified on spermatozoa. CONCLUSIONS:PSA, the main proteinase in seminal fluid, is responsible for the transformation of alpha2-M and for its binding to alpha2-M-R/LRP present on spermatozoa. The binding of alpha2-M-PSA complexes to the spermatozoa receptor may exert an impact on normal sperm-cell functions.
Authors: Maya B Kostova; William Nathaniel Brennen; David Lopez; Lizamma Anthony; Hao Wang; Elizabeth Platz; Samuel R Denmeade Journal: Prostate Date: 2018-04-16 Impact factor: 4.104