Proteolysis of Human Prolactin: Resistance to Cathepsin D and Formation of a Nonangiostatic, C-Terminal 16K Fragment by Thrombin

Abstract

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 Ϯ 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. (Endocrinology 140: [4127][4128][4129][4130][4131][4132] 1999) H UMAN PRL is a single 23-kDa polypeptide hormone composed of four antiparallel ␣-helices with three intrachain disulfide loops between residues 4 -11, 58 -174, and 191-199 (1). Although the position of the disulfide bonds is homologous between PRLs from different species, their primary amino acid sequences may vary considerably. PRL can undergo several posttranslational modifications (2), including cleavage (3, 4), glycosylation (5, 6), and phosphorylation (7, 8) that have been implicated in its functional diversity. However, the N-terminal 16K fragment of PRL is the only well characterized isoform with a unique biological activity as an antiangiogenic agent (9 -11).Angiogenesis, or formation of new blood vessels, is an integral part of tumorigenesis and metastasis and often results from a shift in the balance between angiogenic and angiosta...

Thrombin induces broad spectrum proteolysis in human serum samples

“…First, the parent proteins listed are not functionally exclusive to coagulation or fibrinolysis. This observation is consistent with previous studies, where a thrombin is implicated as a proteolytic agent in other biological processes (30)(31)(32). Second, only a very few of the cleavage sites match the R-G motif commonly expected for a thrombin, and there are a few missed R-G sites included in the observed peptides.…”

“…Enzymes in the clotting cascade, though specific in biological role, have been shown to have promiscuous activity (4,5,(30)(31)(32), outside their expected catalytic function. The data presented here provide direct evidence of this substrate promiscuity, acting broadly upon the protein and peptide content of the plasma/serum itself.…”

Matrix metalloproteases from chondrocytes generate an antiangiogenic 16 kDa prolactin

“…The acidic-aspartyl endoprotease cathepsin-D has been claimed to be the protease responsible for cleaving full-length PRL to 16K-PRL (Baldocchi et al, 1993). However, the relevance of this protease is debatable because human PRL, unlike rat PRL, is resistant (Khurana et al, 1999a) or much less susceptible (Piwnica et al, 2004) to cleavage by cathepsin-D. In search of the biologically relevant PRL-cleaving protease, we reasoned that a likely source for such an enzyme would be an avascular tissue rich in antiangiogenic factors, such as cartilage.…”

Prolactin and Angiogenesis: Biological Implications of Microheterogeneity

“…Although this receptor is still not identified, some of its downstream signaling targets have been elucidated (D'Angelo et al 1999, Corbacho et al 2000).However; many questions related to the biology of 16K PRL remain unanswered. First, although the majority of investigations have used rat 16K PRL, results are much less clear for other species, especially humans, in which PRL was recently reported to be resistant to Cathepsin D (Khurana et al 1999a and1999b). This contrasts with the findings indicating that hPRL yields partial, but reproducible, proteolysis leading to Nterminal 16K like PRL fragments when incubated with this protease.…”

“…This result also demonstrates that Ala45 to Met53 can be the active sites for angiostatic function. The Cterminal 16K peptide (54-199 residues) of hPRL does not appear to have the function of antiangiogenesis (Khurana et al 1999b). In the C-terminal 16K fragment of hPRL, the fourth helix is still close to the partial second loop, which becomes exposed to environment in the N-terminal 16K fragment of hPRL.…”