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Endocrinology Vol. 140, No. 9 4127-4132
Copyright © 1999 by The Endocrine Society

ARTICLES

Proteolysis of Human Prolactin: Resistance to Cathepsin D and Formation of a Nonangiostatic, C-Terminal 16K Fragment by Thrombin1

Sudha Khurana, Karen Liby, Arthur R. Buckley and Nira Ben-Jonathan

Departments of Cell Biology (S.K., K.L., N.B.J.) and Physiology (A.R.B.), University of Cincinnati, Colleges of Medicine and Pharmacy, Cincinnati, Ohio 45267

Address all correspondence and requests for reprints to: Nira Ben-Jonathan, Department of Cell Biology, University of Cincinnati, 231 Bethesda Avenue, Cincinnati, Ohio 45267-0521. E-mail: Nira.Ben-Jonathan{at}uc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HUMAN PRL is a single 23-kDa polypeptide hormone composed of four antiparallel {alpha}-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, 10, 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 angiostatic factors produced by growing tumors (12). In recent years, both rat 16K PRL made by proteolysis (10), and recombinant N-terminal human 16K PRL (11) have been reported to suppress basal and growth factor-stimulated endothelial cell proliferation in vitro and neovascularization in vivo (13). Therefore, the use of 16K PRL as an angiostatic agent could be of valuable therapeutic applications (14).

Witorsch and colleagues first reported that rat tissues, including mammary gland and liver, contain enzymes that can convert rat PRL into a cleaved variant with a nick in its large disulfide loop (15). Upon reduction, the nicked molecule yields 16K and 8K fragments. Subsequent studies by Nicoll and co-workers (16) identified the enzyme that cleaves rat PRL as cathepsin D, a lysosomal aspartyl protease with a pH optimum of 3.2–3.4. Yet, the physiological relevance of PRL cleavage by cathepsin D is unclear because of the highly acidic environment that is required for its activity. Although 16K PRL has been detected by immunoblotting in pituitary extracts and sera from rats, mice, and humans (3, 4, 17, 18), the exact nature of the fragment and the mechanism of its formation remain unknown.

In the present study we sought to identify an enzyme that is capable of cleaving PRL at a physiological pH in the formation of its 16K fragment. Assuming that cleavage of the hormone might occur at the target cell, thrombin, a proteolytic enzyme that is central to endothelial cell biology, was considered. Indeed, thrombin is active at a pH 7.4, is enriched at the endothelial cell surface (19), and has been reported to cleave human GH (20, 21), a structural and functional homologue of human PRL (1).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Highly purified rat (B-7) and human (AFP8982C and SAIFb2) PRLs and human PRL antiserum (IC-5) were a kind gift from the National Hormone and Pituitary Program (NIDDK). Amniotic fluid samples were collected by amniocentesis from pregnant women at 18–25 weeks of gestation at the University of Cincinnati Hospital according to a protocol approved by the institutional review board.

Tissue processing
Microsomal pellets were prepared from lactating rat mammary glands or from human MCF-7 breast cancer cells as described by Clapp (22). Briefly, minced tissues or cell pellets were homogenized in 0.1 M Tris-HCl-0.25 M sucrose buffer, pH 7.5, at 4 C, followed by differential centrifugation to obtain the 25,000 x g subcellular fraction. The protein content of the samples was determined using BCA protein assay reagents (Pierce Chemical Co., Rockford, IL).

Proteolysis of PRL
Hormones were incubated with either of the following: 1) microsomal pellet (16 µg pellet/50 µg PRL) in 0.1 M citrate-phosphate buffer; 2) human cathepsin D (Sigma Chemical Co., St. Louis, MO) in 0.1 M citrate-phosphate buffer (enzyme to protein ratio of 1:160) in the presence or absence of Triton X-100 (0.06%), Triton X-100 plus mercaptoethanol (8%), and mercaptoethanol alone; or 3) human {alpha}-thrombin (2000 NIH U/mg protein; 1 U/5 µg PRL; Sigma Chemical Co.) in 0.1 M Tris-HCl, pH 7.4 in the absence or presence of 2 U of hirudin (Sigma Chemical Co.). In case of amniotic fluid, thrombin was added directly without buffer. Proteolysis was carried out at 37 C at a pH and duration stated for each experiment.

