Membrane-associated Cathepsin Research Articles

Mast cells and neutrophils proteolytically activate chemokine precursor CTAP-III and are subject to counterregulation by PF-4 through inhibition of chymase and cathepsin G

AbstractThe CXC chemokines platelet factor 4 (PF-4/CXCL4) and connective tissue-activating peptide III (CTAP-III) are released by activated human platelets in micromolar concentrations. So far, neutrophils have been recognized to cleave the precursor CTAP-III to form the active chemokine neutrophil-activating peptide 2 (NAP-2/CXCL7) through limited proteolysis by membrane-associated cathepsin G. Here we show for the first time that activated human skin mast cells (MCs) convert CTAP-III into biologically active NAP-2 through proteolytic cleavage by released chymase. A direct comparison on a cell number basis revealed that unstimulated MCs exceed the CTAP-III–processing potency of neutrophils about 30-fold, whereas MCs activated by IgE cross-linking exhibit even 1000-fold higher CTAP-III–processing capacity than fMLP-stimulated neutrophils. Intriguingly, PF-4 counteracted MC- as well as neutrophil-mediated NAP-2 generation at physiologically relevant concentrations. Addressing the underlying mechanism, we obtained evidence that PF-4 acts as an inhibitor of the CTAP-III–processing enzymes cathepsin G and chymase without becoming cleaved itself as a competitive substrate. Because cleavage of the CTAP-III–unrelated substrate substance P was also affected by PF-4, our results suggest a regulatory role for PF-4 not only in NAP-2 generation but also in neutrophil- and MC-mediated processing of other physiologically relevant inflammatory mediators.

Therapeutic effects of cysteine protease inhibition in allergic lung inflammation: inhibition of allergen-specific T lymphocyte migration.

We have evaluated the effects of the broad-spectrum cysteine protease inhibitor E64 on allergic lung inflammation in the mouse ovalbumin model of human asthma. We have also characterised membrane-associated cathepsin enzyme activity on a range of cell types. Balb/C mice, E64 and CA074, various cell lines. E64 was administered by subcutaneous minipump into ovalbumin-sensitised mice prior to intranasal ovalbumin challenge. The effect of E64 on ovalbumin-induced inflammation in vivo and ovalbumin-specific T cell proliferation in vitro and ex vivo was examined. Membrane-associated cathepsin activity on various cell types was measured. E64 treatment (0.36-0.48 mg/day) led to a significant reduction in eosinophil numbers and lung weights in the mouse model. Histological examination of lungs confirmed the anti-inflammatory effect. E64 greatly reduced ovalbumin-specific T cell numbers in the lymph nodes draining the lung following intranasal challenge whilst an accumulation of these T cells was found in the 'priming' lymph nodes. An analysis of various cells involved in lymphocyte priming and migration revealed that monocytes, dendritic cells and endothelial cells express high levels of membrane-associated cathepsin B activity. Since E64 is not cell permeable and does not inhibit antigen-induced T cell proliferation in vitro or in vivo, the data indicate that membrane-associated cysteine proteases, possibly cathepsin B, may regulate T lymphocyte migration in vivo.

β-Cyclodextrin/epoxysuccinyl peptide conjugates: a new drug targeting system for tumor cells

β-Cyclodextrin is known to form inclusion complexes with hydrophobic drugs. Several tumor cell lines are known to secrete and/or contain membrane-associated cathepsin B which is possibly involved in invasion and metastasis. Based on these informations, our recently developed endo-epoxysuccinyl peptide inhibitor MeO-Gly-Gly-Leu-(2S,3S)-tEps-Leu-Pro-OH for cathepsin B was conjugated with β-cyclodextrin to obtain a site-directed drug carrier system. Furthermore, the conjugate was shown to form an inclusion complex with the cytotoxic drug methotrexate.

