Possible role of procathepsin D in human cancer.

For the past ten years, our research has been focused on elucidating the mechanism by which procathepsin D (pCD) impacts cancer development. Various studies have shown that pCD is overexpressed and secreted by numerous cancer cell lines. After secretion, it exhibits "growth hormone-like" activity on cancerous cells but the exact mechanism of this mitogenic activity is not yet understood. The activation peptide of pCD (APpCD) (which is cleaved off upon activation of the zymogen) is responsible for the mitogenic function of pCD. Various in vitro and in vivo studies support our theory that the APpCD interacts with both parent and neighborhood cancer cells and thus functions as an autocrine mitogen. We propose a model of pCD mitogenic function and also some possible approaches for treatment and prevention of certain types of cancer.

Detection of procathepsin D in rat milk.

The presence of procathepsin D, a zymogen of the soluble lysosomal aspartic proteinase cathepsin D, was detected in rat milk using Western blot analysis and assay of proteolytic activity in acidic buffers. No other forms of cathepsin D were found. Two different polyclonal anti-procathepsin D antibodies were used for immunochemical detection of procathepsin D. Both antibodies we found to recognize rat procathepsin D. Proteolytic activity in acidic buffers was detected using a fluorogenic substrate specific for cathepsin D and was abolished by pepstatin A, a specific inhibitor of aspartic proteinases. This study represents third demonstration of presence of procathepsin D in mammal breast milk. Potential sources and physiological functions are discussed.

Role of procathepsin D activation peptide in prostate cancer growth.

BACKGROUND: Enzymatically inactive procathepsin D secreted from cancer cells has been confirmed to play a role in breast cancer development. We focused on prostate cancer and the role of activation peptide in mitogenic activity. METHODS: Synthetic peptides and monoclonal antibodies raised against individual fragments of activation peptide were employed. Cell proliferation was measured by MTT (3-[4,5-dimethylthiatol-2-yl]-2,5-diphenyl tetrazolium bromide) assay or by in vivo growth in nude mice. RESULTS: We demonstrated that the growth factor activity of activation peptide is localized in amino-acid region 27-44. In addition, both anti-activation peptide and anti-27-44 peptide antibodies administered in vivo inhibited the growth of human prostate tumors in mice. CONCLUSIONS: Based on these data, we hypothesize that the interaction of procathepsin D activation peptide with an unknown receptor is mediated by amino-acid sequence 27-44. This interaction leads in certain types of tumor to a proliferation and higher motility. Blocking of this interaction by antibodies or antagonists might be a valuable tool in prostate cancer inhibition.

Anti-human procathepsin D activation peptide antibodies inhibit breast cancer development.

Enzymatically inactive procathepsin D secreted from cancer cells has been confirmed to play a role in development of human breast cancer. In the present study, we focused on the role of activation peptide which was in our preliminary studies suggested to be most probably responsible for mitogenic activity of procathepsin D. Using synthetic fragments and antibodies raised against individual fragments, we demonstrated that the growth factor activity of activation peptide is localized in a nine amino acid stretch (AA 36-44) of activation peptide and moreover both anti-activation peptide and anti-27-44 peptide antibodies administered in vivo inhibited the growth of human breast tumors in athymic nude mice. Taking into account our previous results and presented data, we hypothesize that the interaction of procathepsin D activation peptide with an unknown surface receptor is mediated by a sequence 36-44 plus close vicinity. We also propose that this interaction leads in certain types of tumor derived cell lines to proliferation and higher motility. Blocking of the interaction of activation peptide by specific antibodies or antagonists might be a valuable tool in breast cancer inhibition.

Effect of procathepsin D and its activation peptide on prostate cancer cells.

Cathepsin D, a lysosomal aspartic proteinase, is secreted in the form of enzymatically inactive precursor in some cancer cells. This precursor, called procathepsin D, was found to exhibit growth factor activity toward breast cancer cell lines and this activity was later shown to be mediated by its activation peptide. In the present investigation we have used human procathepsin D and a synthetic 44 amino acid peptide corresponding to the activation peptide of procathepsin D to test its growth factor activity for human prostate cancer-derived cell lines PC3, DU145 and LNCaP. We have tested the level of proliferation of these cell lines depending on the presence of either procathepsin or activation peptide in the medium. In parallel, we have also measured the time dependency of this growth and established the optimal dose of activation peptide. These findings represent the first experimental data showing the direct effects of procathepsin D on prostate cancer cells.