SDS-PAGE and Western blotting
Samples were resolved by electrophoresis on polyacrylamide gels (15%) containing SDS in the absence or presence of 5% ß-mercaptoethanol (ß-ME). This was followed by either silver staining (Sigma Chemical Co. Silver Stain Kit) or Western immunoblotting, using anti-hPRL serum (1:2000) and enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL).

Preparation and purification of PRL fragments
To prevent reformation of disulfide bonds, 16K PRL in the digest was carbamidomethylated as described by Clapp and co-workers (10). Briefly, the digest was reduced with dithiothreitol and then alkylated with excess iodoacetamide in the presence of guanidine HCl. After extensive dialysis, the carbamidomethylated 16K fragment (CAM-16K) was purified by gel permeation HPLC. In case of the thrombin digests, the enzyme was first removed by passage on a para-aminobenzidine (PAB)-Sepharose (Amersham Pharmacia Biotech, Piscataway, NJ) column in 0.1 M Tris buffer, pH 7.4. Hormone fragments in the unbound fraction were purified by reversed-phase HPLC on a C-4 column, using a gradient of acetonitrile (50%–90% over 40 min) containing 0.1% TFA.

Mass spectrometry and N-terminal sequencing
For size determination, liquid chromatography/mass spectrometry was performed on human 16K PRL using a Micromass Platform Single Quadruple Instrument (Micromass, Wythenshawe, UK). Protein sequencing was done on a Perkin-Elmer PE Applied Biosystems 494 (Foster City, CA).

Endothelial cell bioassay
Primary human umbilical vein endothelial cells (HUVEC; Clonetics, San Diego, CA), were used to examine the angiostatic activity of 16K PRL. The cells were maintained in an endothelial cell growth medium (Clonetics), and were seeded in 96-well plates at a density of 5000 cells/well in 200 µl of the same growth medium. After 24 h, the cells were incubated with different hormones in the absence (basal conditions) or presence (stimulated conditions) of basic fibroblast growth factor (bFGF; 1 nM) in 200 µl of medium containing 1% bovine serum. After 3 days, cell number was determined by the MTT optical density method (23).

Nb2 assay
Rat Nb2 lymphoma cells were maintained at 37 C in Fischer’s medium supplemented with 10% FBS and 10% horse serum (BioWhittaker, Inc., Walkersville, MD), ß-ME (100 µM), penicillin (50 U/ml), and streptomycin (50 µg/ml) as described by Gout et al. (24). After arresting cell growth by incubation in lactogen-free medium for 18–24 h, the mitogenic activity of PRL was determined by incorporation of [3H] thymidine as described before (25).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Angiostatic activity of rat 16K PRL
The angiostatic activities of rat 23K and 16K PRLs were compared using the HUVEC proliferation assay. The rat 16K PRL was generated by incubating intact PRL with a rat mammary microsomal pellet, followed by carbamidomethylation. As shown in Fig. 1Go, both basal- and bFGF-stimulated proliferation of HUVEC were inhibited by 16K PRL but not 23K PRL. Over 40% inhibition was observed in response to 60 nM of CAM-16K PRL (P < 0.05).



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  		Figure 1. Inhibition of basal- and bFGF-stimulated proliferation of human umbilical vein endothelial cells (HUVEC) by rat 16K PRL. Note the dose-dependent inhibition of cell proliferation by rat 16K PRL and the lack of effect by rat 23K PRL. Each value is a mean ± SEM of five replications.

