The substrate range of tripeptidyl-peptidase I.

Tripeptidyl-peptidase I (TPP-I) is an exopeptidase which removes tripeptides from the N-terminus of peptides. Mutations in TPP-I are responsible for late infantile neuronal ceroid lipofuscinosis (CLN2). The nature of the physiological substrates and the range and specificity of the enzyme are unclear. Previous experiments suggest that the enzyme can degrade small peptides but not proteins. Digestion of a range of peptides of different size by TTP-I suggests that the enzyme will degrade small peptides with an extended N-terminal domain but not structured peptides. In general, this cut-off occurs between masses of 4.5 kDa and 6 kDa. Reference to the structures of other peptidases suggests a mechanism for this size selectivity.

The specificity of lysosomal tripeptidyl peptidase-I determined by its action on angiotensin-II analogues.

Tripeptidyl peptidase-I (TPP-I) is a lysosomal peptidase which cleaves tripeptides from the N-terminus of peptides. The function of the enzyme is unclear but its importance is demonstrated by the fact that mutations in TPP-I are responsible for late infantile neuronal ceroid lipofuscinosis, a lethal lysosomal storage disease. As a step towards identifying its natural substrates, we have used a series of synthetic peptides, based on angiotensin-II, to explore the effects of peptide chain length and the effects of amino acid substitutions at the P1 and P1' positions on the rate of catalysis. With the exception of angiotensin-(1-8) (angiotensin-II), which is a relatively poor substrate for TPP-I, the rate of catalysis increases with increasing chain length. K(cat)/K(m) values increase 50-fold between angiotensin-(1-5) and angiotensin-(1-14). TPP-I shows little specificity for the nature of the amino acids in the P1 and P1' positions, K(cat)/K(m) values varying only 5-fold for a range of substitutions. However, Pro or Lys in the P1 position and Pro in the P1' positions are incompatible with TPP-I activity. These observations suggest that TPP-I is a non-specific, but essential, peptidase involved in the latter stages of lysosomal protein degradation.

Tripeptidyl-peptidase I deficiency in classical late-infantile neuronal ceroid lipofuscinosis brain tissue. Evidence for defective peptidase rather than proteinase activity.

Brain tissue from patients with classical late-infantile neuronal ceroid lipofuscinosis (LINCL, an infantile form of Batten disease) is deficient in the lysosomal enzyme tripeptidyl-peptidase I (EC 3.4.14.9). The activities of other lysosomal enzymes are either increased or decreased. Tripeptidyl-peptidase I is a pepstatin-insensitive exo-tripeptidase, with little or no endo-proteolytic activity, that is active on small peptides but not on large proteins. Using haemoglobin and casein as substrates for proteolytic activity, we were unable to demonstrate any significant defect in pepstatin-sensitive or pepstatin-insensitive proteinase activity in brain tissue or cultured skin fibroblasts of LINCL patients. These observations suggest that the lysosomal storage of undegraded, small peptides in LINCL results from the absence of peptidase rather than proteinase activity.

Classical late infantile neuronal ceroid lipofuscinosis fibroblasts are deficient in lysosomal tripeptidyl peptidase I.

Tripeptidyl peptidase I (TPP-I) is a lysosomal enzyme that cleaves tripeptides from the N-terminus of polypeptides. A comparison of TPP-I amino acid sequences with sequences derived from an EST database suggested that TPP-I is identical to a pepstatin-insensitive carboxyl proteinase of unknown specificity which is mutated in classical late infantile neuronal ceroid lipofuscinosis (LINCL), a lysosomal storage disease. Both TPP-I and the carboxyl proteinase have an M(r) of about 46 kDa and are, or are predicted to be, resistant to inhibitors of the four major classes of proteinases. Fibroblasts from LINCL patients have less than 5% of the normal TPP-I activity. The activities of other lysosomal enzymes, including proteinases, are in the normal range. LINCL fibroblasts are also defective at degrading short polypeptides and this defect can be induced in normal fibroblasts by treatment with a specific inhibitor or TPP-I. These results suggest that the cell damage, especially neuronal, observed in LINCL results from the defective degradation and consequent lysosomal storage of small peptides.

