Proteolysis of human prolactin: Resistance to cathepsin D and formation ofa nonangiostatic, C-terminal 16K fragment by thrombin

The N-terminal 16K fragments of rat and human PRLs possess angiostatic acti vity. 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 i n the generation of rat 16K PRL. However, the proteolytic cleavage of human PRL has not been demonstrated. Our objective was to identify an enzyme tha t is capable of forming an angiostatic human 16K PRL. To confirm the angios tatic action of rat 16K PRL, the fragment was generated by incubating 23K P RL with rat mammary microsomal fraction at pH 3.2. Upon incubation with hum an umbilical vein endothelial cells (HUVEC), rat 16K PRL, but not 23K PRL, inhibited basal- and basic fibroblast growth factor-stimulated cell prolife ration. Intact rat and human PRLs were then incubated with cathepsin D or a cidified microsomal pellets of MCF-7 human breast cancer cells. Analysis by SDS-PAGE showed cleavage of rat, but not human, PRL. Next, hormones were i ncubated with thrombin at pH 7.4. As shown by SDS-PAGE, digestion of both h uman and rat PRL by thrombin resulted in the formation of 16K fragments. PR L contained within human amniotic fluid was also cleaved by thrombin. Enzym e specificity was supported by prevention of cleavage by the thrombin inhib itor hirudin. When tested with HUVEC, the human 16K PRL was devoid of angio static 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-te rminal sequencing showed that the thrombin cleavage occurred between amino acid residues 53 (Lys) and 54 (Ala), resulting in the formation of a C-term inal, not an N-terminal, 16K fragment. We conclude that, unlike rat PRL, hu man PRL is resistant to cleavage by cathepsin D. Thrombin at a physiologica l pH can generate a C-terminal 16K fragment of human PRL that is not angios tatic and retains little mitogenic activity. We suggest that the precise na ture of endogenous 16K PRL fragments that are present in human tissues and body fluids should be carefully examined.