Human procarboxypeptidase B: three-dimensional structure and implications for thrombin-activatable fibrinolysis inhibitor (TAFI)

Besides their classical role in alimentary protein degradation, zinc-dependant carboxypeptidases also participate in more selective regulatory processes like prohormone and neuropeptide processing or fibrinolysis inhibition in blood plasma. Human pancreatic procarboxypeptidase B (PCPB) is the prototype for those human exopeptidases that cleave off basic C-terminal residues and are secreted as inactive zymogens. One such protein is thrombin-activatable fibrinolysis inhibitor (TAFI), also known as plasma PCPB, which circulates in human plasma as a zymogen bound to plasminogen. The structure of human pancreatic PCPB displays a 95-residue pro-segment consisting of a globular region with an open-sandwich antiparallel-alpha antiparallel-beta topology and a C-terminal alpha-helix, which connects to the enzyme moiety. The latter is a 309-amino acid residue catalytic domain with alpha/beta hydrolase topology and a preformed active site, which is shielded by the globular domain of the pro-segment. The fold of the proenzyme is similar to previously reported procarboxypeptidase structures, also in that the most variable region is the connecting segment that links both globular moieties. However, the empty active site of human procarboxypeptidase B has two alternate conformations in one of the zinc-binding residues, which account for subtle differences in some of the key residues for substrate binding. The reported crystal structure, refined with data to 1.6A resolution, permits in the absence of an experimental structure, accurate homology modelling of TAFI, which may help to explain its properties.     

The complete cDNA sequence of bovine coagulation factor V.

Lack of availability of a primary structure for bovine factor V has hindered detailed analysis of a vast majority of structure-function correlations on this molecule. To determine the primary structure of bovine factor V, we used liver mRNA as a template for the synthesis of three cDNA libraries. The sequences of seven overlapping cDNA clones infer two bovine factor V variants. Variant 1 results in a 6910-basepair (bp) cDNA including 103 bp of 5'-untranslated sequence, 6633 bp of coding sequence and 171 bp of 3'-untranslated sequence with a putative polyadenylation site. Variant 2 differs only in the size of the coding sequence (6618 bp). The open reading frame translates to factor V consisting of 2211 (or 2206) amino acids including a 28-amino acid signal peptide. Comparison of the amino acid sequences with human factor Va reveals 84% identity for the heavy and 86% for the light chains. In contrast, the B domain (connecting region) exhibits only 59% identity relative to the human molecule. The bovine B domain contains two repeats of a 14-amino acid structure that is contained only once in the human sequence. Bovine factor V lacks one of the nine amino acid repeats and one of the 17 amino acid repeats present in the human B domain. Factor V has little homology to the factor VIII molecule in the B domain. The 17-amino acid repeat missing in bovine factor V allows identification of an 18-amino acid sequence that is homologous to the B domain of human factor VIII. These 18 amino acids may either constitute the unique vestige of a divergent evolution between the B domains of factors V and VIII or reveal the convergent evolution toward a critical epitope involved in the activation of both procofactors.     

Cloning and characterization of human pancreatic lipase cDNA

Pancreatic lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) hydrolyzes dietary long chain triacylglycerol to free fatty acids and monoacylglycerols in the intestinal lumen. In the presence of bile acids, the activity of lipase is stimulated by colipase. As a prelude to studying the relationship of the protein structures to the functional properties of lipase and colipase, a cDNA encoding human pancreatic lipase was isolated from a lambda gt11 cDNA library screened with a rabbit polyclonal anti-human pancreatic lipase antibody. The full length cDNA clone of 1477 base pairs contained an open reading frame encoding a 465-amino acid protein, including a 16-amino acid signal peptide. The nucleotide sequence was 69% identical to the dog pancreatic lipase cDNA. The predicted NH2-terminal protein sequence agreed with the published NH2-terminal sequence of human pancreatic lipase and the predicted protein sequence was 85 and 70% identical to the protein sequences of pig and dog pancreatic lipase, respectively. A region of homology around Ser-153 is conserved in a number of lipid-binding proteins. Human hepatic lipase and lipoprotein lipase share extensive homology with pancreatic lipase, suggesting that the three proteins are members of a small gene family. In vitro translation of mRNA transcribed from the cDNA resulted in a protein of the expected molecular size that could be processed by microsomal membranes to yield a glycolated protein with proper signal peptide cleavage. RNA blot analysis demonstrated tissue specificity for pancreatic lipase. Thus, for the first time, a full length human pancreatic lipase cDNA has been isolated and characterized. The demonstrated regions of homology with other lipases will aid definition of interactions with substrate and colipase through site-specific mutagenesis.     

Wound-induced proteinase inhibitors from tomato leaves. I. The cDNA-deduced primary structure of pre-inhibitor I and its post-translational processing

A cDNA containing the coding region for the complete amino acid sequence of wound-induced proteinase Inhibitor I from tomato leaves was constructed in the plasmid pUC9 and characterized. The open reading frame codes for a protein of 111 amino acids. This deduced amino acid sequence revealed the presence of a 42-amino acid N-terminal sequence that is not found in the native protein. This sequence appears to contain a 23-amino acid segment typical of a signal sequence followed by a 19-amino acid sequence containing 9 charged amino acids. The 42-amino acid sequence is apparently lost during maturation to the native Inhibitor I and represents 38% of the translated protein. The Inhibitor I amino acid sequence contains 71% identity with potato tuber Inhibitor I sequence and 35% identity with an inhibitor from the leech.     

Cloning and sequencing of a human pancreatic tumor mucin cDNA

A monospecific polyclonal antiserum against deglycosylated human pancreatic tumor mucin was used to select human pancreatic mucin cDNA clones from a lambda gt11 cDNA expression library developed from a human pancreatic tumor cell line. The full-length 4.4-kilobase mucin cDNA sequence included a 72-base pair 5'-untranslated region and a 307-base pair 3'-untranslated region. The predicted amino acid sequence for this cDNA revealed a protein of 122,071 daltons containing 1,255 amino acid residues of which greater than 60% were serine, threonine, proline, alanine, and glycine. Approximately two-thirds of the protein sequence consisted of identical 20-amino acid tandem repeats which were flanked by degenerate tandem repeats and nontandem repeat sequences on both the amino-terminal and carboxyl-terminal ends. The amino acid sequence also contained five putative N-linked glycosylation sites, a putative signal sequence and transmembrane domain, and numerous serine and threonine residues (potential O-linked glycosylation sites) outside and within the tandem repeat position. The cDNA and deduced amino acid sequence of the pancreatic mucin sequence was over 99% homologous with a mucin cDNA sequence derived from breast tumor mucin, even though the native forms of these molecules are quite distinct in size and degree of glycosylation.     

Cloning and sequencing of cDNA for the rat plasminogen activator inhibitor-1

A cDNA encoding rat plasminogen activator-inhibitor (PAI-1) has been isolated from an HTC rat hepatoma cell cDNA library constructed in phage lambda gt10. The cDNA contains 118 bp of 5'-untranslated sequence, 1206 bp encoding a 402-amino acid (aa) protein and 1747 bp of 3'-untranslated sequence. The protein-coding sequence and the derived amino acid sequence share 82% and 81% identity, respectively, with human PAI-1 cDNA and protein. The rat cDNA encodes a preprotein with a 23-aa leader peptide and a predicted N-terminal serine for the mature protein. Three of four potential N-glycosylation acceptor sites as well as the active site of rat PAI-1 are identical to the human protein. The 3'-untranslated region contains a number of unusual regions, including 80 bp of tandemly repeated GpA dinucleotides, a 115-bp stretch which shares greater than 90% sequence identity with a region within the 3'-untranslated cDNA of human PAI-1, and two 70-bp stretches of highly T-rich sequence located close to the 3'-terminus of the cDNA.     

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.     

Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma

A novel plasminogen-binding protein has been isolated from human plasma utilizing plasminogen-Sepharose affinity chromatography. This protein copurified with alpha 2 antiplasmin when the plasminogen affinity column was eluted with high concentrations of epsilon-aminocaproic acid (greater than 20 mM). Analysis by sodium dodecyl sulfate suggests this protein has an apparent Mr of 60,000. The amino-terminal amino acid sequence showed no similarity to other protein sequences. Based on the amino-terminal amino acid sequence, oligonucleotide probes were designed for polymerase chain reaction primers, and an approximately 1,800 base pair cDNA was isolated that encodes this Mr 60,000 protein. The deduced amino acid sequence reveals a primary translation product of 423 amino acids that is very similar to carboxypeptidase A and B and consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain. This protein shows 44 and 40% similarity to rat procarboxypeptidase B and human mast cell procarboxypeptidase A, respectively. The residues critical for catalysis and zinc and substrate binding of carboxypeptidase A and B are conserved in the Mr 60,000 plasminogen-binding protein. The presence of aspartic acid at position 257 of the catalytic domain suggests that this protein is a basic carboxypeptidase. When activated by trypsin, it hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates, and it is inhibited by the specific carboxypeptidase B inhibitor (DL-5-guanidinoethyl)mercaptosuccinic acid. We propose that the Mr 60,000 plasminogen-binding protein isolated here is a novel human plasma carboxypeptidase B and that it be designated pCPB.     

Cloning, sequencing and functional expression of a cDNA encoding porcine pancreatic preprocarboxypeptidase A1.

A full-length cDNA clone coding for porcine pancreatic preprocarboxypeptidase A1 (prePCPA1) was isolated from a cDNA library. The open reading frame (ORF) of the nucleotide sequence was 1260 nt in length and encoded a protein of 419 amino acids (aa). The cDNA included a short signal peptide of 16 aa and a 94 aa-long activation segment. The calculated molecular mass of the mature proenzyme was 45561 Da, in accordance with that of the purified porcine pancreatic PCPA1. The deduced aa sequence of the corresponding enzyme differed from that predicted by the three-dimensional structure by 40 aa, and showed 85% identity and 55% identity to that of procarboxypeptidases A1 and A2, respectively. Moreover the sequence was identical to that of several independent cDNA clones, suggesting that it is the major transcribed gene. No evidence for a second variant was observed in the cDNA library and PCPA2 is apparently absent from the porcine pancreas. The cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast triose phosphate isomerase promoter. The signal peptide of the PCPA protein efficiently directed its secretion into the culture medium (1.5 mg.L-1) as a protein of the predicted size. The recombinant proenzyme was analyzed by immunological and enzymological methods. Its activation behavior was comparable with that of the native form and led to a 35-kDa active enzyme.     

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.     

Structure of a precursor to human pancreatic polypeptide

We have isolated mRNA from a human pancreatic islet cell tumor and have identified among the cell-free translation products a precursor of pancreatic polypeptide with an approximate Mr = 11,000. Recombinant DNA molecules encoding this precursor were selected from a cDNA library prepared from the islet tumor mRNA. From the nucleotide sequences of cDNAs encoding the precursor, we have deduced the complete amino acid sequence of pre-propancreatic polypeptide. These sequences encode a protein consisting of 95 amino acid residues with a Mr = 10,432. The sequence of human pancreatic polypeptide occurs in the middle of the precursor and is flanked at its carboxyl terminus by a 27-amino acid sequence which is similar to a peptide previously isolated from canine pancreatic islets. At the amino terminus of the precursor is a probable leader sequence which is rich in hydrophobic residues. A smaller pancreatic polypeptide-related protein was generated in cell-free translations of mRNA supplemented with microsomal membranes. Sequential Edman degradations of this smaller peptide indicate that the sequence of pancreatic polypeptide is located at the amino terminus of the prohormone.     

Isolation and molecular cloning of mast cell carboxypeptidase A. A novel member of the carboxypeptidase gene family