Purification and characterisation of a tripeptidyl aminopeptidase I from rat spleen.

A tripeptidyl aminopeptidase I with an M(r) of 47,000 Da has been purified from rat spleen. The N-terminal sequence of the enzyme and internal sequences did not resemble that of any known protein. The enzyme cleaves tripeptides from synthetic substrates provided that the N-terminus is unsubstituted and the amino acid in the P1 position is not charged. The enzyme also cleaves small peptides (angiotensin II and glucagon) releasing tripeptides but does not appear to demonstrate any preference for amino acids on either side of the cleavage site. The enzyme had maximum activity at pH 4 but was unstable above pH 7. Rat spleen tripeptidyl peptidase I was not inhibited by classical inhibitors of serine, cysteine, aspartate or metalloproteinases. The peptidase was potently inhibited by a series of substrate-based tripeptidyl chloromethyl ketones (Ki's of 10(-6)-10(-8) M). Inhibition was rapid and reversible. This mode of inhibition is different to the interaction between chloromethyl ketones and cysteine or serine peptidases. These tripeptidyl chloromethyl ketones were also inhibitors of bone resorption using an in vitro assay suggesting that a tripeptidyl peptidase is involved in the degradation of bone matrix proteins.

Differential expression of type XIV collagen/undulin by human mammary gland intralobular and interlobular fibroblasts.

Immunolocalisation of type XIV collagen/undulin in the human mammary gland revealed greater deposition in the interlobular stroma than in the intralobular stroma. The interlobular stroma is located between the breast lobules and their associated intralobular stroma. Fibroblasts isolated from the interlobular stroma synthesised 3- to 5-fold more type XIV collagen/undulin than intralobular fibroblasts, but synthesised type I and type IV collagens in similar amounts. The differential expression of type XIV collagen/undulin was maintained with passage in culture. The results suggest a role for type XIV collagen/undulin in stabilising dense collagen fibrils. The maintenance of two types of structurally distinct stromas may be important during developmental processes in the mammary gland.

Regulation of urokinase-type plasminogen activator production by rat mammary myoepithelial cells.

Addition of basic fibroblast growth factor (bFGF) to a rat mammary gland myoepithelial cell line (25.5-G4.2.3) resulted in a six- to eightfold increase in cellular and secreted urokinase-type plasminogen activator (uPA) activity after a lag phase of 5-8 h. bFGF had no effect on the uPA activity of mammary epithelial cells. bFGF was active on myoepithelial cells over a narrow concentration range (0.5-2 ng/ml). The bFGF-induced increase in uPA activity was inhibited in a dose-dependent manner by hydrocortisone and transforming growth factor-beta1 (TGF-beta1). Hydrocortisone also inhibited the basal secretion of uPA, as did interleukin-1beta and phorbol myristate acetate, both of which increase uPA levels in other cell systems. The effects of bFGF could also be inhibited by factors which bind bFGF, e.g., heparin and methylamine a2-macroglobulin. TGF-beta1, but not bFGF, induced the synthesis of plasminogen activator inhibitor-1 in the myoepithelial cell line. Mammary gland myoepithelial cells contribute to the synthesis of and are located next to the basement membrane. Myoepithelial-derived uPA is probably associated with basement membrane turnover. The mammary gland basement membrane undergoes many cycles of remodeling and multiple mechanisms may be required to regulate uPA activity.

Intracellular degradation of type I collagen and fibronectin in human lung fibroblasts: evidence against degradation in pre-lysosomal compartments.

