Human cathepsin F. Molecular cloning, functional expression, tissue localization, and enzymatic characterization

A cDNA for a novel human papain-like cysteine protease, designated cathepsin F, has been cloned from a lambdagt10-skeletal muscle cDNA library. The nucleotide sequence encoded a polypeptide of 302 amino acids composed of an 88-residue propeptide and a 214-residue mature protein. Protein sequence comparisons revealed 58% homology with cathepsin W; about 42-43% with cathepsins L, K, S, H, and O; and 38% with cathepsin B. Sequence comparisons of the propeptides indicated that cathepsin F and cathepsin W may form a new cathepsin subgroup. Northern blot analysis showed high expression levels in heart, skeletal muscle, brain, testis, and ovary; moderate levels in prostate, placenta, liver, and colon; and no detectable expression in peripheral leukocytes and thymus. The precursor polypeptide of human recombinant cathepsin F, produced in Pichia pastoris, was processed to its active mature form autocatalytically or by incubation with pepsin. Mature cathepsin F was highly active with comparable specific activities toward synthetic substrates as reported for cathepsin L. The protease had a broad pH optimum between 5.2 and 6.8. Similar to cathepsin L, its pH stability at cytosolic pH (7.2) was short, with a half-life of approximately 2 min. This may suggest a function in an acidic cellular compartment. Transient expression of T7-tagged cathepsin F in COS-7 cells revealed a vesicular distribution of the gene product in the juxtanuclear region of the cells. However, contrary to all known cathepsins, the open reading frame of the cathepsin F cDNA did not encode a signal sequence, thus suggesting that the protease is targeted to the lysosomal compartment via an N-terminal signal peptide-independent lysosomal targeting pathway.     

Molecular cloning and expression of rabbit pancreatic cholesterol esterase

Rabbit pancreatic cholesterol esterase (CEase, carboxyl ester lipase, EC 3.1.1.3) has been cloned from a lambda gt11 library of adult rabbit pancreatic cDNA. The open reading frame consists of 1788 nucleotides which encodes 576 amino acids of the functional protein and a 20 amino acid leader peptide. When compared to other species, the greatest homology is observed between residues 82-248 with little or no homology at the C-terminal end where proline-glutamate-serine-threonine (PEST) segments are a characteristic feature of the human CEase. Rabbit CEase (RCEase) retains the active-site serine (gxsxg), the active-site histidine and the tentative heparin binding site (KKRCLQ) at similar positions in comparison to pancreatic CEases of other species. When rabbit CEase cDNA is expressed in monkey kidney (COS-7) cells, enzymatic hydrolytic activity is detected in the growth medium as is a 67 kDa protein by Western blotting with polyclonal anti-CEase antibody. Northern blot analysis shows two mRNA (2.2 and 3.2 kb) species.     

Molecular cloning, expression, and functional characterization of novel mouse sulfotransferases

STUDY OBJECTIVE: The present study was performed to determine the influence of a perioperative myocardial infarction on long-term mortality in patients who have undergone elective vascular surgery. STUDY DESIGN: This was a 4-year follow-up of patients who had undergone elective vascular procedures at a Veterans Affairs Medical Center. Between January 1989 and December 1990, 115 consecutive patients underwent surgery for either an expanding abdominal aortic aneurysm (AAA) (38%) or for pain in the lower extremities (62%). RESULTS: Vital status at 4 years postsurgery was determined for all patients. Thirty-day postoperative mortality was 3%, while estimates at 1, 2, 3, and 4 years were 19%, 26%, 35%, and 39%, respectively. Of the 45 patients who died within 4 years following surgery, the major causes of death were cardiac (40%), cancer (18%), cerebrovascular (13%), and peripheral vascular disease (11%). Univariate predictors of 1-year mortality on preoperative evaluation were an abnormal ECG, moderate or greater sized exercise thallium defect and left ventricular ejection fraction < or =40%, and a perioperative myocardial infarction. Univariate predictors of 4-year mortality were non-AAA surgery and diabetes mellitus. Perioperative myocardial infarction was a marginally significant independent predictor of 1-year mortality (p=0.06), while the need for non-AAA surgery was a strong independent predictor at 4 years. CONCLUSIONS: Cardiac mortality is the major cause of late death among patients undergoing elective vascular surgery. Although preoperative indicators of symptomatic coronary artery disease and nonfatal perioperative myocardial infarction identified those individuals at increased mortality in the first postoperative year, the extent of vascular disease at presentation may be a more important determinant of long-term survival. A randomized trial in such patients is needed to assess the best strategy for treating patients with coexistent coronary artery and vascular diseases.     