Inhibition of U-937 membrane-associated cathepsin G by GP 120 (IIIB) and V3 loop-derived peptides from several strains of HIV-1

A cell surface-associated cathepsin G has been reported to be a possible complementary factor for HIV-1 infection of U-937 cells. The effect of recombinant gp120 (IIIB) and a series of V3 loop peptides derived from the sequence of different strains of HIV on the activity of U-937 cathepsin G was assayed. The sequence on the N-terminal side of the highly conserved GPGRAF V3 loop segment was required for interaction with cathepsin G. The inhibition was stable for several hours and there was no cleavage of the peptides derived from the HIV-1(IIIB) strain. Recombinant gp120 (IIIB) also remained uncleaved after incubation with cathepsin G for 3 h, but some cleavage occurred, generating 2 fragments (50 kDa and 70 kDa), after 16 h. Linear peptides derived from HIV-1 Mal, ELI, MN, CDC4 and SF162 strains, and consensus V3 peptides all had inhibitory properties towards cathepsin G, although they were significantly cleaved after one hour. The cleavage site was at the carboxy-terminus of Tyr 323 which is conserved in all these HIV-1 strains but not in HIV-1(IIIB). There was no cleavage at the Arg residue of the GPGRAF sequence, whatever the V3 peptide sequence, the amount of proteinase, or the incubation time. We conclude that the inhibition of membrane-associated cathepsin G of U-937 cells by the gp120 V3 loop of HIV-1 does not occur via a Kunitz-type mechanism, and that the proteinase-V3 loop interaction does not result in a significant cleavage of the V3 loop, though it has been suggested that this event is required for the entry phase of the virus.

Cathepsin D associates with lysosomal membranous protein.

The membrane-association of early biosynthetic form of cathepsin D has been demonstrated in hepatoma cells, and this membrane-association is not mediated by mannose 6-phosphate residues, implying that a mannose 6-phosphate receptor-independent mechanism operates in the sorting of cathepsin D. In this paper, to demonstrate whether cathepsin D is associated with the lysosomal membranes, an in vitro binding experiment was carried out employing lysosomal cathepsin D or microsomal procathepsin D isolated from rat liver. Immunoblotting analysis revealed that an intermediate form of cathepsin D was associated with the lysosomal membranes; this lysosomal membrane-associated cathepsin D was released from the membranes by washing with Na2CO3 (pH 10.6) but not with solutions containing mannose 6-phosphate. This suggested that cathepsin D associates with the membranes by ionic-interaction, and that the membrane-associated cathepsin D resides as a peripheral membrane protein in the lysosomal membrane fraction. To confirm that the intermediate form of cathepsin D specifically interacts with the lysosomal integral membrane proteins, the lysosomal membrane fraction was treated with trypsin and the binding experiment was conducted. The result showed that the binding capacity of cathepsin D to the lysosomal membranes was apparently abolished and cathepsin D did not rebind to the membranes. These data suggest that the intermediate form of cathepsin D is preferentially recognized by the lysosomal membranous protein which complements the mannose 6-phosphate receptor-dependent intracellular sorting mechanism.

HIV-1 virion-cell interactions: an electrostatic model of pathogenicity and syncytium formation.