Fibroblasts degrade about 15% of newly synthesised collagen within the cell before it can be secreted. When the helical structure of collagen is disrupted, about 30% is degraded intracellularly. To determine if collagen degradation occurs in a pre-lysosomal compartment, the passage of type 1 collagen out of the endoplasmic reticulum or Golgi was inhibited by incubating human lung fibroblasts with brefeldin A or monensin. In both cases, the type I collagen retained within the cell was stable over a 20 h period. Disrupting the helical structure of collagen with cis-hydroxyproline, 2,2'-bipyridyl or ethyl 3,4-dihydroxybenzoate did not alter the stability of type I collagen in brefeldin or monensin-treated cells. Incubating permeabilised cells in the presence of GTP gamma S (guanosine 5'-(3-O-thio)triphosphate), which blocks transport out of the endoplasmic reticulum, also resulted in the stable retention of type I collagen. Addition of dithiothreitol to permeabilised cells failed to initiate intracellular degradation. Similar results were obtained with fibronectin. Both normal fibronectin and fibronectin in which canavanine replaced arginine were stable for 20 h in cells treated with brefeldin A or monensin. The degradation of native collagen is sensitive to inhibition by a cell-permeable cysteine proteinase inhibitor (ALLN) but is insensitive to chloroquine (which raises the pH of acidic intracellular compartments), whereas the degradation of abnormal collagen was sensitive to both ALLN and chloroquine. These results argue against the intracellular degradation of collagen or fibronectin in a pre-lysosomal compartment.

Ectoenzyme regulation by phenotypically distinct fibroblast sub-populations isolated from the human mammary gland.

Inter- and intralobular mammary fibroblasts have been separated from normal human breast tissue and cultured to study the differential expression of ectoenzymes present within the stroma of the normal gland and associated with breast cancers. Specific ectoenzymes were identified by indirect immunofluorescence and quantified by flow cytometry and semi-quantitative PCR. A consistent difference was noted between the two fibroblast sub-populations at early passage in respect of dipeptidyl peptidase IV (DPP IV) and aminopeptidase N (APN) expression. Early passage intralobular fibroblasts were positive for APN but negative for DPP IV, as seen in the intact tissue. However, with continued sub-culture they gradually began to express DPP IV, until at later passages they became indistinguishable from the interlobular fibroblasts, which were APN and DPP IV-positive at all stages in culture, as they are in intact tissue. Neutral endopeptidase (NEP/CALLA/CD10) is not expressed by normal adult breast fibroblasts but is found in the stroma associated with over 60% of breast cancers. It was up-regulated in vitro on both inter- and intralobular fibroblasts, with final levels that were significantly (< 14 times) higher on the former in all pairs of preparations from individual donors analysed. This difference persisted with continued passage, and levels of the ectoenzyme and its messenger RNA were further up-regulated by hydrocortisone in both populations. These results demonstrate that phenotypically distinct cultures of human mammary fibroblast sub-populations can be used to study the regulation of these stromal ectoenzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of gelatinases by rat mammary epithelial and myoepithelial cell lines.

The synthesis of metalloproteinases capable of degrading the basement membrane is an important factor contributing to the involution of the mammary gland after the young are weaned. To investigate the cellular source of mammary gland metalloproteinases, we have studied the synthesis of gelatinases by rat mammary epithelial and myoepithelial cell lines immortalized with a temperature-sensitive T-antigen. At 39.5 degrees C, the temperature at which the cells display increased differentiated characteristics, myoepithelial cells synthesize 40- to 160-fold greater amounts of three gelatinases (M(r)s-72, 92, and 135 kDa) than epithelial cells. The gelatinases are preferentially secreted through the basolateral surface of myoepithelial cells. Growth of cells in the presence of a variety of growth factors and cytokines demonstrates the differential regulation of the synthesis of the 72- and 92-kDa gelatinases. These results suggest that the myoepithelial cell is a major source of mammary gland metalloproteinases.

Purification and characterization of a novel tyrosyl-aminopeptidase from human osteoclastomas.

1. An aminopeptidase that preferentially releases tyrosine residues from synthetic substrates has been purified from human osteoclastomas. This enzyme also hydrolyses dipeptides having an N-terminal tyrosine and a hydrophobic carboxy-terminal amino acid. 2. The tyrosyl-aminopeptidase consists of two identical subunits with M(r)s of about 100,000. 3. The enzyme is a metallopeptidase and is inhibited by chelating agents, chloromethylketone analogues of hydrophobic amino acids, and bestatin.

Purification and characterization of a tripeptidyl peptidase I from human osteoclastomas: evidence for its role in bone resorption.

