Glycophosphatidylinositol (GPI)-anchored membrane proteins are initially synthesized with a cleav... more Glycophosphatidylinositol (GPI)-anchored membrane proteins are initially synthesized with a cleavable COOH-terminal extension that signals anchor attachment. Overexpression in COS cells of hGH-DAF fusion proteins containing the GPI signal of decay accelerating factor (DAF) fused to the COOH-terminus of human growth hormone (hGH), produces both GPI-anchored hGH-DAF and uncleaved precursors that retain the GPI signal. Using hGH-DAF fusion proteins containing a mutated, noncleavable GPI signal, we show that uncleaved polypeptides are retained inside the cell and accumulate in a brefeldin A-sensitive, Golgi-like juxtanuclear structure. Retention requires the presence of either a functional or a noncleavable GPI signal; hGH-DAF fusion proteins containing only the COOH-terminal hydrophobic domain (a component of the GPI signal) are secreted. Immunofluorescence analysis shows colocalization of the retained, uncleaved fusion proteins with both a Golgi marker and with p53, a marker of the ER...
SummaryTissue factor (TF), the cellular cofactor for the serine protease factor VIIa (F. VIIa), t... more SummaryTissue factor (TF), the cellular cofactor for the serine protease factor VIIa (F. VIIa), triggers blood coagulation and is involved in the pathogenesis of various thrombosis-related disorders. Therefore, agents which specifically target tissue factor, such as monoclonal antibodies, may provide promising new antithrombotic therapy. We mapped the epitopes of several anti-TF antibodies using a panel of soluble TF mutants. They bound to three distinct TF regions. The epitope of the 7G11 antibody included Phe50 and overlapped with a TF-F. VIIa light chain contact area. The common epitope of the antibodies 6B4 and HTF1 included residues Tyr94 and Phe76 both of which make critical contacts to the catalytic domain of F. VIIa. The antibodies D3 and 5G6 had a common epitope outside the TF-F. VIIa contact region. It included residues Lys165, Lys166, Asn199, Arg200 and Lys201 and thus overlapped with the substrate interaction region of tissue factor. The antibodies 5G6 and D3 were potent...
The general features of the glycosylphosphatidylinositol (GPI) signal have been conserved in evol... more The general features of the glycosylphosphatidylinositol (GPI) signal have been conserved in evolution. To test whether the requirements for GPI attachment are indeed the same in mammalian cells and parasitic protozoa, we expressed the prototype GPI-linked protein of Trypanosoma brucei, the variant surface glycoprotein (VSG), in COS cells. Although large amounts of VSG were produced, only a small fraction became GPI linked. This impaired processing is not caused by the VSG ectodomain, since replacement of the VSG GPI signal with that of decay accelerating factor (DAF) produced GPI-linked VSG. Furthermore, whereas fusion of the DAF GPI signal to the COOH terminus of human growth hormone (hGH) produces GPI-linked hGH, an analogous hGH fusion using the VSG GPI signal does not, indicating that the VSG GPI signal functions poorly in mammalian cells. By constructing chimeric VSG-DAF GPI signals and fusing them to the COOH terminus of hGH, we show that of the two critical elements that com...
The homotrimeric human serine protease HtrA1 is homologous to bacterial HtrA proteases regarding ... more The homotrimeric human serine protease HtrA1 is homologous to bacterial HtrA proteases regarding the trypsin-like catalytic and PDZ domains but differs by the presence of an N-terminal domain with IGFBP and Kazal homology. The crystal structures and SAXS analysis presented herein reveal the rare tandem of IGFBP-and Kazal-like modules, a protease active site that adopts a competent conformation in the absence of substrate or inhibitor and a model for the intact protein in solution. Highly sensitive enzymatic assays and binding studies demonstrate that the N-terminal tandem has no apparent effect on protease activity, and in accordance with the structure-based predictions, neither the IGFBPnor Kazal-like module retains the function of their prototype proteins. Our structures of the unliganded HtrA1 active site suggest two-state equilibrium and a ''conformational selection'' model, in which substrate binds to the active conformer.
