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Research paper thumbnail of Interaction of procollagen C-proteinase enhancer-1 (PCPE-1) with cells: identification of target proteins and domains involved

Research paper thumbnail of Folding and activity of recombinant human procollagen C-proteinase enhancer

European Journal of Biochemistry, 2001

Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus sys... more Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains.

Research paper thumbnail of Binding of Procollagen C-Proteinase Enhancer-1 (PCPE-1) to Heparin/Heparan Sulfate: PROPERTIES AND ROLE IN PCPE-1 INTERACTION WITH CELLS

Journal of Biological Chemistry, 2010

Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that c... more Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action.

Research paper thumbnail of Extended interaction network of procollagen C-proteinase enhancer-1 in the extracellular matrix

Biochemical Journal, 2014

PCPE-1 (procollagen C-proteinase enhancer-1) is an extracellular matrix glycoprotein that can sti... more PCPE-1 (procollagen C-proteinase enhancer-1) is an extracellular matrix glycoprotein that can stimulate procollagen processing by procollagen C-proteinases such as BMP-1 (bone morphogenetic protein 1). PCPE-1 interacts with several proteins in addition to procollagens and BMP-1, suggesting that it could be involved in biological processes other than collagen maturation. We thus searched for additional partners of PCPE-1 in the extracellular matrix, which could provide new insights into its biological roles. We identified 17 new partners of PCPE-1 by SPR (surface plasmon resonance) imaging. PCPE-1 forms a transient complex with the β-amyloid peptide, whereas it forms high or very high affinity complexes with laminin-111 (KD=58.8 pM), collagen VI (KD=9.5 nM), TSP-1 (thrombospondin-1) (KD1=19.9 pM, KD2=14.5 nM), collagen IV (KD=49.4 nM) and endostatin, a fragment of collagen XVIII (KD1=0.30 nM, KD2=1.1 nM). Endostatin binds to the NTR (netrin-like) domain of PCPE-1 and decreases the degree of superstimulation of PCPE-1 enhancing activity by heparin. The analysis of the PCPE-1 interaction network based on Gene Ontology terms suggests that, besides its role in collagen deposition, PCPE-1 might be involved in tumour growth, neurodegenerative diseases and angiogenesis. In vitro assays have indeed shown that the CUB1CUB2 (where CUB is complement protein subcomponents C1r/C1s, urchin embryonic growth factor and BMP-1) fragment of PCPE-1 inhibits angiogenesis.

Research paper thumbnail of Interaction of Myosin Subfragment 1 with Two Non-Nucleotide ATP Analogues

Biochemistry, 1998

2-[(2-Nitrophenyl)amino]ethyl triphosphate (NPhAETP) is the smallest ATP analogue that serves as ... more 2-[(2-Nitrophenyl)amino]ethyl triphosphate (NPhAETP) is the smallest ATP analogue that serves as a substrate for the actin-activated ATPase of myosin subfragment 1 (S1) and supports the development of active tension in skinned fibers. 2-(Phenylamino)ethyl triphosphate (PhAETP), in which the nitro group on the phenyl ring of NPhAETP is substituted by a H atom, is also a substrate of the actin-activated ATPase but does not support active tension [Wang, D., Pate, E., Cooke, R., and Yount, R. (1993) J. Muscle Res. Cell Motil. 14, 484-497]. We compared the S1-catalyzed hydrolysis of these analogues, their ability to support the formation of stable complexes with S1 and phosphate analogues, and their effect on S1 conformation. The analogues were hydrolyzed by S1 under various conditions both in the presence and in the absence of actin. In some cases, the effects of the two analogues are similar to each other and to those of ATP; they protect S1 from heat denaturation at 40°C and inhibit the formation of the N-terminal 29 kDa fragment during the tryptic digestion of S1 and the modification of Lys-83 with trinitrobenzene sulfonate. However, in other cases, the effect of the two analogues is different; the effect of NPhAETP resembles that of ATP. NPhAETP and ATP decrease while PhAETP increases the rate of reaction of the SH 1 thiol (Cys-707) with coumarin maleimide. The diphosphate forms of the two analogues induce a much smaller change in the near-UV CD spectrum of S1 than ADP. NPhAEDP forms stable complexes with S1 in the presence of beryllium fluoride (BeF x ), aluminum fluoride (AlF 4 -), or vanadate (Vi) phosphate analogues, while the S1‚PhAEDP complex is stable in the presence BeF x but much less stable with AlF 4and Vi. These results indicate that the S1‚PhAEDP‚P i state is poorly populated during the PhAETP hydrolysis. The models of the atomic structure of S1 complexed by the two analogues show that PhAETP, unlike NPhAETP or ATP, does not form a H bond with Tyr-134 in S1, which is the probable structural reason of the lack of tension development, with PhAETP as the substrate.