Tripeptidyl peptidase I (EC 3.4.14.9), which cleaves tripeptides from the N-terminus of synthetic substrates, has been purified from human osteoclastomas (a bone tumor containing large numbers of normal osteoclasts). The enzyme has an M(r) of 48 kDa but forms aggregates with an M(r) of about 700 kDa. The tripeptidyl peptidase has an acidic pH optimum (approximately pH 5.0), suggesting that it has a lysosomal localization and prefers substrates with a hydrophobic amino acid in the P1 position. There is an absolute requirement for a nonsubstituted N-terminus. The enzyme is inhibited by reagents which modify serine and histidine residues. Lysosomal tripeptidyl peptidase is known to be capable of cleaving Gly-Pro-X triplets from synthetic collagen-like polypeptides. Ala-Ala-Phe-CH2Cl, a potent inhibitor of osteoclastoma tripeptidyl peptidase, inhibits osteoclastic bone resorption in an in vitro test system. This suggests that tripeptidyl peptidase I, secreted by osteoclasts, is involved at some stage in the degradation of bone collagen.

The synthesis of alpha 2-macroglobulin by rat mammary myoepithelial cells is regulated by synergism between glucocorticoids and cytokines.

Addition of hydrocortisone to the rat mammary gland myoepithelial cell line, G4.2.3, induces the synthesis and secretion of alpha 2-macroglobulin. Interleukin-1 beta, interleukin-6 and transforming growth factor-beta synergize with hydrocortisone, increasing the synthesis of alpha 2-macroglobulin 2- to 4-fold, although they have no effect in the absence of hydrocortisone. Interleukin-6 is the most potent inducer having an optimum concentration of 1 ng/ml. Interleukin-6, unlike interleukin-1 beta or transforming growth factor-beta, decreases the lag phase from 10 h to 4 h before alpha 2-macroglobulin synthesis is induced by hydrocortisone. These results suggest that the mechanism of activation of transcription of the alpha 2-macroglobulin gene in mammary myoepithelial cells might differ from that operating in hepatic cells.

Hydrocortisone induces the synthesis of alpha 2-macroglobulin by rat mammary myoepithelial cells.

The effects of lactogenic hormones on protein secretion by 25.5-G4.2.3 cells, a rat mammary myoepithelial cell line immortalised with a temperature-sensitive T-antigen, were investigated. Insulin, prolactin, estradiol and progesterone had no effect but hydrocortisone induced the secretion of two proteins with molecular masses of 175 kDa (p175) and 146 kDa (p146), 10-30-fold and 5-fold respectively. The induction of p175 and p146 synthesis by hydrocortisone was greater at 39.5 degrees C than at 33 degrees C reflecting the increased differentiation of 25.5-G4.2.3 cells at the higher temperature. Rat mammary epithelial cells did not synthesise p175. After addition of hydrocortisone to 25.5-G4.2.3 cells, there was a lag phase of 10 h before the synthesis of p175 was induced. Half-maximal induction of p175 synthesis required a hydrocortisone concentration of 0.5 microM. p175 was identified as alpha 2-macroglobulin by N-terminal amino-acid sequence determination and immunoprecipitation with a specific antibody. Hydrocortisone induced a 5-kb alpha 2-macroglobulin-specific mRNA transcript in 25.5-G4.2.3 cells. Myoepithelial cells are responsible for synthesising the basement membrane around the rapidly expanding mammary alveoli during pregnancy. Myoepithelial cells also secrete metalloproteinases which are probably involved in turnover of the basement membrane. We suggest that increased levels of hydrocortisone during pregnancy induce the synthesis of alpha 2-macroglobulin, which is believed to be a potent inhibitor of metalloproteinases, by rat mammary myoepithelial cells to reduce proteolytic degradation of the basement membrane.

Degradation of bone matrix proteins by osteoclast cathepsins.

1. The degradation of the bone matrix proteins osteocalcin, osteonectin and alpha 2HS-glycoprotein by human cathepsins B and L and human osteoclastoma cathepsins has been investigated. 2. Intermediate degradation products (M(r) > 12 kDa) were not observed during the digestion of alpha 2HS-glycoprotein and osteonectin by cathepsins B and L although they were observed with some of the osteoclastoma cathepsins. Most of the osteoclastoma cathepsins were capable of degrading these two proteins to small peptides at comparable rates. 3. Each cathepsin produced a different pattern of osteocalcin degradation products. 4. The extensive range of non-collagenous proteins in bone matrix may necessitate the production by osteoclasts of cathepsins with different specificities during bone resorption.

