Cathepsin D processes human prolactin into multiple 16K-like N-terminal fragments: study of their antiangiogenic properties and physiological relevance.
16K prolactin (PRL) is the name given to the 16-kDa N-terminal fragment obtained by proteolysis of rat PRL by tissue extracts or cell lysates, in which cathepsin D was identified as the candidate protease. Based on its antiangiogenic activity, 16K PRL is potentially a physiological inhibitor of tumor growth. Full-length human PRL (hPRL) was reported to be resistant to cathepsin D, suggesting that antiangiogenic 16K PRL may be physiologically irrelevant in humans. In this study, we show that hPRL can be cleaved by cathepsin D or mammary cell extracts under the same conditions as described earlier for rat PRL, although with lower efficiency. In contrast to the rat hormone, hPRL proteolysis generates three 16K-like fragments, which were identified by N-terminal sequencing and mass spectrometry as corresponding to amino acids 1-132 (15 kDa), 1-147 (16.5 kDa), and 1-150 (17 kDa). Biochemical and mutagenetic studies showed that the species-specific digestion pattern is due to subtle differences in primary and tertiary structures of rat and human hormones. The antiangiogenic activity of N-terminal hPRL fragments was assessed by the inhibition of growth factor-induced thymidine uptake and MAPK activation in bovine umbilical endothelial cells. Finally, an N-terminal hPRL fragment comigrating with the proteolytic 17-kDa fragment was identified in human pituitary adenomas, suggesting that the physiological relevance of antiangiogenic N-terminal hPRL fragments needs to be reevaluated in humans.
Piwnica
David
D
Institut National de la Santé et de la Recherche Médicale (INSERM) Unit 584, Hormone Targets, Faculté de Médecine Necker, 75730, Paris Cedex 15, France.
Touraine
Philippe
P
Struman
Ingrid
I
Tabruyn
Sébastien
S
Bolbach
Gérard
G
Clapp
Carmen
C
Martial
Joseph A
JA
Kelly
Paul A
PA
Goffin
Vincent
V
D000595:Amino Acid Sequence; D020533:Angiogenesis Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D000818:Animals; D001940:Breast / Q000166:cytology / Q000502:physiology; D002401:Cathepsin B / Q000378:metabolism; D002402:Cathepsin D / Q000378:metabolism*; D002417:Cattle; D004730:Endothelium, Vascular / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology*; D011388:Prolactin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014471:Umbilical Veins
D020533:Angiogenesis Inhibitors; D010446:Peptide Fragments; D011388:Prolactin; D002401:Cathepsin B; D002402:Cathepsin D