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Pro-Carboxypeptidase R is an Acute Phase Protein in the Mouse, Whereas Carboxypeptidase N Is Not^{1 ,2}

Tomoo Sato^*,, Takashi Miwa^*,, Hiroyasu Akatsu, Noriyuki Matsukawa, Kyoko Obata^*, Noriko Okada^*, William Campbell and Hidechika Okada^3,*

^* Department of Molecular Biology, Nagoya City University Medical School, Nagoya, Japan; and Choju Medical Institute, Fukushimura Hospital, Toyohashi, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Carboxypeptidase R (EC 3.4.17.20; CPR) and carboxypeptidase N (EC 3.4.17.3; CPN) cleave carboxyl-terminal arginine and lysine residues from biologically active peptides such as kinins and anaphylatoxins, resulting in regulation of their biological activity. Human proCPR, also known as thrombin-activatable fibrinolysis inhibitor, plasma pro-carboxypeptidase B, and pro-carboxypeptidase U, is a plasma zymogen activated during coagulation. CPN, however, previously termed kininase I and anaphylatoxin inactivator, is present in a stable active form in plasma. We report here the isolation of mouse proCPR and CPN cDNA clones that can induce their respective enzymatic activities in culture supernatants of transiently transfected cells. Potato carboxypeptidase inhibitor can inhibit carboxypeptidase activity in culture medium of mouse proCPR-transfected cells. The expression of proCPR mRNA in murine liver is greatly enhanced following LPS injection, whereas CPN mRNA expression remains unaffected. Furthermore, the CPR activity in plasma increased 2-fold at 24 h after LPS treatment. Therefore, proCPR can be considered a type of acute phase protein, whereas CPN is not. An increase in CPR activity may facilitate rapid inactivation of inflammatory mediators generated at the site of Gram-negative bacterial infection and may consequently prevent septic shock. In view of the ability of proCPR to also inhibit fibrinolysis, an excess of proCPR induced by LPS may contribute to hypofibrinolysis in patients suffering from disseminated intravascular coagulation caused by sepsis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It has been well established that human carboxypeptidase N (CPN),⁴ which consists of two small active subunits and two large glycosylated subunits, is an important inactivator of several potent peptides, including anaphylatoxins, kinins, and fibrinopeptides (1, 2, 3, 4). CPN is present in the active form in plasma (5, 6). Mathews et al. (7) has reported familial partial carboxypeptidase N deficiency resulting in angioedema/chronic urticaria, hay fever/asthma, or both. Over the last decade, a new basic carboxypeptidase that is generated from its zymogen during coagulation was identified independently by a number of groups (8, 9, 10, 11, 12). We designated this enzyme carboxypeptidase R (CPR) and its zymogen, proCPR. The activated form appeared to remove arginine residues (R) more rapidly than lysine residues (K) at the carboxyl terminus of substrates compared with CPN (5, 11, 13). It has also been termed plasma carboxypeptidase B (11), carboxypeptidase U (CPU) (8), and thrombin-activatable fibrinolysis inhibitor (TAFI) (12). It is known that human proCPR can be hydrolyzed by trypsin-like enzymes such as thrombin (11), thrombin/thrombomodulin complex (14), and plasmin (11), thereby acquiring enzymatic activity by which it cleaves carboxyl-terminal arginine and lysine residues from various peptides. This activity can be completely inhibited by potato carboxypeptidase inhibitor (PCI) at concentrations at which CPN is not affected (15). Because the activity of CPR is similar to that of CPN, this enzyme has been implicated in the inactivation of several natural potent peptides, including anaphylatoxins and bradykinin, as well as in the removal of arginine and lysine residues from partially plasmin-degraded fibrin (8, 15, 16). To elucidate the in vivo roles of proCPR and CPN, the establishment of an animal model is essential. For this purpose we identified cDNAs of mouse proCPR and CPN and studied the effects of LPS on the expression of these enzymes during the inflammatory process.