Analysis of the interaction of procathepsin D activation peptide with breast cancer cells.

Cathepsin D, a lysosomal aspartic proteinase, is secreted in the form of enzymatically inactive proenzyme by many types of human breast cancer tissue and exerts mitogenic activity toward these tissues. Flow cytometry was used to test the binding of procathepsin D purified from the secretion of the breast cancer cell line ZR-75-1 to human breast cancer cells. No previously known surface antigens or soluble M6P-R or anti-M6P-R antibodies were found to inhibit the specific binding of procathepsin D-FITC. Similarly, none of these potential inhibitors was found to inhibit growth factor activity of procathepsin D. Our results indicate that procathepsin D growth factor activity is mediated by a new, previously unknown receptor moiety and that the binding activity can be localized in position 27-44 of the activation peptide of procathepsin D. Furthermore, in vivo experiments indicate that treatment with anti-procathepsin D antibodies can reverse the growth of human breast tumors in athymic nude mice.

Human procathepsin D: three-dimensional model and isolation.

Human procathepsin D was isolated from medium of human breast cancer cell line ZR-75-1 potentiated with estrogen. The isolation involved both immunoaffinity chromatography and ion-exchange chromatography. The affinity chromatography employed polyclonal antibodies raised against a synthetic activation peptide of human cathepsin D. We have started preliminary crystallization trials using the isolated material. A model of human procathepsin D was also built using coordinates of human cathepsin D and pig pepsinogen. The model aids understanding of multiple roles played by activation peptides of aspartic proteinases and will be used as a starting model for molecular replacement.

Mitogenic function of human procathepsin D: the role of the propeptide.

The proform of cathepsin D is secreted by some human breast-cancer cell lines upon stimulation with oestrogen. In these cell lines, procathepsin D was described to act as an autocrine mitogen, and a correlation between the cathepsin D concentration in tumour tissues and poor prognosis for the patient was demonstrated in several independent investigations. In the present study, we focused on the mechanism of procathepsin D mitogenic activity. Procathepsin D isolated from secretions of ZR-75-1 breast-cancer cell line was used to test for mitogenic activity on a set of seven human cell lines. For nanomolar procathepsin D concentrations, we found a stronger dose-responsive cellular reaction in the case of several different human breast-cancer-derived cell lines. The mitogenic activity was not blocked by the inhibition of proteolytic activity nor by the inhibition of the interaction of procathepsin D with mannose-6-phosphate receptors. On the other hand, the addition of antibodies raised against the propeptide impaired the mitogenic activity of procathepsin D, and a synthetic peptide alone corresponding to the propeptide of procathepsin D produced similar effects, as did the zymogen molecule. The synthetic propeptide was shown to block partially the interaction of procathepsin D with the cellular surface. Our results indicate that the mitogenic function involves the propeptide of cathepsin D, which appears to be recognized by a surface receptor.

Extracellular aspartic proteinases from Candida albicans, Candida tropicalis, and Candida parapsilosis yeasts differ substantially in their specificities.

Extracellular aspartic proteinases have been implicated for some time as virulence factors associated with Candida opportunistic fungal infections. Our present knowledge of the enzymatic properties of these proteinases is rather limited. Information on their substrate specificity is important for understanding their roles in invasive Candida infections. We have isolated aspartic proteinases from each of the three Candida yeasts, Candida albicans, Candida tropicalis, and Candida parapsilosis, and investigated the specificities of these proteinases using a library of synthetic substrates and testing inhibition by pepstatin A. The specificities of these aspartic proteinases are different from those of major human proteinases, including gastric pepsins, renal renin, and cathepsin D. For the peptide substrate, Lys-Pro-Ala-Leu-Phe*Phe(p-NO2)-Arg-Leu, the values of kcat/Km were 2.95 x 10(6) M-1 s-1 for cleavage by Candida albicans proteinase, 1.60 x 10(6) M-1 s-1 for cleavage by Candida tropicalis proteinase, and 0.59 x 10(6) M-1 s-1 for Candida parapsilosis proteinase. Substantial differences in specificity among the Candida yeast proteinases were identified. For example, Candida tropicalis shows large changes in the kcat/Km value depending on the acidobasic character of the residue occupying the P2 position (1.6 x 10(6) M-1 s-1 for Leu, 0.47 x 10(6) M-1 s-1 for Lys, and 0.05 x 10(6) M-1 s-1 for Asp at P2, respectively). Candida parapsilosis by comparison is tolerant of these substitutions at P2 and is highly restrictive at position P4.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of peripheral blood neutrophils and lymphocytes by human procathepsin D and insulin-like growth factor II.