Molecular cloning, heterologous expression, and characterization of human glyoxalase II

A clone encoding glyoxalase II has been isolated from a human adult liver cDNA library. The sequence of 1011 base pairs consists of a full-length coding region of 780 base pairs, corresponding to a protein with a calculated molecular mass of 28,861 daltons. Identities (50-60%) were found to partial 5' and 3' cDNA sequences from Arabidopsis thaliana as well as within a limited region of glutathione transferase I cDNA from corn. A vector was constructed for heterologous expression of glyoxalase II in Escherichia coli. For optimal yield of enzyme, silent random mutations were introduced in the 5' coding region of the cDNA. A yield of 25 mg of glyoxalase II per liter of culture medium was obtained after affinity purification with immobilized glutathione. The recombinant enzyme had full catalytic activity and kinetic parameters indistinguishable from those of the native enzyme purified from human erythrocytes.     

Molecular cloning, expression and enzymatic activity of a thioredoxin peroxidase from Dirofilaria immitis

A Dirofilaria immitis cDNA clone encoding a nucleic acid homolog of thioredoxin peroxidase (nDiTPx) was isolated from a fourth-stage larval cDNA library, using serum from dogs vaccinated by chemotherapeutically-abbreviated D. immitis larval infections. The protein encoded by nDiTPx had a predicted molecular mass of 22.1 kDa and the deduced amino acid sequence was homologous to thioredoxin peroxidase-like sequences described in other filarial nematodes, yeast, bacteria and mammals. As is true for other members of this peroxiredoxin family, the nDiTPx-encoded protein had the conserved cysteine near the amino terminus, considered to be essential for enzyme activity. nDiTPx was expressed in E. coli and the resulting recombinant fusion protein was shown to have thioredoxin peroxidase (TPx) activity, by its ability to protect DNA from oxidative-nicking in a metal-catalyzed oxidation system. A polyclonal antibody to the DiTPx fusion protein detected a 22-kDa native protein in D. immitis larval and adult parasite extracts.     

Molecular cloning and expression of human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein sulfotransferase homologue in Caenorhabditis elegans

Tyrosine O-sulfation, a common post-translational modification in eukaryotes, is mediated by Golgi enzymes that catalyze the transfer of the sulfuryl group from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues in polypeptides. We recently isolated cDNAs encoding human and mouse tyrosylprotein sulfotransferase-1 (Ouyang, Y. B., Lane, W. S., and Moore, K. L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 2896-2901). Here we report the isolation of cDNAs encoding a second tyrosylprotein sulfotransferase (TPST), designated TPST-2. The human and mouse TPST-2 cDNAs predict type II transmembrane proteins of 377 and 376 amino acid residues, respectively. The cDNAs encode functional N-glycosylated enzymes when expressed in mammalian cells. In addition, preliminary analysis indicates that TPST-1 and TPST-2 have distinct specificities toward peptide substrates. The human TPST-2 gene is on chromosome 22q12.1, and the mouse gene is in the central region of chromosome 5. We have also identified a cDNA that encodes a TPST in the nematode Caenorhabditis elegans that maps to the right arm of chromosome III. Thus, we have identified two new members of a class of membrane-bound sulfotransferases that catalyze tyrosine O-sulfation. These enzymes may catalyze tyrosine O-sulfation of a variety of protein substrates involved in diverse physiologic functions.     