AIDS Research and Human RetrovirusesVol. 10, No. 3 HIV-1 Virion-Cell Interactions: An Electrostatic Model of Pathogenicity and Syncytium FormationLARRY CALLAHANLARRY CALLAHANSearch for more papers by this authorPublished Online:16 Mar 2009https://doi.org/10.1089/aid.1994.10.231AboutSectionsPDF/EPUB ToolsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail FiguresReferencesRelatedDetailsCited ByIn vitro methods for testing antiviral drugsBiotechnology Advances, Vol. 36, No. 3The ins and outs of eukaryotic viruses: Knowledge base and ontology of a viral infection16 February 2017 | PLOS ONE, Vol. 12, No. 2Age and sex or gender (sex/gender) and HIV vaccine preparedness29 October 2015 | Psychology, Health & Medicine, Vol. 21, No. 4Motivators of enrolment in HIV vaccine trials: a review of HIV vaccine preparedness studies4 July 2011 | AIDS Care, Vol. 23, No. 11Rapid Dissociation of HIV-1 from Cultured Cells Severely Limits Infectivity Assays, Causes the Inactivation Ascribed to Entry Inhibitors, and Masks the Inherently High Level of Infectivity of VirionsJournal of Virology, Vol. 84, No. 6Could CD4 Capture by CD8+ T Cells Play a Role in HIV Spreading?Journal of Biomedicine and Biotechnology, Vol. 2010Primary HIV-1 R5 isolates from end-stage disease display enhanced viral fitness in parallel with increased gp120 net chargeVirology, Vol. 379, No. 1Enrollment and Retention of HIV Discordant Couples in Lusaka, ZambiaJAIDS Journal of Acquired Immune Deficiency Syndromes, Vol. 47, No. 1Amino Functionalized Novel Cholic Acid Derivatives Induce HIV-1 Replication and Syncytia Formation in T Cells25 March 2006 | Journal of Medicinal Chemistry, Vol. 49, No. 8The Role of Community Advisory Boards: Involving Communities in the Informed Consent ProcessAmerican Journal of Public Health, Vol. 91, No. 12LFA-1 Expression on Target Cells Promotes Human Immunodeficiency Virus Type 1 Infection and TransmissionJournal of Virology, Vol. 75, No. 2Efficacy of Fusion Peptide Homologs in Blocking Cell Lysis and HIV-Induced Fusion KATHERINE A. SILBURN, DALE A. McPHEE, ANNE L. MAERZ, PANTELIS POUMBOURIOS, ROBERT G. WHITTAKER, ALAN KIRKPATRICK, WAYNE G. REILLY, MICHAEL K. MANTHEY, and CYRIL C. CURTAIN15 March 2009 | AIDS Research and Human Retroviruses, Vol. 14, No. 5Inhibition of HIV-1, HIV-2 and SIV envelope glycoprotein-mediated cell fusion by calmodulinVirus Research, Vol. 50, No. 2Anti-HIV Type 1 Activity of Sulfated Derivatives of Dextrin against Primary Viral Isolates of HIV Type 1 in Lymphocytes and Monocyte-Derived Macrophages CAROLINE M. JAVAN, NIGEL J. GOODERHAM, ROBERT J. EDWARDS, DONALD S. DAVIES, and SUNIL SHAUNAK15 March 2009 | AIDS Research and Human Retroviruses, Vol. 13, No. 10Role of the Amino-Terminal Extracellular Domain of CXCR-4 in Human Immunodeficiency Virus Type 1 EntryVirology, Vol. 231, No. 1Mechanism of Anti-HIV Activity of Negatively Charged Albumins: Biomolecular Interaction with the HIV-1 Envelope Protein gp120Journal of Acquired Immune Deficiency Syndromes and Human Retrovirology, Vol. 11, No. 5Positioning of Positively Charged Residues in the V3 Loop Correlates with HIV Type 1 Syncytium-Inducing Phenotype DHANANJAY BHATTACHARYYA, BERNARD R. BROOKS, and LARRY CALLAHAN15 March 2009 | AIDS Research and Human Retroviruses, Vol. 12, No. 2Inhibition of U-937 membrane-associated cathepsin G by GP 120 (IIIB) and V3 loop-derived peptides from several strains of HIV-17 March 2000 | FEBS Letters, Vol. 367, No. 3Adaptation to persistent growth in the H9 cell line renders a primary isolate of human immunodeficiency virus type 1 sensitive to neutralization by vaccine seraJournal of Virology, Vol. 69, No. 1 Volume 10Issue 3Mar 1994 To cite this article:LARRY CALLAHAN.HIV-1 Virion-Cell Interactions: An Electrostatic Model of Pathogenicity and Syncytium Formation.AIDS Research and Human Retroviruses.Mar 1994.231-233.http://doi.org/10.1089/aid.1994.10.231Published in Volume: 10 Issue 3: March 16, 2009PDF download