 
Cleavage of rat PRL, but not human PRL, by cathepsin D
Since cathepsin D has been implicated in the formation of 16K PRL, rat 23K PRL was subjected to digestion by cathepsin D at pH 3.2 for different times. As evident in Fig. 2Go, cleavage of the hormone by cathepsin D was rapid, reaching near completion within 1 h. The major product present in the reaction mixture was a 16K protein band that was not further fragmented up to 24 h. To determine whether human breast cancer cells, which express large amounts of cathepsin D (26), can cleave PRL, both human and rat PRLs were incubated with acidified microsomal pellets from MCF-7 cells. Unexpectedly, cleavage of only rat, but not human PRL, was observed (Fig. 3Go). Cleavage of rat PRL only was also observed when the two hormones were incubated with microsomal pellets from either normal or cancerous human breast tissues (data not shown).



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  		Figure 2. Time-course analysis of the cleavage of rat PRL by human cathepsin D. Cleavage was performed in citrate-phosphate buffer, pH 3.2, and the products were resolved by SDS-PAGE under reducing (R) or nonreducing (NR) conditions. M-marker proteins. Note the rapid cleavage of 23K PRL to 16K and 8K fragments without further degradation.

 


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  		Figure 3. Cleavage of rat PRL (rPRL), but not human PRL (hPRL), by microsomal pellets from MCF-7 cells. Digestion was carried out overnight in citrate-phosphate buffer at pH 3.2. Reduced (R) and nonreduced (NR) samples of hPRL and rPRL were resolved by SDS-PAGE.

 
Next, human PRL was subjected to digestion with purified cathepsin D. However, the enzyme failed to generate 16K PRL at any pH tested or upon prolonged digestion. The presence of a reducing agent (ß-ME), a mild denaturing agent (Triton X-100) or both in the digestion mixture did not facilitate cleavage of human PRL by cathepsin D but resulted in formation of smaller fragments (Fig. 4Go). Faint 16K bands were occasionally seen in undigested human PRL preparations (see, for example Fig. 3Go). Under similar acidic conditions, cathepsin D failed to cleave human PRL from the amniotic fluid (data not shown).



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  		Figure 4. Failure of cathepsin D to cleave human PRL. Incubation was carried out in citrate-phosphate buffer at the designated pH and times. Lanes T, TM, and M show digestion in the presence of Triton X-100, Triton X-100 plus mercaptoethanol, and mercaptoethanol alone, respectively. Note the enhanced degradation of PRL in the presence of the thiol and Triton X-100.

 
Cleavage of both rat PRL and human PRL by thrombin
As shown in Fig. 5Go, thrombin at pH 7.4 cleaved both rat and human PRL. Formation of a 16K fragment from an endogenous PRL was also observed when human amniotic fluid samples, which contain very large amounts of PRL (27, 28) were incubated with thrombin (Fig. 6Go). The specificity of the enzymatic reaction was verified by inhibition of cleavage in the presence of hirudin, a potent and specific inhibitor of thrombin activity. Notably, the undigested PRL in the amniotic fluid contains a 16K fragment.



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  		Figure 5. Cleavage of both rat PRL and human PRL by thrombin (pH 7.4) but only rat PRL by cathepsin D (pH 3.2). Each sample was incubated with the enzyme for 4 h and then separated by SDS-PAGE (15%) under reducing conditions, followed by silver staining. C, Control; D, digest.

 


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  		Figure 6. Western immunoblot analysis of thrombin-cleaved (T) samples of human PRL (hPRL) and PRL present in human amniotic fluid (AF). The specificity of thrombin cleavage was examined by co-incubation with hirudin (T+H). Note the presence of both 23K and 16K hPRL in the undigested AF. C-undigested controls.