The transmembrane (TM) subfamily of Eph ligands and their receptors have been implicated in axon ... more The transmembrane (TM) subfamily of Eph ligands and their receptors have been implicated in axon pathfinding and in pattern formation during embryogenesis. These functions are thought to involve repulsive interactions but this has not been demonstrated directly. In this study we used a growth cone collapse assay to determine if the TM ligands Lerk2 and HtkL have repellant guidance activity. We show that Lerk2, but not HtkL, is a collapsing factor for a subset of embryonic cortical neurons. Analysis of the effects of Lerk2 on both the morphology and the cytoskeleton of cortical neurons suggests a mechanism of action different from that of AL-1, a GPI-linked Eph ligand having similar repellant activity. Treatment with Lerk2 disrupts the organization of both the actin cytoskeleton and the microtubules and induces the formation of swellings in the center of the growth cone and along the axon. Measurement of the relative F-actin concentrations in the neurites and soma indicated that F-actin levels in the neurites decrease while those in the soma increase, with the net F-actin content of the neuron remaining unchanged. In contrast, we show that prolonged treatment with AL-1 leads to a net loss of F-actin, consistent with the hypothesis that AL-1 acts by perturbing actin polymerization. These results provide evidence that the ectodomain of Lerk2 functions as a repellant guidance cue and show that, despite overlapping specificities in vitro, the biological activities of related ligands are not necessarily overlapping. Further, TM and GPI-linked Eph ligands appear to exert repellant activity by different mechanisms, opening up the possibility that they may have different effects on growth cones in vivo.
6A6 is a murine monoclonal antibody raised against the humanized antitissue factor antibody D3H44... more 6A6 is a murine monoclonal antibody raised against the humanized antitissue factor antibody D3H44. 6A6 is able to completely neutralize the anticoagulant activity of D3H44 in tissue factor-dependent functional assays, such as endotoxin-induced whole blood clotting, prothrombin time, as well as factor X and factor IX activation. ELISA-type assays further showed that 6A6 binds to an epitope with critical determinants on the V L domain of D3H44. The possibility that the anti-idiotypic 6A6 might carry an "internal image" of the original antigen (tissue factor) was examined using the X-ray structure of the 6A6-Fab/D3H44-Fab complex determined at 2.5 Å resolution. We find that 6A6 structurally mimics tissue factor only so far as it combines with the antigen recognition surface of D3H44. While 6A6 contacts both V L and V H domains of D3H44, as does tissue factor, there is more contact with the D3H44 V L domain and less with the D3H44 V H domain relative to the tissue factor contacts on D3H44. Additionally, there is an almost total lack of correspondence between 6A6 and tissue factor at the level of amino acid side-chain functional groups. Despite the fact that both tissue factor and 6A6 are composed largely of b-sheets, they present fundamentally different elements of secondary structure to D3H44; tissue factor presents b-sheets edge-on, while 6A6 uses mostly loops. Finally, the finding that 6A6 competes with tissue factor for D3H44 binding raises the possibility of using 6A6 as an antidote for D3H44 anticoagulant therapy. To this end, we constructed a chimeric murine/human 6A6-Fab, which effectively neutralized D3H44 and fully restored tissue factor function in enzymatic assays.
Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a rec... more Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a receptor tyrosine kinase implicated in development, tissue regeneration, and invasive tumor growth. HGF acquires signaling activity only upon proteolytic cleavage of single-chain HGF into its ␣/ heterodimer, similar to zymogen activation of structurally related serine proteases. Although both chains are required for activation, only the ␣-chain binds Met with high affinity. Recently, we reported that the protease-like HGF -chain binds to Met with low affinity (
Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of... more Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of an ␣-chain containing four Kringle domains (K1-K4) and a serine protease domain-like -chain. Receptor activation by HGF is contingent upon prior proteolytic conversion of the secreted inactive single chain form (pro-HGF) into the biologically active two chain form by a single cleavage at the Arg 494-Val 495 bond. By screening a panel of serine proteases we identified two new HGF activators, plasma kallikrein and coagulation factor XIa (FXIa). The concentrations of kallikrein and FXIa to cleave 50% (EC 50) of 125 I-labeled pro-HGF during a 4-h period were 10 and 17 nM. Unlike other known activators, both FXIa and kallikrein processed pro-HGF by cleavage at two sites. Using N-terminal sequencing they were identified as the normal cleavage site Arg 494-Val 495 and the novel site Arg 424-His 425 located in the K4 domain of the ␣-chain. The identity of this unusual second cleavage site was firmly established by use of the double mutant HGF(R424A/R494E), which was completely resistant to cleavage by kallikrein and FXIa. Experiments with another mutant form, HGF(Arg 494 3 Glu), indicated that cleavage at the K4 site was independent of a prior cleavage at the primary, kinetically preferred Arg 494-Val 495 site. The cleavage at the K4 site had no obvious consequences on HGF function, because it was fully capable of phosphorylating the c-Met receptor of A549 cells. This may be explained by the disulfide bond network in K4, which holds the cleaved ␣-chain together. In conclusion, the ability of plasma kallikrein and FXIa to activate pro-HGF in vitro raises the possibility that mediators of inflammation and blood coagulation may also regulate processes that involve the HGF/ c-Met pathway, such as tissue repair and angiogenesis.
Background: Two forms of PCSK9, an intact and a furin cleaved form, circulate in blood. Results: ... more Background: Two forms of PCSK9, an intact and a furin cleaved form, circulate in blood. Results: Both forms, as highly purified recombinant proteins, are able to bind to and trigger degradation of LDL receptors and elevate serum cholesterol levels. Conclusion: Furin cleavage is not associated with a loss of PCSK9 polypeptides or a significant loss of function. Significance: LDL-c levels are controlled by both forms of PCSK9. Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg 218-Gln 219 in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg 218-Gln 219 more efficiently than furin but also cleaves at Arg 215-Phe 216. Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser 153-Arg 218), which remained attached to the catalytic domain. Both furin-and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furincleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.
The transmembrane serine protease hepsin is one of the most highly upregulated genes in prostate ... more The transmembrane serine protease hepsin is one of the most highly upregulated genes in prostate cancer. Here, we investigated its tumor-promoting activity by use of a mouse orthotopic prostate cancer model. First, we compared the tumor growth of low hepsin-expressing LnCaP-17 cells with hepsin-overexpressing LnCaP-34 cells. After implantation of cells into the left anterior prostate lobe, LnCaP-34 tumors not only grew faster based on increased serum prostate-specific antigen levels but also metastasized to local lymph nodes and, most remarkably, invaded the contralateral side of the prostate at a rate of 100% compared with only 18% for LnCaP-17 tumors. The increased tumor growth was not due to nonspecific gene expression changes and was not predicted from the unaltered in vitro growth and invasion of LnCaP-34 cells. A likely explanation is that the in vivo effects of hepsin were mediated by specific hepsin substrates present in the tumor stroma. In a second study, mice bearing LnCa...
Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme tran... more Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme transition. In particular, the region of the protease domain that must contact TF has a conformation that is altered from that of VIIa, suggesting that the VII protease domain interacts with TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTF mutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have been evaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmon resonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensor chip surface. Dissociation constants (K D) measured for binding to wild-type sTF are 7.5 (2.4 nM for VII and 5.1 (2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significant decrease (>2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding with substitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VII and VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII can readily adopt a VIIa-like conformation required for binding to TF.
The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue f... more The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue factor (TF). TF binds factor VIIa with high affinity and, in addition, participates in substrate interaction through its C-terminal fibronectin type III domain. We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF) mutants were expressed in E. coli, and their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface. According to the degree of activity loss of the sTF mutants, this TF region can be divided into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 A(2) and an extended region which comprises an additional 7-8 residues, including the distally positioned Asn199, Arg200, and Asp204. Some of the identified TF residues, such as Trp158 and those within the loop Lys159-Lys165, are near the factor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the factor VIIa Gla-domain may also participate in substrate interaction. Moreover, the surface identified as important for substrate interaction carries a net positive charge, suggesting that charge interactions may significantly contribute to TF-substrate binding. The calculated surface-exposed area of this substrate interaction region is about 1100 A(2), which is approximately half the size of the TF area that is in contact with factor VIIa. Therefore, a substantial portion of the TF surface (3000 A(2)) is engaged in protein-protein interactions during substrate catalysis.
The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphat... more The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphatidylinositol (GPI) membrane anchor attachment in a process involving concerted proteolytic removal of 28 COOH-terminal residues. At least two elements are required for anchor addition: a COOH-terminal hydrophobic domain and a cleavage/attachment site located NH2-terminal to it, requiring a small amino acid as the acceptor for GPI addition. We previously showed that the last 29-37 residues of DAF, making up the COOH-terminal hydrophobic domain plus 20 residues of the adjacent serine/threonine-rich domain (including the anchor addition site), when fused to the COOH terminus of human growth hormone (hGH) will target the fusion protein to the plasma membrane via a GPI anchor. In contrast, a similar fusion protein (hGH-LDLR-DAF17, abbreviated HLD) containing a fragment of the serine/threonine-rich domain of the LDL receptor (LDLR) in place of the DAF-derived serine/threonine-rich sequences, d...
The serine protease hepsin is highly upregulated in prostate cancer and is implicated in tumor pr... more The serine protease hepsin is highly upregulated in prostate cancer and is implicated in tumor progression. Therefore, specific inhibition of hepsin enzymatic activity by an antibody constitutes an attractive therapeutic approach. Here, we report the identification of the anti-hepsin antibody Fab25 by screening of a Fab phage display library with a restricted chemical diversity at the complementary determining regions. Hepsin with its S1 pocket occupied by 3,4dichloro-isocoumarin was used as the 'bait' for library screening. Fab25 was highly specific and it potently inhibited hepsin activity toward a panel of synthetic and macromolecular substrates. Biochemical and enzymatic studies with synthetic substrates of variable length suggested that Fab25 acts as an allosteric inhibitor based on non-competitive inhibition kinetics. Isothermal titration calorimetric experiments showed that the high-affinity (K D 6.1 nM) binding of Fab25 with hepsin is enthalpically driven. Despite an unusually long CDR-H3 loop with several potential hepsin cleavage sites (Lys, Arg residues), Fab25 was not processed by hepsin. Antibody-25 should be valuable for investigating hepsin's role in cancer progression and for potential therapeutic applications. Furthermore, the herein presented phage display strategy using an active site-modified protease should be widely applicable for identifying potential allosteric anti-protease antibodies.
Glycophosphatidylinositol (GPI)-anchored membrane proteins are initially synthesized with a cleav... more Glycophosphatidylinositol (GPI)-anchored membrane proteins are initially synthesized with a cleavable COOH-terminal extension that signals anchor attachment. Overexpression in COS cells of hGH-DAF fusion proteins containing the GPI signal of decay accelerating factor (DAF) fused to the COOH-terminus of human growth hormone (hGH), produces both GPI-anchored hGH-DAF and uncleaved precursors that retain the GPI signal. Using hGH-DAF fusion proteins containing a mutated, noncleavable GPI signal, we show that uncleaved polypeptides are retained inside the cell and accumulate in a brefeldin A-sensitive, Golgi-like juxtanuclear structure. Retention requires the presence of either a functional or a noncleavable GPI signal; hGH-DAF fusion proteins containing only the COOH-terminal hydrophobic domain (a component of the GPI signal) are secreted. Immunofluorescence analysis shows colocalization of the retained, uncleaved fusion proteins with both a Golgi marker and with p53, a marker of the ER...
SummaryTissue factor (TF), the cellular cofactor for the serine protease factor VIIa (F. VIIa), t... more SummaryTissue factor (TF), the cellular cofactor for the serine protease factor VIIa (F. VIIa), triggers blood coagulation and is involved in the pathogenesis of various thrombosis-related disorders. Therefore, agents which specifically target tissue factor, such as monoclonal antibodies, may provide promising new antithrombotic therapy. We mapped the epitopes of several anti-TF antibodies using a panel of soluble TF mutants. They bound to three distinct TF regions. The epitope of the 7G11 antibody included Phe50 and overlapped with a TF-F. VIIa light chain contact area. The common epitope of the antibodies 6B4 and HTF1 included residues Tyr94 and Phe76 both of which make critical contacts to the catalytic domain of F. VIIa. The antibodies D3 and 5G6 had a common epitope outside the TF-F. VIIa contact region. It included residues Lys165, Lys166, Asn199, Arg200 and Lys201 and thus overlapped with the substrate interaction region of tissue factor. The antibodies 5G6 and D3 were potent...
The general features of the glycosylphosphatidylinositol (GPI) signal have been conserved in evol... more The general features of the glycosylphosphatidylinositol (GPI) signal have been conserved in evolution. To test whether the requirements for GPI attachment are indeed the same in mammalian cells and parasitic protozoa, we expressed the prototype GPI-linked protein of Trypanosoma brucei, the variant surface glycoprotein (VSG), in COS cells. Although large amounts of VSG were produced, only a small fraction became GPI linked. This impaired processing is not caused by the VSG ectodomain, since replacement of the VSG GPI signal with that of decay accelerating factor (DAF) produced GPI-linked VSG. Furthermore, whereas fusion of the DAF GPI signal to the COOH terminus of human growth hormone (hGH) produces GPI-linked hGH, an analogous hGH fusion using the VSG GPI signal does not, indicating that the VSG GPI signal functions poorly in mammalian cells. By constructing chimeric VSG-DAF GPI signals and fusing them to the COOH terminus of hGH, we show that of the two critical elements that com...
The homotrimeric human serine protease HtrA1 is homologous to bacterial HtrA proteases regarding ... more The homotrimeric human serine protease HtrA1 is homologous to bacterial HtrA proteases regarding the trypsin-like catalytic and PDZ domains but differs by the presence of an N-terminal domain with IGFBP and Kazal homology. The crystal structures and SAXS analysis presented herein reveal the rare tandem of IGFBP-and Kazal-like modules, a protease active site that adopts a competent conformation in the absence of substrate or inhibitor and a model for the intact protein in solution. Highly sensitive enzymatic assays and binding studies demonstrate that the N-terminal tandem has no apparent effect on protease activity, and in accordance with the structure-based predictions, neither the IGFBPnor Kazal-like module retains the function of their prototype proteins. Our structures of the unliganded HtrA1 active site suggest two-state equilibrium and a ''conformational selection'' model, in which substrate binds to the active conformer.
The transmembrane (TM) subfamily of Eph ligands and their receptors have been implicated in axon ... more The transmembrane (TM) subfamily of Eph ligands and their receptors have been implicated in axon pathfinding and in pattern formation during embryogenesis. These functions are thought to involve repulsive interactions but this has not been demonstrated directly. In this study we used a growth cone collapse assay to determine if the TM ligands Lerk2 and HtkL have repellant guidance activity. We show that Lerk2, but not HtkL, is a collapsing factor for a subset of embryonic cortical neurons. Analysis of the effects of Lerk2 on both the morphology and the cytoskeleton of cortical neurons suggests a mechanism of action different from that of AL-1, a GPI-linked Eph ligand having similar repellant activity. Treatment with Lerk2 disrupts the organization of both the actin cytoskeleton and the microtubules and induces the formation of swellings in the center of the growth cone and along the axon. Measurement of the relative F-actin concentrations in the neurites and soma indicated that F-actin levels in the neurites decrease while those in the soma increase, with the net F-actin content of the neuron remaining unchanged. In contrast, we show that prolonged treatment with AL-1 leads to a net loss of F-actin, consistent with the hypothesis that AL-1 acts by perturbing actin polymerization. These results provide evidence that the ectodomain of Lerk2 functions as a repellant guidance cue and show that, despite overlapping specificities in vitro, the biological activities of related ligands are not necessarily overlapping. Further, TM and GPI-linked Eph ligands appear to exert repellant activity by different mechanisms, opening up the possibility that they may have different effects on growth cones in vivo.
6A6 is a murine monoclonal antibody raised against the humanized antitissue factor antibody D3H44... more 6A6 is a murine monoclonal antibody raised against the humanized antitissue factor antibody D3H44. 6A6 is able to completely neutralize the anticoagulant activity of D3H44 in tissue factor-dependent functional assays, such as endotoxin-induced whole blood clotting, prothrombin time, as well as factor X and factor IX activation. ELISA-type assays further showed that 6A6 binds to an epitope with critical determinants on the V L domain of D3H44. The possibility that the anti-idiotypic 6A6 might carry an "internal image" of the original antigen (tissue factor) was examined using the X-ray structure of the 6A6-Fab/D3H44-Fab complex determined at 2.5 Å resolution. We find that 6A6 structurally mimics tissue factor only so far as it combines with the antigen recognition surface of D3H44. While 6A6 contacts both V L and V H domains of D3H44, as does tissue factor, there is more contact with the D3H44 V L domain and less with the D3H44 V H domain relative to the tissue factor contacts on D3H44. Additionally, there is an almost total lack of correspondence between 6A6 and tissue factor at the level of amino acid side-chain functional groups. Despite the fact that both tissue factor and 6A6 are composed largely of b-sheets, they present fundamentally different elements of secondary structure to D3H44; tissue factor presents b-sheets edge-on, while 6A6 uses mostly loops. Finally, the finding that 6A6 competes with tissue factor for D3H44 binding raises the possibility of using 6A6 as an antidote for D3H44 anticoagulant therapy. To this end, we constructed a chimeric murine/human 6A6-Fab, which effectively neutralized D3H44 and fully restored tissue factor function in enzymatic assays.
Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a rec... more Hepatocyte growth factor (HGF), a plasminogen-related growth factor, is the ligand for Met, a receptor tyrosine kinase implicated in development, tissue regeneration, and invasive tumor growth. HGF acquires signaling activity only upon proteolytic cleavage of single-chain HGF into its ␣/ heterodimer, similar to zymogen activation of structurally related serine proteases. Although both chains are required for activation, only the ␣-chain binds Met with high affinity. Recently, we reported that the protease-like HGF -chain binds to Met with low affinity (
Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of... more Hepatocyte growth factor (HGF), the ligand for the receptor tyrosine kinase c-Met, is composed of an ␣-chain containing four Kringle domains (K1-K4) and a serine protease domain-like -chain. Receptor activation by HGF is contingent upon prior proteolytic conversion of the secreted inactive single chain form (pro-HGF) into the biologically active two chain form by a single cleavage at the Arg 494-Val 495 bond. By screening a panel of serine proteases we identified two new HGF activators, plasma kallikrein and coagulation factor XIa (FXIa). The concentrations of kallikrein and FXIa to cleave 50% (EC 50) of 125 I-labeled pro-HGF during a 4-h period were 10 and 17 nM. Unlike other known activators, both FXIa and kallikrein processed pro-HGF by cleavage at two sites. Using N-terminal sequencing they were identified as the normal cleavage site Arg 494-Val 495 and the novel site Arg 424-His 425 located in the K4 domain of the ␣-chain. The identity of this unusual second cleavage site was firmly established by use of the double mutant HGF(R424A/R494E), which was completely resistant to cleavage by kallikrein and FXIa. Experiments with another mutant form, HGF(Arg 494 3 Glu), indicated that cleavage at the K4 site was independent of a prior cleavage at the primary, kinetically preferred Arg 494-Val 495 site. The cleavage at the K4 site had no obvious consequences on HGF function, because it was fully capable of phosphorylating the c-Met receptor of A549 cells. This may be explained by the disulfide bond network in K4, which holds the cleaved ␣-chain together. In conclusion, the ability of plasma kallikrein and FXIa to activate pro-HGF in vitro raises the possibility that mediators of inflammation and blood coagulation may also regulate processes that involve the HGF/ c-Met pathway, such as tissue repair and angiogenesis.
Background: Two forms of PCSK9, an intact and a furin cleaved form, circulate in blood. Results: ... more Background: Two forms of PCSK9, an intact and a furin cleaved form, circulate in blood. Results: Both forms, as highly purified recombinant proteins, are able to bind to and trigger degradation of LDL receptors and elevate serum cholesterol levels. Conclusion: Furin cleavage is not associated with a loss of PCSK9 polypeptides or a significant loss of function. Significance: LDL-c levels are controlled by both forms of PCSK9. Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg 218-Gln 219 in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg 218-Gln 219 more efficiently than furin but also cleaves at Arg 215-Phe 216. Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser 153-Arg 218), which remained attached to the catalytic domain. Both furin-and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furincleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.
The transmembrane serine protease hepsin is one of the most highly upregulated genes in prostate ... more The transmembrane serine protease hepsin is one of the most highly upregulated genes in prostate cancer. Here, we investigated its tumor-promoting activity by use of a mouse orthotopic prostate cancer model. First, we compared the tumor growth of low hepsin-expressing LnCaP-17 cells with hepsin-overexpressing LnCaP-34 cells. After implantation of cells into the left anterior prostate lobe, LnCaP-34 tumors not only grew faster based on increased serum prostate-specific antigen levels but also metastasized to local lymph nodes and, most remarkably, invaded the contralateral side of the prostate at a rate of 100% compared with only 18% for LnCaP-17 tumors. The increased tumor growth was not due to nonspecific gene expression changes and was not predicted from the unaltered in vitro growth and invasion of LnCaP-34 cells. A likely explanation is that the in vivo effects of hepsin were mediated by specific hepsin substrates present in the tumor stroma. In a second study, mice bearing LnCa...
Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme tran... more Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme transition. In particular, the region of the protease domain that must contact TF has a conformation that is altered from that of VIIa, suggesting that the VII protease domain interacts with TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTF mutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have been evaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmon resonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensor chip surface. Dissociation constants (K D) measured for binding to wild-type sTF are 7.5 (2.4 nM for VII and 5.1 (2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significant decrease (>2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding with substitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VII and VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII can readily adopt a VIIa-like conformation required for binding to TF.
The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue f... more The enzymatic activity of coagulation factor VIIa is controlled by its cellular cofactor tissue factor (TF). TF binds factor VIIa with high affinity and, in addition, participates in substrate interaction through its C-terminal fibronectin type III domain. We analyzed surface-exposed residues in the C-terminal TF domain to more fully determine the area on TF important for substrate activation. Soluble TF (sTF) mutants were expressed in E. coli, and their ability to support factor VIIa-dependent substrate activation was measured in the presence of phospholipid vesicles or SW-13 cell membranes. The results showed that factor IX and factor X interacted with the same TF region located proximal to the putative phospholipid surface. According to the degree of activity loss of the sTF mutants, this TF region can be divided into a main region (residues Tyr157, Lys159, Ser163, Gly164, Lys165, Lys166, Tyr185) forming a solvent-exposed patch of 488 A(2) and an extended region which comprises an additional 7-8 residues, including the distally positioned Asn199, Arg200, and Asp204. Some of the identified TF residues, such as Trp158 and those within the loop Lys159-Lys165, are near the factor VIIa gamma-carboxyglutamic acid (Gla) domain, suggesting that the factor VIIa Gla-domain may also participate in substrate interaction. Moreover, the surface identified as important for substrate interaction carries a net positive charge, suggesting that charge interactions may significantly contribute to TF-substrate binding. The calculated surface-exposed area of this substrate interaction region is about 1100 A(2), which is approximately half the size of the TF area that is in contact with factor VIIa. Therefore, a substantial portion of the TF surface (3000 A(2)) is engaged in protein-protein interactions during substrate catalysis.
The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphat... more The COOH terminus of decay-accelerating factor (DAF) contains a signal that directs glycophosphatidylinositol (GPI) membrane anchor attachment in a process involving concerted proteolytic removal of 28 COOH-terminal residues. At least two elements are required for anchor addition: a COOH-terminal hydrophobic domain and a cleavage/attachment site located NH2-terminal to it, requiring a small amino acid as the acceptor for GPI addition. We previously showed that the last 29-37 residues of DAF, making up the COOH-terminal hydrophobic domain plus 20 residues of the adjacent serine/threonine-rich domain (including the anchor addition site), when fused to the COOH terminus of human growth hormone (hGH) will target the fusion protein to the plasma membrane via a GPI anchor. In contrast, a similar fusion protein (hGH-LDLR-DAF17, abbreviated HLD) containing a fragment of the serine/threonine-rich domain of the LDL receptor (LDLR) in place of the DAF-derived serine/threonine-rich sequences, d...
The serine protease hepsin is highly upregulated in prostate cancer and is implicated in tumor pr... more The serine protease hepsin is highly upregulated in prostate cancer and is implicated in tumor progression. Therefore, specific inhibition of hepsin enzymatic activity by an antibody constitutes an attractive therapeutic approach. Here, we report the identification of the anti-hepsin antibody Fab25 by screening of a Fab phage display library with a restricted chemical diversity at the complementary determining regions. Hepsin with its S1 pocket occupied by 3,4dichloro-isocoumarin was used as the 'bait' for library screening. Fab25 was highly specific and it potently inhibited hepsin activity toward a panel of synthetic and macromolecular substrates. Biochemical and enzymatic studies with synthetic substrates of variable length suggested that Fab25 acts as an allosteric inhibitor based on non-competitive inhibition kinetics. Isothermal titration calorimetric experiments showed that the high-affinity (K D 6.1 nM) binding of Fab25 with hepsin is enthalpically driven. Despite an unusually long CDR-H3 loop with several potential hepsin cleavage sites (Lys, Arg residues), Fab25 was not processed by hepsin. Antibody-25 should be valuable for investigating hepsin's role in cancer progression and for potential therapeutic applications. Furthermore, the herein presented phage display strategy using an active site-modified protease should be widely applicable for identifying potential allosteric anti-protease antibodies.
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Papers by Paul Moran