Isolation of a Membrane-Associated Cathepsin D-like Enzyme from the Model Antigen Presenting Cell, A20, and Its Ability to Generate Antigenic Fragments from a Protein Antigen in a Cell-Free System

The biochemical mechanisms and enzymes involved in the processing of protein antigens for presentation by major histocompatibility complex class II molecules are poorly understood. This work describes the purification of a cathepsin D-like enzyme isolated from the murine B lymphoma cell line A20, a model antigen presenting cell. Two forms of cathepsin D-like enzyme were detected. One is soluble and located in the lysosome-enriched sub-cellular fraction. The other is membrane-associated and located in the endosome-enriched fraction. The membrane-associated form was purified to apparent homogeneity by affinity chromatography on pepstatin A-Sepharose. Its apparent molecular weight is 48,000, and its pH optimum is pH 4.0. Endosomal cathepsins are known to be involved in antigen processing in vivo, and the purified membrane-associated cathepsin D-like enzyme from A20 cells was used to study antigen processing in vitro. The enzyme cleaved a model protein antigen, Staphylococcus aureus nuclease (Nase) and thereby generated antigenic fragments recognized by a Nase-specific T cell hybridoma. Such studies have allowed us to begin to understand the role of protease specificity and T cell determinant selection.

Effects of membrane-associated cathepsin B on the activation of receptor-bound prourokinase and subsequent invasion of reconstituted basement membranes

The present study was undertaken to assess the role of membrane-associated cathepsin B as an activator of receptor-bound single-chain urokinase-type plasminogen activator (pro-uPA) and to determine the importance of receptor-bound uPA activity in the destruction of extracellular matrix by tumor cells with subsequent invasion through basement membranes. Ovarian cancer HOC-I cells express pro-uPA/HMW-uPA and cathepsin B on their surface. uPAs are bound to a specific surface receptor, about 30% of which is saturated. 60% of the receptor-bound uPA is pro-uPA. No reduction in the specific binding of biotinylated DFP-HMW-uPA was observed when cells were cultivated in the presence of E-64, a cysteine proteinase inhibitor, for 24 h. Inhibition of cell-surface cathepsin B activity was associated with a decrease in cell-bound uPA activity to undetectable levels, and > 95% of the membrane-associated uPA was pro-uPA in cells cultivated with E-64. This suggested that receptor-bound pro-uPA cannot be converted to HMW-uPA in the absence of enzymatically-active cathepsin B. The significance of the expression of cell-surface uPA activity regarding invasive potential was examined in an in-vitro Matrigel invasion assay. Decreased cell-surface uPA activity was associated with a decrease in invasive potential. These data support our hypothesis that membrane-associated cathepsin B may be important for the conversion of pro-uPA to HMW-uPA and that receptor-bound uPA activity constitutes an efficient mechanism which contributes to tumor cell invasion. As HOC-I cells produce both uPA and cathepsin B, the implications of tumor-cell-derived pro-uPA activation by cellular proteinase cathepsin B should be considered.

Membrane-associated cathepsin L: A role in metastasis of melanomas

Subcellular distribution of cathepsin L, the major protein released by transformed or ras transfected fibroblasts, was examined in murine liver, murine B16 amelanotic melanoma and human A2058 melanoma after sequential differential and Percoll density gradient centrifugation. In both murine and human melanomas, cathepsin L activity was found to be enriched in plasma membrane fractions; cathepsin L in these fractions was in both native and acid activatable forms. Plasma membrane fractions from B16 melanoma subpopulations of “low” and “high” metastatic potential were assayed for activity of cathepsin L and of heat stable endogenous inhibitors. The relative specific activity of cathepsin L was 7-fold greater in the subpopulation of “high” metastatic potential, whereas cysteine proteinase inhibitory activity was 5-fold less. Since cathepsin L can degrade intact basement membrane, this membrane-associated cathepsin L may well contribute to metastatic spread of melanomas.

Degradation of laminin by human tumor cathepsin B.