Molecular cloning of three sulfotransferase cDNAs from mouse liver

Sulfate conjugation plays an important role in the biotransformation of not only xenobiotics but also many endogenous substances. Sulfotransferases, the enzymes that are responsible for this process, exist as a superfamily of genes. It has long been recognized that significant species differences exist among drug and carcinogen metabolizing enzymes such as cytochrome P450. Species differences in both regulation and catalytic activities of sulfotransferases may also exist. To investigate this, we conducted cDNA cloning and cDNA expression studies of sulfotransferase in the mouse. Three sulfotransferase cDNA clones were isolated from a female B6CBA mouse liver. Two of the clones, mSTa1 and mSTa2, were highly homologous to each other. Alignment of mSTa1 and mSTa2 cDNAs' nucleotide sequences with those of other sulfotransferase cDNAs revealed the greatest sequence identity with the rat STsmp cDNA. This analysis suggests that mSTa1, mSTa2 and rSTsmp cDNAs are derived from orthologous genes belonging to the alcohol/hydroxysteroid sulfotransferase gene family. The third clone, mSTp1 showed high identity to rSTp, hSTp1, hSTp3, and rSTp1C1, suggesting that mSTp1 belongs to the phenol family.     

Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor

Previous studies have shown that tissue-factor-pathway inhibitor (TFPI) is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa and factor VIIa-tissue factor activity. We describe the molecular cloning and expression of a full-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino acid sequence homology to TFPI. After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines and two canonical N-linked glycosylation sites. The deduced sequence of mature TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxyl-terminal tail highly enriched in basic amino acids. Northern analysis indicates that TFPI-2 is transcribed in umbilical vein endothelial cells, liver, and placenta. TFPI-2 was expressed in baby hamster kidney cells and purified from the serum-free conditioned medium by a combination of heparin-agarose chromatography, Mono Q FPLC, Mono S FPLC, and Superose 12 FPLC. Purified TFPI-2 migrated as a single band in SDS/PAGE and exhibited a molecular mass of 32 kDa in the presence and absence of reducing agent. The amino-terminal sequence of recombinant TFPI-2 was identical to that predicted from the cDNA. Despite its structural similarity to TFPI, the purified recombinant TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary studies indicated that purified recombinant TFPI-2 strongly inhibited the amidolytic activities of trypsin and the factor VIIa-tissue factor complex. In addition, the inhibition of factor VIIa-tissue factor amidolytic activity by recombinant TFPI-2 was markedly enhanced in the presence of heparin. TFPI-2 at high concentrations weakly inhibited the amidolytic activity of human factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.     

Extremely thermostable serine-type protease from Aquifex pyrophilus. Molecular cloning, expression, and characterization

A gene encoding a serine-type protease has been cloned from Aquifex pyrophilus using a sequence tag containing the consensus sequence of proteases as a probe. Sequence analysis of the cloned gene reveals an open reading frame of 619 residues that has three canonical residues (Asp-140, His-184, and Ser-502) that form the catalytic site of serine-type proteases. The size of the mature form (43 kDa) and its localization in the cell wall fraction indicate that both the NH2- and COOH-terminal sequences of the protein are processed during maturation. When the cloned gene is expressed in Escherichia coli, it is weakly expressed as active and processed forms. The pH optimum of this protease is very broad, and its activity is completely inactivated by phenylmethylsulfonyl fluoride. The half-life of the protein is 6 h at 105 degreesC, suggesting that it is one of the most heat-stable proteases. The cysteine residues in the mature form may form disulfide bonds that are responsible for the strong stability of this protease, because the thermostability of the protein is significantly reduced in the presence of reducing reagent.     

Molecular cloning, expression and characterization of an Ascaris inhibitor for pepsin and cathepsin E

Molecular cloning of a cDNA for a pepsin inhibitor in the round worm, Ascaris suum, was achieved. The ORF was found to encode a 20-residue potential signal peptide and a 149-residue inhibitor moiety. Northern analysis showed the mRNA for the inhibitor to be expressed in the body wall and not in the viscera. To obtain the active inhibitor, we constructed a yeast expression vector, pYES2API, containing the inducible galactosidase promoter and a DNA fragment encoding a fusion protein of the yeast alpha-factor leader and the Ascaris pepsin inhibitor. The active inhibitor was secreted in the culture medium, the yield being approximately 3 mg x l(-1) x day(-1), and purified by a two-step procedure that included HPLC. The inhibitor inactivated pepsin A and cathepsin E almost completely at amounts equimolar with the enzymes, but was 100-fold less effective against pepsin C and did not act on cathepsin D and renin. Ki values for the inhibition of pepsin A and cathepsin E were in the nanomolar range below pH 5. Since the inhibitor activity was lost by modification of specific Lys residues, including Lys110, an electrostatic interaction between these Lys residues and Asp/Glu residues of pepsin A or cathepsin E was thought to be essential for the inhibition.     