	Materials and Methods

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Probes for screening a cDNA library and Northern blot analysis

We obtained two partial mouse sequences similar to that of human plasma pro-carboxypeptidase B (proCPR) using Entrez (http://www.ncbi.nlm.nih.gov/Entrez/) from the National Center of Biotechnology Information. The GenBank accession numbers are AA244760 and AA254734. In the same manner we obtained data on three partial mouse sequences from clones similar to the human small subunit of CPN. The numbers for these sequences are AA238838, AA285540, and AA396985. The sets of primers generated based on the above were: mCPR86⁺, 5'-CTGCTCTTCCAAGAACCTCC-3'; mCPR292^-, 5'-CCACGTTGTTCATCAGAACG-3'; mCPR533⁺, 5'-GAATCCATGCCAGAGAATGG-3'; mCPR740^-, 5'-TTGTTCTTGTGAGCAGAGCG-3'; mCPN108⁺, 5'-CAAGGTGCACAACCAATGC-3'; mCPN510^-, 5'-GAAGTAGGTGTTGAGATCCGG-3'; mCPN609⁺, 5'-GATTCGCTCCTTGAACTTCG-3'; and mCPN1118^-, 5'-GTTCACCTGAAGTGACGTCG-3'. RT-PCR was performed with these sets of primers using mouse liver total RNA. PCR products were subcloned with a TA cloning kit (Invitrogen, San Diego, CA). The presence of these inserts was confirmed by sequencing. Clones were digested with several restriction enzymes followed by electrophoresis on a 1.0% agarose gel. The inserts were purified using a Prep-A-Gene kit (Bio-Rad, Hercules, CA) and the probes were labeled CPR1, CPR2, CPN1, and CPN2, respectively.

Molecular cloning and base sequence analysis

Dr. Mayumi Nonaka of our laboratory provided the cDNA library, which was constructed using the ZAP II vector with poly(A)⁺ RNA isolated from adult BALB/c mouse liver. Recombinants (1 x 10⁶) were plated at 50,000 plaques/137-mm plate. Screening was conducted under stringent conditions by the plaque hybridization method using each of the [-³²P]dCTP-labeled probes (CPR1, CPR2, CPN1, and CPN2) described above. Hybridization was performed at 65°C for 16 h in a buffer containing 50 mM Tris (pH 7.4), 10x Denhardt’s solution, 1 M NaCl, 10 mM EDTA, 0.1% SDS, and 0.1 mg/ml denatured salmon sperm DNA (Wako, Osaka, Japan). Filters were washed twice with 2x SSC (0.3 M NaCl and 30 mM sodium citrate, pH 7.0) containing 0.1% SDS at 65°C for 30 min and once with 0.2x SSC containing 0.1% SDS at room temperature for 60 min. The dried filters were then exposed for 16 h at -80°C to x-ray films (X-OMAT AR, Eastman Kodak, Rochester, NY). The pBluescript phagemids were excised from positive clones using R408 helper phage (Stratagene, La Jolla, CA). The nucleotide sequence was determined by means of an A.L.F. sequence system (Pharmacia Biotech, Uppsala, Sweden). Sequence alignments and analyses were performed using GENETYX-MAC software.

Mice

Male BALB/c mice, 8–10 wk of age, were purchased from Japan SLC (Shizuoka, Japan). They were allowed free access to food and water. Animal experiments were conducted according to the Guidelines for the Care and Use of Laboratory Animals of Nagoya City University Medical School. Experimental protocols were approved by the institutional animal care and use committee of Nagoya City University Medical School.

RT-PCR

Total RNA extracts were prepared from normal BALB/c mouse brain, lung, liver, stomach, intestine, spleen, and kidney using Trizol reagent (Life Technologies, Gaithersburg, MD). Each cDNA was synthesized from 1 µg of total RNA treated with DNase I using Superscript II reverse transcriptase (Life Technologies) and random hexamer; 2.5% of this reaction product was used as a template for PCR. To confirm the integrity of each total RNA obtained, GAPDH mRNA was amplified from the same cDNA preparation. The following primers were used: mCPN, mCPN1⁺ (5'-ATGCCAGACCTGCCCTCAG-3') and mCPN510^-; mouse proCPR, mCPR86⁺ and mCPR740^-; and mouse GAPDH (5'-CATCACCATCTTCCAGGA-3' and 5'-TTGTCATGGATGACCTTGGC-3'). DNA was amplified by PCR with AmpliTaq Gold DNA polymerase (Perkin-Elmer Applied Biosystems, Foster City, CA) for 30 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min, followed by 72°C for 5 min. PCR products were separated by electrophoresis on a 1.2% agarose gel and visualized by ethidium bromide staining.

Northern blot analysis

Total RNA was extracted from normal mouse brain, lung, liver, stomach, intestine, spleen, and kidney using Trizol reagent (Life Technologies). A 15-µg amount of each total RNA preparation was electrophoresed in 1% formaldehyde-agarose gel and transferred to a nylon membrane (Hybond-N, Amersham, Arlington Heights, IL). The membrane was hybridized with ³²P-labeled mouse proCPR and CPN cDNA probes (CPR2 and CPN2) in QuikHyb solution (Stratagene). It was then washed twice with 2x SSC containing 0.1% SDS at room temperature for 15 min and once with 0.1x SSC containing 0.1% SDS at 60°C for 10 min and exposed to X-OMAT AR film (Kodak) at -80°C overnight. In another experiment, BALB/c mice, 9–12 wk of age, were injected i.p. with 0.5 or 4 mg/kg of LPS (from Escherichia coli 0111:B4, Sigma, St. Louis, MO) in 0.5 ml of saline or with 0.5 ml of saline alone and killed under anesthesia at 4, 8, 12, and 24 h after LPS injection. Total RNA of the livers was prepared using Trizol reagent. The rest of the procedure was conducted as described above except for the use of another mouse proCPR probe (CPR1).

Construction of eukaryotic expression vectors for transfection of mouse proCPR and mCPN

The eukaryotic expression vector pDR2EF1 synthesized by Dr. I. Anegon (Institut National de la Santé et de la Recherche Médicale, Unité 437, Nantes, France) was a gift from Dr. B. P. Morgan (University of Wales College of Medicine, Cardiff, U.K.) (17). pDR2EF1 contains the powerful polypeptide chain elongation factor-1 promoter. Mouse proCPR cDNA in pBluescript plasmid vector was subcloned into BamHI and EcoRV sites of pDR2EF1, which is termed m-proCPR/pDR2EF1. Mouse CPN cDNA in the pBluescript vector was digested with BamHI, and an 1.6-kb fragment containing an open reading frame was purified using a Prep-A-Gene kit (Bio-Rad). The mCPN BamHI fragment was also subcloned into BamHI sites of pDR2EF1, and this form was designated m-CPN/pDR2EF1. The presence and fidelity of m-proCPR/pDR2EF1 and m-CPN/pDR2EF1 were confirmed by restriction enzyme digestion and sequencing, respectively.

Transient expression of the cloned cDNAs in COS cells

COS-7 cells derived from the kidney of an African green monkey were cultured in 25-cm² flasks in DMEM containing 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 U/ml penicillin, 50 µg/ml streptomycin, and 1 µg/ml fungizone in a 37°C humidified incubator (95% air/5% CO₂ atmosphere). The m-proCPR/pDR2EF1 and m-CPN/pDR2EF1 expression vectors were transfected into COS-7 cells using Lipofectamine reagent (Life Technologies) in a 24-well culture plate. When the cells were 60–80% confluent, the conditioned medium in each well was replaced with 300 µl of Opti-MEM I Reduced Serum Medium (Life Technologies) containing 5 µl of Lipofectamine and 1.6 µg of an appropriate plasmid (pDR2EF1 alone as a control, m-proCPR/pDR2EF1, or m-CPN/pDR2EF1). Five hours after transfection, the DNA/lipid complex medium was replaced with 1 ml of normal growth medium containing 10% FBS. To prevent the FBS from affecting the assay for carboxypeptidase (CP) activity, the medium was exchanged for serum-free DMEM after 12 h. Cells were cultured for 4 days post-transfection. The harvested culture medium was clarified by passing through a 0.22-µm pore size membrane filter and was concentrated from 500 to 20 µl using a microconcentrator (Microcon-10, Amicon, Beverly, MA). The concentrated medium was used to measure CP activity.

Preparation of fresh serum and plasma samples to measure CP activity

BALB/c mice, 9–12 wk of age, were injected i.p. with 0.5 or 4 mg/kg of LPS (E. coli O111:B4) in sterile saline. They were anesthetized with pentobarbital before cardiac puncture at 0, 1, 12, 24, 36, and 48 h after LPS injection. Each blood sample was immediately separated into a 1.5-ml plastic tube containing 10 µl of heparin and a silicone-coated glass tube kept on ice. The sample in the plastic tube was immediately mixed and then centrifuged at 8000 rpm for 10 min at 4°C. This was used as the source of mouse plasma. The glass tube sample was incubated for 12 h at 4°C and then centrifuged at 3000 rpm for 15 min at 4°C. This was used as the source of mouse serum. Results were obtained from three mice at each time point.

Measurement of CP activity

CP activity was determined using hippuryl-L-arginine (Sigma) as a synthetic substrate. The amount of hippuric acid generated by the enzyme was determined by means of a modification of a liquid chromatography method described previously (18). For concentrated culture medium, 20 µl of each sample and 40 µl of 30 mM hippuryl-L-arginine in 50 mM HEPES (pH 8.2) as the substrate solution were mixed with or without 20 µl of 1 mg/ml trypsin solution (Sigma) and then incubated at 37°C for 45 min. With mouse fresh serum and plasma, the sample volume was 10 µl. After incubation, 20 µl of 2.5 M HCl was added to stop the enzyme reaction. After extraction with 300 µl of ethyl acetate, the top 200 µl was removed and evaporated, and the residue was dissolved in 200 µl of double-distilled water. The OD₂₂₈ of the reaction product (hippuric acid) was compared with that of dilutions of a standard solution of hippuric acid. When the potato carboxypeptidase inhibitor (PCI; Calbiochem, La Jolla, CA) was used, each sample was incubated with 1 µl of PCI solution (adjusted to the appropriate concentration with 50 mM Tris buffer, pH 7.5) for >5 min before measurement of CP activity.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of mouse proCPR and mouse CPN cDNAs

Based on several partial mouse sequences similar to those of human plasma procarboxypeptidase B (=proCPR) and the human CPN active subunit acquired from the mouse EST database, two sets of primers for each enzyme were synthesized. Using these primers, we performed PCR amplification on cDNA that had been reverse transcribed from mouse liver total RNA. Probes were then made of the PCR products. These probes were labeled with [-³²P]dCTP and used to screen 1,000,000 plaques from the mouse liver cDNA library. The screening identified two clones containing a full-length cDNA of either the mouse homologue of human proCPR or the mouse homologue of the active subunit of human CPN. We tentatively designated the isolated cDNAs as mouse proCPR and mouse CPN, respectively. These were completely sequenced using both universal primers and specific internal primers of our own design. The DNA sequences of these clones are shown in Fig. 1. Sequence analysis of the mouse proCPR clone revealed an insert of 1432 bp with an open reading frame of 1266 bp coding for a protein of 422 aa (Fig. 1A). The 3'-untranslated sequence includes the canonical polyadenylation signal, AATAAA, 33 bp upstream from the poly(A) tail. The NH₂-terminal of 21 aa probably represents a portion of the signal peptide as determined by the weight-matrix method (19). The deduced protein sequence for mouse proCPR has 84% identity to human proCPR. Based on analysis of human proCPR, mouse proCPR appears to consist of a 92-aa activation peptide and a 309-aa mature enzyme. Four N-linked glycosylation sites (Asn-X-Ser/Thr) are probably present in the activation peptide as in human proCPR. From the amino acid sequence, two glycosylation sites may exist in the catalytic domain. Sequence analysis of the mouse CPN clone revealed an insert of 1774 bp with an open reading frame of 1371 bp coding for a protein of 457 aa in length (Fig. 1B). There is no canonical polyadenylation signal preceding the poly(A) tail at the appropriate distance as is present in the cDNA of human CPN. However, the sequence ATTAAA, 22 bp upstream from the poly(A) tail, probably serves this function. The 20 aa of the NH₂-terminal were suggested to be a portion of the signal peptide, as determined using the weight-matrix method (19). The deduced amino acid sequence for mouse CPN has 83% identity to the human CPN small subunit. Although it had been shown that the small subunits of human CPN are not glycosylated, two N-linked glycosylation sites were predicted for mouse CPN.

View larger version (86K): [in this window] [in a new window]   FIGURE 1. Nucleotide sequences of mouse proCPR (A) and mouse CPN (B) cDNAs and their deduced amino acid sequences. A, Nucleotides are numbered from the Met initiation codon. The first arrowhead indicates the signal peptidase cleavage site, and others indicate trypsin cleavage sites deduced from human plasma proCPB (=proCPR) sequence analysis (11 ). The polyadenylation site is underlined. Residues implicated in zinc (circles) and substrate (squares) binding previously determined for human proCPR are indicated. Predicted N-glycosylation sites are shown by asterisks. B, The arrowhead indicates the signal peptidase cleavage site. The other symbols are the same in A.

We confirmed that the products of our isolated genes have the same properties as human proCPR and CPN. Since we detected little CP activity in the culture medium of COS-7 cells alone (data not shown), we used these cells as host cells for cDNA transfection. COS-7 cells were transiently transfected with the following expression vectors: pDR2EF1 alone as a control, m-proCPR/pDR2EF1, or m-CPN/pDR2EF1. Because mouse proCPR and CPN cDNAs both harbor a portion of the signal peptide, it was predicted that the recombinant COS-7 cells would secrete the gene products. Therefore, serum-free medium was used to measure the CP activity of recombinant proteins secreted from the transfected cells, since its use ensured that there would be little interference from FBS CPs. Enzyme activity was determined from the amount of hippuric acid generated by the cleavage of hippuryl-L-arginine as substrate. To demonstrate whether the gene product is a proenzyme, enzyme activities were also measured after trypsin treatment (16). As shown in Fig. 2A, the medium harvested from COS-7 cells transiently transfected with m-proCPR/pDR2EF1 plasmid showed much higher activity than medium without trypsin. The medium used for the control transfectant (vector alone) showed little CP activity even with trypsin. In addition, we demonstrated that PCI (50 µg/ml) could inhibit 92% of the trypsin-generated CP activity in the culture medium of mouse proCPR-transfected cells (Fig. 2A). In the culture medium of COS-7 cells transfected with m-CPN/pDR2EF1 plasmid, significant CP activity was observed without trypsin treatment (Fig. 2B). However, treatment with trypsin reduced the CP activity in the supernatant of mouse CPN cDNA transfectant cells to about half that without trypsin treatment despite the fact that limited trypsin treatment of human CPN was shown to enhance activity (W. Campbell, unpublished observations). This may be due to a difference in experimental conditions, and additional experiments will establish whether there is a difference in sensitivity to trypsin between mouse CPN and human CPN.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. CP activity in culture media of COS-7 cells transfected with m-proCPR/pDR2EF1 (A) or m-CPN/pDR2EF1 (B) and with pDR2EF1 vector alone as a control. CP activity was measured with or without 0.25 mg/ml trypsin to establish whether each of our isolated gene products is a proenzyme. Preincubation with 50 µg/ml of PCI can inhibit the CP activity of trypsin-treated culture medium of m-proCPR cDNA-transfected cells. Activity was determined with hippuryl-L-arginine as substrate. One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 µmol of substrate/min at 37°C. Data are the mean ± SD of triplicate samples.

By means of RT-PCR, we detected mRNA for mouse proCPR and CPN in all tissues studied except mouse brain CPN or mouse spleen proCPR (Fig. 3A). A comparison of mouse proCPR and CPN expression showed that they shared almost the same pattern, in that liver and stomach showed stronger expression than did other tissues. Northern blot analysis of mouse CPN mRNA revealed two species of 1.6 and 1.9 kb that were expressed abundantly in the liver and more weakly in the stomach (Fig. 3B), and on longer exposure were also seen in the lung and kidney; however, only the 1.6-kb species was seen in the lung. Expression of the 1.9-kb species was much stronger than that of the 1.6-kb species in the liver and stomach. The 1.9-kb species appears to be from the mouse CPN gene isolated in the present study. Northern blot analysis of mouse proCPR mRNA showed only a 1.5-kb species that was expressed abundantly in the liver and was undetected in other tissues. This 1.5-kb Northern blot species corresponded to the length of our isolated gene.

View larger version (48K): [in this window] [in a new window]   FIGURE 3. Expression of mouse proCPR and CPN transcripts in tissues. A, RT-PCR amplification of mouse CPN, mouse proCPR, and GAPDH cDNAs from total RNA of mouse tissues. The primers for mouse CPN, mouse proCPR, and GAPDH generated amplification products of 510, 655, and 282 bp, respectively. B, Northern blot analysis of mouse tissues. Total RNA (15 µg/lane) isolated from the indicated mouse tissues was electrophoresed and hybridized with probes for mouse proCPR and CPN.

We examined how LPS influences mouse proCPR and CPN gene expression in murine liver, which is the major site of synthesis. As shown in Fig. 4, i.p. administration of LPS (0.5 mg/kg) caused an up-regulation of mouse proCPR gene expression, which reached a maximal level at 12 h after LPS treatment. The level at 24 h was still higher than that in the control. On the other hand, expression of both 1.6- and 1.9-kb mouse CPN mRNAs remained unchanged following LPS injection (0.5 mg/kg). A high dose (4 mg/kg) and a low dose (0.5 mg/kg) of LPS elicited the same response (data not shown). Furthermore, to examine whether the levels of expression of mouse proCPR and CPN mRNAs in various other tissues are affected by LPS treatment, we compared levels at 12 h after LPS or saline injection by Northern blot analysis. Northern blotting did not detect mouse proCPR mRNA in brain, lung, stomach, intestine, or kidney despite LPS treatment. However, LPS stimulation reduced transcription of mouse CPN mRNA in the stomach to half that in the untreated control, although it did not change the level in the kidney or lung (data not shown).

View larger version (44K): [in this window] [in a new window]   FIGURE 4. Time course of induction of mouse proCPR and CPN mRNAs in liver after LPS injection. Mice received an i.p. injection of 0.5 ml of saline alone as the control or LPS (E. coli O111: B4, 0.5 mg/kg) in 0.5 ml of saline. Total RNA was isolated from mouse liver at 4, 8, 12, and 24 h after LPS injection and at 12 h after saline injection. Each total RNA (15 µg/lane) was loaded onto a formaldehyde agarose gel. Blots were probed with 32P-labeled cDNA for the mouse proCPR or mouse CPN fragment. Lower bands contain 18S ribosomal RNA in the total RNA loaded onto the gel.

Human CPR was initially identified following the observation that the CP activity of fresh serum is higher than that of plasma (8), and we have now observed the same phenomenon in the mouse. The CP activity of fresh mouse serum was approximately twice that of plasma. This augmented activity decreased in a time-dependent manner at 37°C and returned to the plasma level within 1 h. Furthermore, PCI could lower this enhanced activity in a dose-dependent fashion, and activity was completely suppressed to the plasma level using 50 µg/ml of this inhibitor (data not shown). These results agree with the finding that PCI inhibits human CPR but not human CPN. Therefore, we determined that the CP activity in fresh serum and plasma of LPS-treated mice is representative of the CP activity of CPR plus CPN and of CPN alone, respectively. At 24 h following LPS administration, the CP activity of fresh serum was significantly increased to 5 times the plasma level (Fig. 5). The CP activity of serum did not rise above the plasma level in the presence of 100 µg/ml of PCI (preliminary results). The CP activity of fresh serum was reduced at 6 h following a transient increase at 1 h, but it increased again at 24 h to a level about 2.5 times higher than that in sera of untreated mice. The CPR activity, therefore, was 4 times greater than that in untreated mouse serum, as calculated by subtracting the plasma CP activity, corresponding to CPN activity, from the total activity in serum. ProCPR consumption during inflammation induced by LPS may be responsible for the decrease in serum CP activity observed in the early stage after injection. Similarly, plasma CP activity corresponding to CPN had decreased slightly at 6 and 12 h after LPS injection. This difference in activity between serum and plasma was the same whether mice were injected with 0.5 or 4 mg/kg of LPS.

View larger version (22K): [in this window] [in a new window]   FIGURE 5. Induction of arginine CP activity by LPS. The CP activity of trypsin-treated plasma () and trypsin-treated plasma plus 100 µg/ml of PCI () was measured before and after i.p. injection of 0.5 mg/kg of LPS. The CP activity of mouse plasma and serum at 0, 1, 6, 12, 24, 36, and 48 h following treatment with LPS is indicated (mouse plasma, 0.5 mg/kg () and 4 mg/kg () of LPS; mouse serum, 0.5 mg/kg () and 4 mg/kg () of LPS). Data represent the mean ± SD of three mice at the indicated times. During these experiments, a total of seven mice injected with 4 mg/kg of LPS died from 12 to 36 h following injection. These mice were replaced with seven animals who survived the 4 mg/kg challenge to analyze results for three mice from each group.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study we isolated mouse homologues of human proCPR cDNA and human CPN small active subunit cDNA. The deduced amino acid sequences revealed >80% homology between mice and humans. Substrate and zinc binding sites were conserved. Culture media of COS-7 cells transfected with these homologues had enzyme activities similar to those of the human enzymes, as mentioned below. The trypsin-treated culture medium of the proCPR transfectant had high CP activity, and that of the CPN transfectant also had significant carboxypeptidase activity without trypsin treatment. Furthermore, PCI inhibited the CP activity of mouse CPR as is seen with human CPR; therefore, we considered the isolated genes to be those of mouse proCPR and CPN, respectively. Northern blot analysis revealed that mouse CPN mRNA consists of two species of 1.6 and 1.9 kb. The mouse CPN cDNA that we isolated was regarded as the 1.9-kb species because it was more abundantly expressed than was the 1.6-kb species. It has been demonstrated that human proCPR and human CPN are synthesized in the liver (11, 20, 21). This organ appears to be the major site of synthesis in the mouse as well. Although mouse CPN mRNA expression was not affected by LPS, mouse proCPR mRNA expression steadily increased for at least 12 h after LPS treatment. It is conceivable that the CP activity inhibited by PCI of trypsin-treated plasma reflects the level of proCPR in plasma. Its value increased 2-fold at 24 h after LPS injection, suggesting that proCPR expression was up-regulated at the protein level in addition to the mRNA level. Furthermore the CPR activity (fresh serum CP activity minus the plasma CP activity) at 24 h after LPS stimulation increased 4-fold, whereas the plasma CP activity was essentially unaffected. This increase in CPR activity demonstrates that LPS induces activation of proCPR as well as enhanced expression, and CPN is not affected. These results indicate that mouse proCPR is an acute phase protein, whereas mouse CPN is not. CPN may be required for homeostasis to remove terminal lysine and arginine from a variety of peptides. This was supported by the finding that CPN is related to the pathophysiology of chronic diseases such as chronic urticaria or asthma (7). An increase in CPR activity may facilitate rapid inactivation of inflammatory mediators such as anaphylatoxins and kinins generated at sites of Gram-negative bacterial infections and may consequently prevent septic shock. Based on these findings, it is conceivable that administration of proCPR could prevent death from septic shock.

However, several recent reports suggest that excess activation of proCPR may exacerbate a state of disseminated intravascular coagulation (DIC) resulting from a Gram-negative bacterial infection (14, 15, 22, 23, 24, 25, 26, 27). Following activation by thrombin, proCPR, otherwise known as TAFI, exerts an antifibrinolytic effect by removing carboxyl-terminal lysines on fibrin, preventing plasminogen binding and activation (15). On the other hand, it has been established that CPN does not remove carboxyl-terminal lysine residues from the clot surface despite the preference for lysine residues rather than arginine residues (15, 26). Furthermore, according to the present study, LPS stimulation did not essentially alter CPN expression or consumption of CPN compared with proCPR following LPS injection (Fig. 5), suggesting that CPN may not be actively involved in the pathophysiology of DIC. Activated protein C specifically inactivates factors VIIIa and Va, two essential cofactors in the intrinsic coagulation pathway, thereby attenuating further thrombin formation and consequent activation of TAFI (proCPR) (23). Expression of protein C mRNA in the mouse, mainly in liver, dropped to a minimal level at 8 h after LPS treatment (24). Analysis of serial plasma samples from patients with DIC revealed that the activity of protein C and its Ag decreases progressively during the initial stages of DIC and remains at a low level for 24–48 h (25). Our studies with a murine model demonstrated that proCPR increases and reaches a maximum level 24 h after LPS treatment. Therefore, endotoxin induces down-regulation of protein C and up-regulation of proCPR. Reduced protein C activity favors the intrinsic pathway of coagulation, with a relative increase in thrombin formation and a consequent activation of proCPR, which is up-regulated following LPS stimulation. Endotoxin thereby causes the hypercoagulability and hypofibrinolysis seen in DIC. Thrombomodulin (TM) is expressed on the surface of endothelial cells, and soluble functional proteolytic fragments of TM are also present in circulating plasma. It has been reported that the thrombin/TM complex activates TAFI (proCPR) 1250 times more rapidly than does free thrombin (14) and that plasma TM, including soluble TM fragments, inhibits fibrinolysis in a dose-dependent manner via activation of plasma proCPB (=proCPR) (26). Plasma levels of soluble TM fragments are elevated in patients with DIC (27). These findings suggest that high concentrations of these fragments in patients with DIC facilitate the activation of proCPR.

Our results suggest that CPR may actually play two important roles in vivo: first, as an inactivator of inflammatory mediators to prevent excessive inflammation, and secondly, as an inhibitor of fibrinolysis. Therefore, although a higher level of CPR activity may reduce susceptibility to shock, an increase in its activity could also facilitate DIC by preventing fibrinolysis. Further research on CPR both in vitro and in vivo should provide insight into its important dual function as a regulatory enzyme.

	Acknowledgments

 
We thank Yasushige Ishikawa, Chiharu Naito, Yoshiaki Tani, and Norihiro Ogawa for their excellent technical assistance. We also thank Catherine Campbell for her help in editing this manuscript.

	Footnotes

 
¹ This work was supported by a research grant from the Organization for Pharmaceutical Safety and Research.

² The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases under accession numbers AB021968 and AB021969.

³ Address correspondence and reprint requests to Dr. H. Okada, Department of Molecular Biology, Nagoya City University Medical School, Mizuho-cho, Nagoya 467-8601, Japan.

⁴ Abbreviations used in this paper: CPN, carboxypeptidase N; mCPN, mouse CPN; CPR, carboxypeptidase R; TAFI, thrombin-activatable fibrinolysis inhibitor; CP, carboxypeptidase; EST, expressed sequence tags; TM, thrombomodulin; DIC, disseminated intravascular coagulation; PCI, potato carboxypeptidase inhibitor.

Received for publication December 6, 1999. Accepted for publication May 1, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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