Cathepsin D, a lysosomal aspartic proteinase, is well known to be overexpressed and secreted in the form of its zymogen by many types of human breast cancer tissues. In the cell lines derived from these tissues, cathepsin D functions as an autocrine mitogen, and it was suggested that its secretion might pose some physiological functions. Recently we have identified the presence of procathepsin D in human breast milk and similar findings were reported for bovine milk which imply also some physiological function. Thus, we have tested the influence of procathepsin D and insulin-like growth factor II on the expression of CD11a, CD11b, FcRI, CD62L, and HLA-DR surface determinants on neutrophils and lymphocytes. We have used procathepsin D purified from the secretions of breast cancer cell line ZR-75-1 and commercially available IGF II. Our results showed that both studied factors significantly influence the expression of tested surface molecules.

Effect of human procathepsin D on proliferation of human cell lines.

We have used human procathepsin D isolated from supernatant of human breast cancer cell line ZR-75-1 to test its mitogenic activity for a broad spectrum of human-derived cell lines. These cell lines included: breast cancer cell lines ZR-75-1, MDA-MB-436, MBA-MD-483 and MDA-MB-231, B lymphoblastoid cell line Raji, the monocytoid cell line U937, T lymphoblastoid cell line 8402, epitheloid carcinoma cell line HELA, hepatocellular carcinoma cell line Hep G2, breast milk epithelial cell line HBL-100 and angiosarcoma cell line HAEND-1. We have tested the level of proliferation of these cell lines depending on the presence of procathepsin D in the medium. In parallel we have also measured the effect of insulin-like growth factor II under the same experimental conditions. We have found a significant difference between the influence of IGF II and that of procathepsin D. While IGF II promoted in practically the same way the proliferation of all cell lines tested, procathepsin D had a very pronounced effect on breast cancer cell lines only. This finding might help to explain some contradictory results of prognostic significance of procathepsin D in human breast cancer.

Multiple functions of pro-parts of aspartic proteinase zymogens.

The importance of aspartic proteinases in human pathophysiology continues to initiate extensive research. With burgeoning information on their biological functions and structures, the traditional view of the role of activation peptides of aspartic proteinases solely as inhibitors of the active site is changing. These peptide segments, or pro-parts, are deemed important for correct folding, targeting, and control of the activation of aspartic proteinase zymogens. Consequently, the primary structures of pro-parts reflect these functions. We discuss guidelines for formation of hypotheses derived from comparing the physiological function of aspartic proteinases and sequences of their pro-parts.

Human breast milk contains procathepsin D--detection by specific antibodies.

The presence of the zymogen of cathepsin D in human milk was detected using antibodies specific for the proenzyme and by the proteolytic activity at low pH. The antibodies were raised against a synthetic propeptide of human cathepsin D and were tested using immunoprecipitations and western blots of samples from different breast cancer cell lines as well as cytosol fractions of human breast cancer tissues. In all experiments these antibodies recognized specifically procathepsin D. Procathepsin D from human milk was partially activated at low pH. The activity was monitored using hemoglobin 14C proteolytic assay, and it was abolished by pepstatin A--a specific inhibitor of aspartic proteinases. Western blots did not reveal presence of cathepsin B or cathepsin H. These data indicate specific secretion of cathepsin D in human breast milk.

Candida parapsilosis expresses and secretes two aspartic proteinases.