Cathepsin B activity, including that of a plasma membrane-associated cathepsin B, has been linked to tumor malignancy. As cathepsin B at the tumor cell surface has been hypothesized to play a role in the focal degradation of basement membrane during the metastatic cascade, we have examined the ability of human tumor cathepsin B to degrade laminin, an adhesive glycoprotein found exclusively in the basement membrane. We report that at pH 6.5 and 7.0 tumor cathepsin B degraded by specific, limited proteolysis both subunits of native laminin. The disappearance of both subunits and the appearance of lower Mr protein bands could be observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Accumulation of degradation products was also observed using gel filtration chromatography and a fluorescamine assay. The proteolysis of laminin by tumor cathepsin B could be inhibited by an active site titrant for cysteine proteinases or stefin B, an endogenous low-Mr cysteine proteinase inhibitor.

Activation mechanism of erythrocyte cathepsin E. evidence for the occurrence of the membrane-associated active enzyme.

Activation of the erythrocyte cathepsin E located on the cytoplasmic surface of the membrane in a latent form was studied in stripped inside-out membrane vesicles prepared from human erythrocyte membranes. Incubation of the vesicles at 40 degrees C at pH 4 resulted in increased degradation of the membrane proteins, especially band 3. This proteolysis was selectively inhibited by the inclusion of pepstatin (isovaleryl-Val-Val-statyl-Ala-statine) or H 297 [Pro-Thr-Glu-Phe(CH2-NH)Nle-Arg-Leu] in the incubation mixtures, indicating that cathepsin E, as the only aspartic proteinase in erythrocytes, is responsible for the proteolysis. Two potential active-site-directed inhibitors of aspartic proteinases, pepstatin and H 297, were used to prove the occurrence of the membrane-associated active enzyme. To minimize potential errors arising from non-specific binding, the concentrations of the inhibitors used in the binding assay (pepstatin, 5 x 10(-8) M; H 297, 1 x 10(-5) M) were determined by calibration for purified and membrane-associated cathepsin E. The inhibition of the membrane-associated cathepsin E by each inhibitor, which showed the binding of the inhibitor to the activated enzyme, was temperature- and time-dependent. The binding of each inhibitor to the enzyme on the exposed surface of the membrane at pH 4 was highly specific, saturable, and reversible. The present study thus provides the first evidence that cathepsin E tightly bound to the membrane is converted to the active enzyme in the membrane-associated form, and suggests that this enzyme may be responsible for the degradation of band 3.

Immunodetection of cathepsins B and L present in and secreted from human pre-malignant and malignant colorectal tumour cell lines.

Pre-malignant and malignant human colorectal tumour epithelial cell lines both secreted precursor forms of the 2 cysteine proteinases, cathepsins B and L. The amount of proteinases secreted by these cell lines varied according to the cell density. Comparison at similar cell densities showed that the pre-malignant, adenoma-derived cell line (PC/AA) secreted as much, or more, of both cathepsin B and L precursors as did the malignant, carcinoma-derived cell line (PC/JW/FI). However, mature forms of cathepsins B and L were detected in the culture media of only the carcinoma-derived cell line, thus indicating that the invasive potential of a tumour may be related to its ability to process extracellularly the secreted precursor enzyme to a mature and consequently active enzyme, rather than to the amount of proteinase synthesized and/or secreted. Similar results were obtained using 2 other epithelium-derived tumour cell lines, HT/29 (carcinoma) and SP/AN (adenoma). Immunolocation studies showed that cathepsin B was lysosomal while cathepsin L appeared to have a distribution more consistent with a plasma membrane association. Purified human cathepsins B and L (mature form) were capable of solubilizing an isolated basement membrane matrix (bovine anterior lens capsule) in vitro, thus indicating that the secreted mature enzymes and the membrane-associated cathepsin L could potentially degrade basal laminae or sub-endothelial basement membranes in vivo.

Age-related and phenylhydrazine-induced activation of the membrane-associated cathepsin E in human erythrocytes.