 
Lack of angiostatic activity and loss of bioactivity of the thrombin-cleaved human 16K PRL
Next, the human PRL digest was stripped of thrombin by PAB-Sepharose chromatography that specifically binds to serine proteases. Both 16K and 8K fragments were purified by reversed-phase HPLC. The angiostatic activity of the thrombin-cleaved 16K PRL, after carbamidimethylation, was then analyzed using the HUVEC proliferation assay. Surprisingly, the fragment had no inhibitory effect on the growth of HUVEC, either under basal or FGF-stimulated conditions (data not shown). The bioactivity of the fragments was then evaluated using the rat Nb2 lymphoma bioassay. As shown in Fig. 7Go, as little as 1 ng/ml of 23K human PRL stimulated cell proliferation 6- to 8-fold, whereas the 16K PRL retained less than 10% of the potency of the native hormone (P < 0.05). Neither the 8K PRL nor thrombin alone had any detectable bioactivity in the bioassay (Fig. 7Go).



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  		Figure 7. Comparison of the mitogenic activities of human PRL (hPRL) and its purified thrombin-cleaved 16K and 8K fragments in the rat NB2 lymphoma bioassay. Thrombin alone (0.0005–0.5 U/ml) had no effect on cell proliferation. Each value is a mean ± SEM of triplicate determinations.

 
Structural characterization of the thrombin-cleaved human 16K PRL
The lack of angiostatic activity of the thrombin-cleaved human 16K PRL raised the possibility that it differs from the recombinant N-terminal 16K PRL, which exhibits a strong angiostatic activity (11). Therefore, the HPLC-purified 16K fragment was first subjected to mass spectrometry for size determination, followed by N-terminal analysis. The exact size of the fragment was 16,878.30 ± 15.8 Daltons. N-terminal sequencing revealed the following stretch of amino acid residues: Ala-Ile-Asn-Ser-Cys-His-Thr-Ser-Ser-Leu . . . These data unequivocally showed that the thrombin cleavage took place between amino acid residues 53 (Lys) and 54 (Ala), thereby generating a C- rather than an N-terminal 16K fragment.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have confirmed that rat 16K PRL, made by incubating intact PRL with the acidified rat mammary microsomal fraction, suppresses basal- and bFGF-stimulated proliferation of endothelial cells. Whereas rat PRL was cleaved by cathepsin D at pH 3.2, human PRL was resistant to such cleavage under various conditions. On the other hand, thrombin at pH 7.4 was capable of generating 16K fragments from both hormones. Unexpectedly, the thrombin cleavage resulted in the formation of a C-terminal human 16K PRL that was devoid of the angiostatic property of the N-terminal fragment. A diagram illustrating the formation of the N-terminal and C-terminal 16K PRL by cathepsin D and thrombin, respectively, is shown in Fig. 8Go.



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  		Figure 8. A model depicting the cleavage of 23K PRL by cathepsin D and thrombin in the formation of its N- and C-terminal 16K fragments respectively.

 
16K fragments of PRL have been detected in the pituitary and circulation of both humans and rats (3, 4, 18). Here we provided evidence for the existence of such fragment in human amniotic fluid (Fig. 6Go). The exact nature of this 16K PRL is unknown, however, and is currently under investigation. Based on in vitro studies, formation of the rat 16K PRL has been attributed to cathepsin D. Cathepsin D is an aspartyl endoproteinase that is present in lysosomes and endosomes and degrades proteins at a very acidic pH. The synthesis and secretion of cathepsin D is increased in breast cancer, is stimulated by estrogens, and is correlated with the metastatic potential of tumors (26). Consistent with these reports, we observed cleavage of rat PRL by both purified human cathepsin D at pH 3.2 (Fig. 2Go) and acidified microsomal pellets of MCF-7 breast cancer cells (Fig. 3Go). Neither of these was capable of cleaving human PRL.

Two cleavage sites for cathepsin D have been identified within the middle disulfide loop of rat PRL, one between Tyr145 and Leu146 and the other between Trp148 and Ser149. The nicked molecule in the presence of a reducing agent yields N-terminal 16,364 Daltons and C-terminal 5,808 Dalton fragments (16). Comparison of the primary amino acid sequences revealed presence of only one site, Trp 148-Ser 149, in human PRL, whereas Leu 146 is replaced by Pro. The failure of cathepsin D to cleave human PRL, even under reducing and/or denaturing conditions, suggests that the presence of the two putative cleavage sites is required for cleavage of the hormone by cathepsin D.

