Proteolysis of human prolactin: resistance to cathepsin D and formation of a nonangiostatic, C-terminal 16K fragment by thrombin.
The N-terminal 16K fragments of rat and human PRLs possess angiostatic activity. 16K PRL has also been detected in vivo in both humans and rats. Based on an in vitro study, cathepsin D, an acid protease, has been implicated in the generation of rat 16K PRL. However, the proteolytic cleavage of human PRL has not been demonstrated. Our objective was to identify an enzyme that is capable of forming an angiostatic human 16K PRL. To confirm the angiostatic action of rat 16K PRL, the fragment was generated by incubating 23K PRL with rat mammary microsomal fraction at pH 3.2. Upon incubation with human umbilical vein endothelial cells (HUVEC), rat 16K PRL, but not 23K PRL, inhibited basal- and basic fibroblast growth factor-stimulated cell proliferation. Intact rat and human PRLs were then incubated with cathepsin D or acidified microsomal pellets of MCF-7 human breast cancer cells. Analysis by SDS-PAGE showed cleavage of rat, but not human, PRL. Next, hormones were incubated with thrombin at pH 7.4. As shown by SDS-PAGE, digestion of both human and rat PRL by thrombin resulted in the formation of 16K fragments. PRL contained within human amniotic fluid was also cleaved by thrombin. Enzyme specificity was supported by prevention of cleavage by the thrombin inhibitor hirudin. When tested with HUVEC, the human 16K PRL was devoid of angiostatic activity. The activity of this fragment in the Nb2 lymphoma bioassay was 10- to 15-fold lower than that of 23K PRL. Mass spectrometry revealed that the fragment has a mass of 16,878.30+/-15.8 Daltons. Subsequent N-terminal sequencing showed that the thrombin cleavage occurred between amino acid residues 53 (Lys) and 54 (Ala), resulting in the formation of a C-terminal, not an N-terminal, 16K fragment. We conclude that, unlike rat PRL, human PRL is resistant to cleavage by cathepsin D. Thrombin at a physiological pH can generate a C-terminal 16K fragment of human PRL that is not angiostatic and retains little mitogenic activity. We suggest that the precise nature of endogenous 16K PRL fragments that are present in human tissues and body fluids should be carefully examined.
Khurana
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Department of Cell Biology, University of Cincinnati, Colleges of Medicine and Pharmacy, Ohio 45267, USA.
Liby
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K
Buckley
A R
AR
Ben-Jonathan
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N
D000818:Animals; D002402:Cathepsin D / Q000494:pharmacology*; D002455:Cell Division / Q000187:drug effects; D004351:Drug Resistance; D004730:Endothelium, Vascular / Q000166:cytology; D005260:Female; D006801:Humans; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000737:chemistry; D010447:Peptide Hydrolases / Q000096:biosynthesis / Q000378:metabolism*; D011388:Prolactin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D051381:Rats; D013917:Thrombin / Q000494:pharmacology*
D010446:Peptide Fragments; D011388:Prolactin; D010447:Peptide Hydrolases; D013917:Thrombin; D002402:Cathepsin D