We examined the effects of cell aging and phenylhydrazine-induced oxidant damage on erythrocyte cathepsin E, which is present as a latent, membrane-associated enzyme in normal human erythrocytes. When young erythrocytes isolated from human mature erythrocytes by Percoll density gradient centrifugation were aged in vitro, the membrane-associated cathepsin E was progressively released from the membrane as an active enzyme. During the cell aging up to 100 h, about 40% of the membrane-associated enzyme was activated and solubilized. When phenylhydrazine was incubated with the erythrocytes, it also caused the activation and solubilization of cathepsin E in a dose-dependent and time-dependent manner. Exposure of erythrocytes to 2.5 mM phenylhydrazine for up to 2 h led to about 40% activation of the membrane-associated enzyme. Both aging and phenylhydrazine-treatment were accompanied with an increase in the association of the cytosolic proteins, primarily hemoglobin, with the membrane, which occurred prior to the release of cathepsin E from the membrane. A similar activation for the membrane-associated enzyme was observed with in vitro-aged hemoglobin-free membrane ghosts. Thus, the primary mechanism for activation of cathepsin E in the intact cells seems to be through lesion of the membrane framework that results from increased binding of hemoglobin to the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting employing polyclonal IgG antibodies for human spectrin and band 3 revealed that breakdown of the membrane proteins was enhanced in both aged and phenylhydrazine-treated cells. The relation between the cathepsin E activation and the membrane protein breakdown is discussed.

Cathepsin D is membrane-associated in macrophage endosomes.

Previously we identified an acid protease activity which was located in the endosomes of rabbit alveolar macrophages (Diment, S., and Stahl, P.D. (1985) J. Biol. Chem. 260, 15311-15317). In this study, the endosomal protease is identified as cathepsin D by immunoprecipitation with polyclonal antibodies raised against rabbit cathepsin D and by NH2-terminal sequence. In order to elucidate the mechanism for targeting of cathepsin D to endosomes, we first examined the membrane association of cathepsin D with light (rho = 1.05 g/ml) and heavy density (rho = 1.1 g/ml) vesicles from Percoll density gradients. After sequential washes, 8.4 and 21.9% of cathepsin D activity remained associated with heavy and light density vesicles, respectively. This membrane-associated cathepsin D could not be solubilized in either buffer at pH 5.0 containing mannose 6-phosphate and EDTA or in buffer at pH 10.6. Solubilization required the detergent Triton X-100. To determine whether membrane-associated cathepsin D was found in endosomes, the enzyme was radioiodinated within endosomes and lysosomes with internalized lactoperoxidase. The membrane-associated form was detected in endosomes, but much less in lysosomes. Biosynthetic studies combined with the same extraction procedure revealed that macrophage cathepsin D is first synthesized as an inactive membrane-associated precursor. The precursor is processed to an active, membrane-associated form and then to the active soluble form found in lysosomes. Our studies provide evidence that 1) cathepsin D is in endosomes of macrophages; 2) cathepsin D is transported to endosomes as a membrane-associated form; and 3) the membrane-associated form is a biosynthetic precursor for the soluble form found in endosomes and lysosomes.

Plasma membrane-associated cysteine proteinases in human and animal tumors.

The ability of tumor cells to invade into and through normal tissue during the metastatic cascade has been attributed to tumor-associated degradative enzymes including proteinases of the metallo, serine and cysteine classes. Work from several laboratories has established that the cysteine proteinases cathepsins L and B are released from tumor cells, primarily as latent precursor forms. In addition, a cathepsin B-like cysteine proteinase has been shown to be associated with the plasma membrane fraction of several animal and human tumors. This form of the enzyme retains activity under physiologic (or pathologic) conditions including at neutral pH and in the presence of low Mr inhibitors. Since we have established that cathepsin B can degrade the basement membrane attachment glycoprotein laminin, we speculate that plasma membrane-associated cathepsin B may participate in focal dissolution of the basement membrane during tumor cell extravasation.