Characterisation of chondroitin/dermatan sulphate proteoglycans synthesised by rat mammary myoepithelial and fibroblastic cell lines.

Chondroitin sulphate proteoglycans were isolated from the culture medium of rat mammary gland fibroblast (Rama 27) and myoepithelial (Rama 401) cell lines which had been labelled with [35S]sulphate. Chromatography on Sepharose CL-4B indicated that the Rama 401 proteoglycan was larger than the Rama 27 proteoglycan (Kav values 0.47 and 0.56, respectively). Treatment of the proteoglycans with alkaline NaBH4 yielded chondroitin sulphate chains with average M(r) values of 37,000 (Rama 401) and 21,000 (Rama 27). Structural analysis of the glycosaminoglycan chains indicated that both were co-polymers of chondroitin and dermatan sulphate although there were differences in the amounts and distribution of the disaccharide repeating units. The M(r) values of the core proteins, determined by immunoblotting, were about 43,000 and 46,000 (Rama 27) and 44,500 (Rama 401). Using an antibody to chondroitin sulphate proteoglycan in immunofluorescence experiments, the proteoglycan was demonstrated on the surface of both cell lines. Rama 27 cells additionally possessed an extensive fibrous extracellular matrix which also stained with the antibody. Staining of sections of lactating mammary gland suggested that the proteoglycan was present in the basement membrane as well as the stromal connective tissue. The presence of chondroitin sulphate proteoglycan in the basement membrane was confirmed by ultrastructural immunolocalisation.

Immunocytochemical localization of the ectoenzyme aminopeptidase N in the human breast.

The ectoenzyme aminopeptidase N (APN) was localized in the normal human breast at both the light microscopic and the ultrastructural level. APN was expressed on intralobular and interlobular fibroblasts and on the apical surface of some luminal epithelial cells. This enzyme was not detected on either myoepithelial cells and their associated basement membrane or capillary endothelium. Furthermore, the staining pattern was maintained in benign and malignant breast disease. APN belongs to a family of enzymes that hydrolyze peptides in the extracellular space. As with other ectoenzymes present in the breast, APN expression is restricted to specific cell types. This pattern of expression may indicate a role for these enzymes in the biology of the normal breast.

Enhanced synthesis of gelatinase and stromelysin by myoepithelial cells during involution of the rat mammary gland.

During the involution of the mammary gland there is destruction of the basement membrane as the secretory alveolar structures degenerate. Immunofluorescence staining of sections of rat mammary gland with antibodies to 72 KD gelatinase (MMP-2) and stromelysin (MMP-3) revealed increased production of these two proteinases during involution. This increased expression was mostly restricted to myoepithelial cells. Increased expression during involution was also demonstrated by immunoblotting techniques. Gelatin zymography indicated that the predominant metalloproteinase present in involuting rat mammary glands was a 66 KD gelatinase.

Role of fibronectin in the organisation of the cytoskeleton during the spreading of rat mammary epithelial cells.

The correlation between the extracellular deposition of fibronectin and the development of the actin-containing cytoskeleton was studied during the attachment and spreading of the rat mammary epithelial cell line Rama 25. During the initial phase of cell spreading, actin is localised in peripheral microfilament bundles. As cell spreading increases, the peripheral ring is displaced towards the perinuclear region. Fibronectin, deposited beneath the basal surface, co-localises with the actin-containing peripheral ring. The peripheral ring subsequently disappears and is replaced by a system of radial microfilaments that extend from the perinuclear region to the cell periphery. At this stage, there is no correlation between the distribution of fibronectin and actin. As cells form colonies, radial microfilament bundles are replaced by peripheral microfilament bundles which do not co-localise with fibronectin. Cells at the edges of colonies extend lamellae that contain microfilament stress fibres. In these structures there is co-localisation of actin, fibronectin and the a5 beta 1-integrin fibronectin receptor.