Research paper thumbnail of Interaction of procollagen C-proteinase enhancer-1 (PCPE-1) with cells: identification of target proteins and domains involved

Research paper thumbnail of Folding and activity of recombinant human procollagen C-proteinase enhancer

European Journal of Biochemistry, 2001

Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus sys... more Recombinant human procollagen C-proteinase enhancer (rPCPE) was expressed using a baculovirus system and purified to homogeneity using a three-step procedure including heparin affinity chromatography. Heparin binding was dependent on the C-terminal netrin-like domain. The recombinant protein was found to be active, increasing the activity of procollagen C-proteinase/bone morphogenetic protein-1 on type I procollagen in a manner comparable to the native protein. Enhancing activity was dependent on intact disulfide bonding within the protein. By circular dichroism, the observed secondary structure of rPCPE was consistent with the known three-dimensional structures of proteins containing homologous domains.

Research paper thumbnail of Binding of Procollagen C-Proteinase Enhancer-1 (PCPE-1) to Heparin/Heparan Sulfate: PROPERTIES AND ROLE IN PCPE-1 INTERACTION WITH CELLS

Journal of Biological Chemistry, 2010

Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that c... more Procollagen C-proteinase enhancer-1 (PCPE-1) is an extracellular matrix (ECM) glycoprotein that can stimulate procollagen processing by procollagen C-proteinases (PCPs) such as bone morphogenetic protein-1 (BMP-1). The PCPs can process additional extracellular protein precursors and play fundamental roles in developmental processes and assembly of the ECM. The stimulatory activity of PCPE-1 is restricted to the processing of fibrillar procollagens, suggesting PCPE-1 is a specific regulator of collagen deposition. PCPE-1 consists of two CUB domains that bind to the procollagen C-propeptides and are required for PCP enhancing activity, and one NTR domain that binds heparin. To understand the biological role of the NTR domain, we performed surface plasmon resonance (SPR) binding assays, cell attachment assays as well as immunofluorescence and activity assays, all indicating that the NTR domain can mediate PCPE-1 binding to cell surface heparan sulfate proteoglycans (HSPGs). The SPR data revealed binding affinities to heparin/HSPGs in the high nanomolar range and dependence on calcium. Both 3T3 mouse fibroblasts and human embryonic kidney cells (HEK-293) attached to PCPE-1, an interaction that was inhibited by heparin. Cell attachment was also inhibited by an NTR-specific antibody and the NTR fragment. Immunofluorescence analysis revealed that PCPE-Flag binds to mouse fibroblasts and heparin competes for this binding. Cell-associated PCPE-Flag stimulated procollagen processing by BMP-1 several fold. Our data suggest that through interaction with cell surface HSPGs, the NTR domain can anchor PCPE-1 to the cell membrane, permitting pericellular enhancement of PCP activity. This points to the cell surface as a physiological site of PCPE-1 action.

Research paper thumbnail of Extended interaction network of procollagen C-proteinase enhancer-1 in the extracellular matrix

Biochemical Journal, 2014

PCPE-1 (procollagen C-proteinase enhancer-1) is an extracellular matrix glycoprotein that can sti... more PCPE-1 (procollagen C-proteinase enhancer-1) is an extracellular matrix glycoprotein that can stimulate procollagen processing by procollagen C-proteinases such as BMP-1 (bone morphogenetic protein 1). PCPE-1 interacts with several proteins in addition to procollagens and BMP-1, suggesting that it could be involved in biological processes other than collagen maturation. We thus searched for additional partners of PCPE-1 in the extracellular matrix, which could provide new insights into its biological roles. We identified 17 new partners of PCPE-1 by SPR (surface plasmon resonance) imaging. PCPE-1 forms a transient complex with the β-amyloid peptide, whereas it forms high or very high affinity complexes with laminin-111 (KD=58.8 pM), collagen VI (KD=9.5 nM), TSP-1 (thrombospondin-1) (KD1=19.9 pM, KD2=14.5 nM), collagen IV (KD=49.4 nM) and endostatin, a fragment of collagen XVIII (KD1=0.30 nM, KD2=1.1 nM). Endostatin binds to the NTR (netrin-like) domain of PCPE-1 and decreases the degree of superstimulation of PCPE-1 enhancing activity by heparin. The analysis of the PCPE-1 interaction network based on Gene Ontology terms suggests that, besides its role in collagen deposition, PCPE-1 might be involved in tumour growth, neurodegenerative diseases and angiogenesis. In vitro assays have indeed shown that the CUB1CUB2 (where CUB is complement protein subcomponents C1r/C1s, urchin embryonic growth factor and BMP-1) fragment of PCPE-1 inhibits angiogenesis.

Research paper thumbnail of Interaction of Myosin Subfragment 1 with Two Non-Nucleotide ATP Analogues

Biochemistry, 1998

2-[(2-Nitrophenyl)amino]ethyl triphosphate (NPhAETP) is the smallest ATP analogue that serves as ... more 2-[(2-Nitrophenyl)amino]ethyl triphosphate (NPhAETP) is the smallest ATP analogue that serves as a substrate for the actin-activated ATPase of myosin subfragment 1 (S1) and supports the development of active tension in skinned fibers. 2-(Phenylamino)ethyl triphosphate (PhAETP), in which the nitro group on the phenyl ring of NPhAETP is substituted by a H atom, is also a substrate of the actin-activated ATPase but does not support active tension [Wang, D., Pate, E., Cooke, R., and Yount, R. (1993) J. Muscle Res. Cell Motil. 14, 484-497]. We compared the S1-catalyzed hydrolysis of these analogues, their ability to support the formation of stable complexes with S1 and phosphate analogues, and their effect on S1 conformation. The analogues were hydrolyzed by S1 under various conditions both in the presence and in the absence of actin. In some cases, the effects of the two analogues are similar to each other and to those of ATP; they protect S1 from heat denaturation at 40°C and inhibit the formation of the N-terminal 29 kDa fragment during the tryptic digestion of S1 and the modification of Lys-83 with trinitrobenzene sulfonate. However, in other cases, the effect of the two analogues is different; the effect of NPhAETP resembles that of ATP. NPhAETP and ATP decrease while PhAETP increases the rate of reaction of the SH 1 thiol (Cys-707) with coumarin maleimide. The diphosphate forms of the two analogues induce a much smaller change in the near-UV CD spectrum of S1 than ADP. NPhAEDP forms stable complexes with S1 in the presence of beryllium fluoride (BeF x ), aluminum fluoride (AlF 4 -), or vanadate (Vi) phosphate analogues, while the S1‚PhAEDP complex is stable in the presence BeF x but much less stable with AlF 4and Vi. These results indicate that the S1‚PhAEDP‚P i state is poorly populated during the PhAETP hydrolysis. The models of the atomic structure of S1 complexed by the two analogues show that PhAETP, unlike NPhAETP or ATP, does not form a H bond with Tyr-134 in S1, which is the probable structural reason of the lack of tension development, with PhAETP as the substrate.