Mast cell carboxypeptidase A has been isolated from the secretory granules of mouse peritoneal connective tissue mast cells (CTMC) and from a mouse Kirsten sarcoma virus-immortalized mast cell line (KiSV-MC), and a cDNA that encodes this exopeptidase has been cloned from a KiSV-MC-derived cDNA library. KiSV-MC-derived mast cell carboxypeptidase A was purified with a potato-derived carboxypeptidase-inhibitor affinity column and was found by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be a Mr 36,000 protein. Secretory granule proteins from KiSV-MC and from mouse peritoneal CTMC were then resolved by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transblotted to polyvinylidine difluoride membranes. Identical aminoterminal amino acid sequences were obtained for the prominent Mr 36,000 protein present in the granules of both cell types. Based on the amino-terminal sequence, an oligonucleotide probe was synthesized and used to isolate a 1,470-base pair cDNA that encodes this mouse exopeptidase. The deduced amino acid sequence revealed that, after cleavage of a 15-amino acid hydrophobic signal peptide and a 94-amino acid activation peptide from a 417-amino acid preproenzyme, the mature mast cell carboxypeptidase A protein core has a predicted Mr of 35,780 and a high positive charge [Lys + Arg) - (Asp + Glu) = 17) at neutral pH. Although critical zinc-binding amino acids (His67, Glu70, His195), substrate-binding amino acids (Arg69, Asn142, Arg143, Tyr197, Asp255, Phe278), and cysteine residues that participate in intrachain disulfide bonds (Cys64-Cys77, Cys136-Cys159) of pancreatic carboxypeptidases were also present in mast cell carboxypeptidase A, the overall amino acid sequence identities for mouse mast cell carboxypeptidase A relative to rat pancreatic carboxypeptidases A1, A2, and B were only 43, 41, and 53%, respectively. RNA and DNA blot analyses revealed that mouse peritoneal CTMC, KiSV-MC, and bone marrow-derived mast cells all express a prominent 1.5-kilobase mast cell carboxypeptidase A mRNA which is transcribed from a single gene. We conclude that mouse mast cell carboxypeptidase A is a prominent secretory granule enzyme of mast cells of the CTMC subclass and represents a novel addition to the carboxypeptidase gene family.     

Mosaic evolution of prepropancreatic polypeptide

Pancreatic polypeptide, a 36-amino acid peptide hormone, is synthesized in pancreatic islets of Langerhans and acts as a regulator of pancreatic and gastrointestinal functions. We isolated cDNA clones encoding rat pancreatic polypeptide precursor from an islet cDNA library and determined their nucleic acid sequences. Rat pancreatic polypeptide was found to be flanked on the amino terminus by a putative signal peptide and on the carboxyl terminus by Gly-Lys-Arg followed by a 30-amino acid peptide. Nucleotide and amino acid sequences of the signal peptide and the pancreatic polypeptide of the rat were highly homologous to those of the human (Boel, E., Schwartz, T. W., Norris, K. E., and Fill, N. P. (1984) EMBO J. 3, 909-912). On the other hand, the rat carboxyl-terminal peptide differed markedly from the corresponding domain of the human precursor and did not contain any sequence similar to the icosapeptide, which has so far been known to be a second stable product from mammalian pancreatic polypeptide precursors (Schwartz, T. W., Hansen, H. F., Hakanson, R., Sundler, F., and Tager, H. S. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 708-712). The mosaicism of sequence conservation and divergence in prepropancreatic polypeptides may be a unique example in the evolution of prohormones.     

A collagen-binding protein in the endoplasmic reticulum of myoblasts exhibits relationship with serine protease inhibitors.

Several cDNA clones encoding a 46-kDa collagen-binding glycoprotein (gp46) from rat skeletal myoblasts were isolated and sequenced. The cDNA encoded a 17-amino acid signal peptide and a 400-amino acid mature protein, containing three potential N-linked oligosaccharide attachment sites. The cDNA sequence of gp46 shows 93% identity in the coding region with J6, a retinoic acid-inducible gene coding for a protein of unknown function described from embryonal carcinoma F9 cells. The first 41 NH2-terminal amino acids of the predicted J6 sequence are, however, different from the gp46 sequence as a result of a 7-base pair insertion in the gp46 cDNA. In addition, the NH2-terminal amino acid sequence of hsp47, a collagen-binding protein found in chick embryo fibroblasts, shows 64% identity to gp46 over 36 residues. Interestingly, this alignment begins 10 residues inward from the first amino acid in the mature form of gp46. A significant sequence similarity was observed between gp46 and members of the serine protease inhibitor (serpin) family. Unlike other serpins, however, gp46 is both a heat shock and a collagen-binding protein and is localized to the lumen of the endoplasmic reticulum, as suggested by the presence of the RDEL sequence at the COOH terminus. This sequence is similar to other proposed endoplasmic reticulum retention signals.     

Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily

A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.