We have isolated and characterized a second aspartic proteinase secreted by the CHUV E-18 strain of Candida parapsilosis. This proteinase is produced at a level corresponding to approximately 25% of the production of the main proteinase described earlier [1]. This minor proteinase has similar molecular weight and pH optimum but differs in the isoelectric point and in the specificity when compared with the major secreted form. The determination of the amino terminal amino acid sequence identified this minor form of Candida parapsilosis aspartic proteinase as a protein which corresponds to the sequence deduced from genomic DNA originally reported as a pseudogene [1]. We conclude that strain CHUV E-18 of Candida parapsilosis expresses and secretes two different aspartic proteinases.

Two crystal structures for cathepsin D: the lysosomal targeting signal and active site.

Two crystal structures are described for the lysosomal aspartic protease cathepsin D (EC 3.4.23.5). The molecular replacement method was used with X-ray diffraction data to 3 A resolution to produce structures for human spleen cathepsin D and for bovine liver cathepsin D complexed with the 6-peptide inhibitor pepstatin A. The lysosomal targeting region of cathepsin D defined by previous expression studies [Barnaski et al. (1990) Cell, 63, 281-219] is located in well defined electron density on the surface of the molecules. This region includes the putative binding site of the cis-Golgi phosphotransferase which is responsible for the initial sorting step for soluble proteins destined for lysosomes by phosphorylating the carbohydrates on these molecules. Carbohydrate density is visible at both expected positions on the cathepsin D molecules and, at the best defined position, four sugar residues extend towards the lysosomal targeting region. The active site of the protease and the active site cleft substrate binding subsites are described using the pepstatin inhibited structure. The model geometry for human cathepsin D has rms deviations from ideal of bonds and angles of 0.013 A and 3.2 degrees respectively. For bovine cathepsin D the corresponding figures are 0.014 A and 3.3 degrees. The crystallographic residuals (R factors) are 16.1% and 15.8% for the human and inhibited bovine cathepsin D models respectively. The free R factors, calculated with 10% of the data reserved for testing the models and not used for refinement, are 25.1% and 24.1% respectively.

Crystallization and initial crystallographic results for pepstatin A inhibited bovine cathepsin D.

Cathepsin D was purified from bovine liver by a method using two pepstatin A affinity columns. The eluted protein was combined with pepstatin A and the complex crystallized from 15% polyethylene glycol 8000 at pH 5.9. The crystals diffract to a resolution of 3.0 A and have space group P2(1)2(1)2(1) with unit cell dimensions a = 74.8 A, b = 76.0 A, c = 157.7 A. There are two molecules in the asymmetric unit. The structure was solved by molecular replacement using a pepsin search model and both molecules showed clearly interpretable density in the position expected for pepstatin A in a preliminary difference map. The refined model has r.m.s. deviations from ideal bond lengths and angles of 0.014 A and 3.2 degrees, respectively, and a crystallographic R factor of 17%.

pH dependence of kinetic parameters of pepsin, rhizopuspepsin, and their active-site hydrogen bond mutants.

The pH dependence of the kinetic parameters of pepsin, rhizopuspepsin, and their active-site hydrogen bond mutants has been determined. These data have permitted the calculation of two active-site ionization constants in the free enzymes (pKe1 and pK32) and in the enzyme-substrate complexes (pKes1 and pKes2). The pKe1 of rhizopuspepsin (2.8) is near that of a normal carboxyl group and near the pKe1 of human immunodeficiency virus type 1 (HIV-1) protease (3.32) (Ido, E., Han, H. P., Kezdy, F. J., and Tang, J. (1991) J. Biol. Chem. 266, 24359-24366). The pKe1 of pepsin (1.57) is thus abnormally low. The pKe2 of rhizopuspepsin (4.44) is lower than that of pepsin (5.02) and HIV protease (6.80). The binding of substrate to rhizopuspepsin causes the lowering of pKes1 to 1.8 and the elevating of pKes2 to above 6. The pK alpha shifts due to substrate binding are much less pronounced in pepsin. Thus, the two enzyme-substrate complexes have similar pK alpha values. For both pepsin and rhizopuspepsin, the removal of hydrogen bonds to the active-site carboxyls by mutagenesis results in negligible changes in the four pK alpha values. The major alteration caused by these mutations is the decrease in kcat values, while there is little change in Km. These observations suggest that these hydrogen bonds to the active-site aspartyls contribute little to the pH-activity relationships of the aspartic proteases. The role of the active-site hydrogen bonds may well be to preserve the conformational rigidity of the catalytic apparatus.