Presumably, an angiostatic 16K PRL can be generated by the producing cell, e.g. the pituitary lactotroph, or by a proteolytic enzyme(s) at the target site, e.g. the endothelial cell. Recent evidence suggests that several angiostatic factors such as angiostatin (29) and endostatin (30) are fragments of heparin-binding proteins that are cleaved at the target sites. We recently reported that human PRL, but not PRLs from several other species including the rat, binds to heparin (31). Binding of the hormone to heparan sulfate glycosaminoglycans on endothelial cells may enable its immobilization at the cell surface, thereby facilitating proteolytic cleavage. This raises the question of the nature of the cleaving enzyme. The inability of cathepsin D to cleave human PRL, combined with the fact that the pericellular environment is unlikely to be acidic enough for cathepsin D action, prompted us to look for another enzyme. Because thrombin is present at high concentrations at the endothelial surface (19), is active at a physiological pH, and has been reported to cleave several hormones and growth factors (20, 21, 32, 33, 34), we reasoned that thrombin might be a promising candidate for cleaving PRL.

Thrombin is a serine protease that plays a central role in the coagulation cascade and endothelial cell biology. Thrombin is activated by estrogen (35) and its levels in the amniotic fluid increase toward the end of gestation in humans (36). Thrombin-like activity has also been detected in the rat pituitary gland (37, 38) and in several other endocrine tissues (39). Both GH (20, 21) and IGF-binding proteins (32, 33, 34) are cleaved by thrombin. In fact, increased breakdown of IGFBP-5 by thrombin during term pregnancy, diabetes, and cancer, which is accompanied by a loss of its capacity to bind to IGF-1, is believed to play a role in controlling its bioavailability.

Our studies reveal that both rat and human PRLs are cleaved by thrombin (Figs. 5Go and 6Go). As judged by electrophoretic mobility on the gels, the major digestion product migrated as a 16K protein band. The size of the fragment, as determined by mass spectrometry, was 16,878.30 ± 15. Subsequent N-terminal sequencing established both the C-terminal nature of the fragment and a thrombin cleavage site between Lys 53 and Ala 54. This site is present in both rat and human PRLs at a homologous position. This 16K product lacked the angiostatic activity of an N-terminal 16K fragment and was accompanied by a substantial loss of mitogenicity in the Nb2 bioassay (Fig. 7Go).

The ability of thrombin to generate a C-terminal 16K fragment of PRL raises the question as to whether 16K fragment(s) present in pituitary, mammary gland, serum, amniotic fluid, and purified hormone preparations (2, 40) represent N- and/or a C-terminal fragments. Unlike the case of rat PRL, the existence of a "nicked" form of human PRL, capable of yielding the 16K and 8K fragments upon reduction, has not been reported. Sinha et al. have reported the existence of an "unknown 16K PRL" in human pituitary extracts, which was not detected by an antiserum raised against the N-terminal region of human PRL (41) suggesting that it may not be the N-terminal fragment of the hormone. In summary, our findings suggest a role for thrombin in the formation of a nonangiostatic 16K form of PRL. Therefore, the issue of the existence of an endogenous N-terminal 16K PRL in human tissues and body fluids should be carefully revisited. Whether thrombin cleavage of PRL confers a unique biological activity on this functionally diverse hormone or serves to limit its bioactivity, remains to be determined.


   Acknowledgments
 
We thank the National Hormone and Pituitary Program, NIDDK for providing reagents for this study. The technical assistance of Donna Buckley in carrying out the Nb2 assay is greatly appreciated.


   Footnotes
 
1 This work was supported by NIH Grant NS-13243 (to N.B.J.) and DK-53452 (to A.R.B.). Part of this work was presented at the 1998 Annual Meeting of The Endocrine Society, New Orleans, Louisiana, Abstract P3–560. Back

Received February 5, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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