cDNA cloning and expression of a new form of human aryl sulfotransferase

Twenty-eight patients (with 30 primary and 8 revision total hip arthroplasties) completed a standardized questionnaire containing the Western Ontario and McMaster Universities (WOMAC) osteoarthritis index and Harris hip score questions prior to an office visit a minimum of 1 year after surgery. The range of hip motion measured by an orthopaedic surgeon was compared with the responses to questions on stiffness and function as well as with global scores in the WOMAC osteoarthritis index. Patient responses to the questions asking if they could cut their toenails on the operated side and the Harris hip score question asking if they could put on socks and tie a shoe correlated significantly with postoperative hip motion (P < .005). The WOMAC global pain and stiffness scores did not correlate with range of motion. The WOMAC physical function score correlated significantly only with hip flexion (P < .05). Of the WOMAC physical function questions, difficulty bending to pick an object off the floor (P < .05) and getting on and off the toilet (P < .05) correlated with the sum of the range of motion in all planes and weighted Harris hip score range of motion calculation. These data suggest that the points allocated in the Harris hip score for range of motion can be estimated reasonably accurately from questionnaire or phone response to a series of questions on a standardized questionnaire. The question on ability to cut toenails or the Harris hip score question regarding ability to put on socks and tie a shoe correlated with the most individual planes of motion, but several WOMAC physical function questions also correlated with total and weighted range of motion calculations.     

Molecular cloning, expression, and characterization of the authentic hyaluronan synthase from group C Streptococcus equisimilis

We previously reported the first cloning of a functional glycosaminoglycan synthase, the hyaluronan synthase (HAS) from Group A Streptococcus pyogenes (spHAS) (DeAngelis, P. L., Papaconstantinou, J., and Weigel, P. H. (1993) J. Biol. Chem. 268, 19181-19184). Group A spHAS was unrelated to a putative Group C HA synthase reported by others (Lansing, M., Lellig, S., Mausolf, A., Martini, I. , Crescenzi, F., Oregon, M., and Prehm, P. (1993) Biochem. J. 289, 179-184). Here we report the isolation of a bona fide HA synthase gene from a highly encapsulated strain of Group C Streptococcus equisimilis. The encoded protein, designated seHAS, is 417 amino acids long (calculated molecular weight, 47,778; calculated pI, 9.1) and is the smallest member of the HAS family identified thus far. The enzyme migrates anomalously fast in SDS-polyacrylamide gel electrophoresis (approximately 42,000 Da). The seHAS protein shows no similarity (<2% identity) to the previously reported Group C gene, which is not an HA synthase. The seHAS and spHAS protein and coding sequences are 72 and 70% identical, respectively. seHAS is also similar to eukaryotic HAS1 (approximately 31% identical), HAS2 (approximately 28% identical), and HAS3 (28% identical). The deduced protein sequence of seHAS was confirmed by reactivity with a synthetic peptide antibody. Recombinant seHAS expressed in Escherichia coli was recovered in membranes as a major protein (approximately 10% of the total protein) and synthesized very large HA (Mr >7 x 10(6)) in the presence of UDP-GlcNAc and UDP-GlcA. The product contained equimolar amounts of both sugars and was degraded by the specific Streptomyces hyaluronidase. Comparison of the two recombinant streptococcal enzymes in isolated membranes showed that seHAS and spHAS are essentially identical in the steady-state size distribution of HA chains they synthesize, but seHAS has an intrinsic 2-fold faster rate of chain elongation (Vmax) than spHAS. seHAS is the most active HA synthase identified thus far; it polymerizes HA at an average rate of 160 monosaccharides/s. The two bacterial HA synthase genes may have arisen from a common ancient gene shared with the early evolving vertebrates.     

Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone

The heavy-chain and kappa light-chain variable region genes of an antizearalenone hybridoma cell line (2G3-6E3-2E2) were isolated by PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a single-chain Fv (scFv) DNA fragment. The scFv DNA fragment was cloned into a phagemid (pCANTAB5E) and expressed as a fusion protein with E tag and phage M13 p3 in Escherichia coli TG1. In the presence of helper phage M13K07, the scFv fusion protein was displayed on the surfaces of recombinant phages. High-affinity scFv phages were enriched through affinity selection in microtiter wells coated with zearalenone-ovalbumin conjugate. The selected recombinant phages were used to infect E. coli HB2151 for the production of soluble scFv antibodies. One selected clone (pQY1.5) in HB2151 secreted a soluble scFv antibody (QY1.5) with a high zearalenone-binding affinity (concentration required for 50% inhibition of binding, 14 ng/ml), similar to that of parent monoclonal antibody in a competitive indirect enzyme-linked immunosorbent assay. However, scFv QY1.5 exhibited higher cross-reactivity with zearalenone analogs and had greater sensitivity to methanol destabilization than the parent monoclonal antibody did. Nucleotide sequence analyses revealed that the light-chain portion of scFv QY1.5 had a nucleotide sequence identity of 97% to a mouse germ line gene VK23.32 in mouse kappa light-chain variable region subgroup V, whereas the heavy-chain nucleotide sequence was classified as mouse heavy-chain subgroup III (D) but without any closely related members having highly homologous complementarity-determining region sequences. The potential of soluble scFv QY1.5 for routine screening of zearalenone and its analogs was demonstrated with zearalenone-spiked corn extracts.     

Immunocytochemical localization and biological activity of hydroxysteroid sulfotransferase in the frog brain

Biosynthesis of the neuroactive steroids pregnenolone sulfate (delta5PS) and dehydroepiandrosterone sulfate (DHEAS) is catalyzed by the enzyme hydroxysteroid sulfotransferase (HST), which transfers the sulfonate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) on the 3-hydroxy site of steroids. Although high concentrations of delta5PS and DHEAS have been detected in the rat brain, the anatomical localization of HST in the CNS has never been determined. Using an antiserum against rat liver HST, we have investigated the distribution of HST-like immunoreactivity in the CNS of the frog Rana ridibunda. Two populations of HST-immunoreactive neurons were observed in the hypothalamus, and several bundles of positive nerve fibers were visualized in the telencephalon and diencephalon. Incubation of frog brain homogenates with [35S]PAPS and [3H]pregnenolone yielded the formation of several 3H,35S-labeled compounds, including delta5PS and testosterone sulfate. When [3H]dehydroepiandrosterone and [35S]PAPS were used as precursors, one of the 3H,35S-labeled metabolites coeluted with DHEAS. Neosynthesis of [3H]delta5PS and [3H]DHEAS was reduced significantly by 2,4-dichloro-6-nitrophenol, a specific inhibitor of sulfotransferases. The present study provides the first immunocytochemical mapping of HST in the brain. Our data also demonstrate for the first time that biosynthesis of the highly potent neuroactive steroids delta5PS and DHEAS occurs in the CNS of nonmammalian vertebrates.     

Purification and characterization of two amine N-sulfotransferases, AST-RB1 (ST3A1) and AST-RB2 (ST2A8), from liver cytosols of male rabbits

Two sulfotransferases (STs), designated as AST-RB1 (ST3A1) and AST-RB2 (ST2A8), with high a amine N-sulfonating activity, were purified from male rabbit liver cytosols. AST-RB1 and AST-RB2 were purified to homogeneity by the anion-exchange, affinity, and hydroxyapatite chromatography. The N-terminus of both enzymes were blocked. The subunit molecular mass of both enzymes was estimated to be 34 kDa on SDS-PAGE. AST-RB1 efficiently catalyzed N-sulfonation of alicyclic, alkyl, and arylamines such as 4-phenyl-1,2,3, 6-tetrahydropyridine, 1-[(5-chloro-2-oxo-3(2H)-benzothiazolyl)acetyl]-piperazine, desipramine, and aniline, whereas its catalytic activities toward 2-naphthol and dehydroepiandrosterone (DHEA) were very low. On the other hand, AST-RB2 efficiently catalyzed sulfonation of desipramine and DHEA, but had no activity toward 2-naphthol. