Structural characterization and anti-angiogenic properties of prostate-specific antigen isoforms in seminal fluid.
BACKGROUND
The prostate produces high levels of prostate-specific antigen (PSA), which has been shown to exert anti-angiogenic properties and thus might slow down prostate tumor growth. It has been suggested that the protease activity of PSA is not needed for its anti-angiogenic function. We have previously shown that seminal fluid contains both active and inactive, internally cleaved forms of PSA. The precise structural differences between these isoforms and their function are not known.


METHODS
To elucidate the structures, we purified PSA from seminal fluid and separated it by anion-exchange chromatography into six different isoforms, which were characterized by mass spectrometry. The anti-angiogenic activity of these PSA-isoforms was analyzed by human umbilical vein endothelial cell (HUVEC) tube formation assay.


RESULTS
The enzymatically active PSA-isoforms had an intact peptide moiety but could be separated into three isoforms based on differences in glycosylation. The major isoform contained PSA with a biantennary carbohydrate with terminal sialic acids on both antennae. The other active isoforms showed significant carbohydrate heterogeneity, containing one or no sialic acid. The inactive isoforms were internally cleaved at several different positions, but the fragments were held together by disulphide bonds. The enzymatic activity of PSA correlated with its inhibitory effect on the endothelial cell tube formation and the inhibition was dose-dependent at physiological concentrations, whereas enzymatically inactive internally cleaved PSA-isoforms had no effect.


CONCLUSIONS
Our results show that the anti-angiogenic effect of PSA is based on its proteolytic activity.
Mattsson
Johanna M
JM
Department of Clinical Chemistry, Biomedicum Helsinki, Helsinki University Central Hospital and University of Helsinki, Helsinki, Finland.
Valmu
Leena
L
Laakkonen
Pirjo
P
Stenman
Ulf-Håkan
UH
Koistinen
Hannu
H
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017430:Prostate-Specific Antigen / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology*; D020033:Protein Isoforms; D012661:Semen / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D014471:Umbilical Veins / Q000187:drug effects
D020533:Angiogenesis Inhibitors; D020033:Protein Isoforms; D017430:Prostate-Specific Antigen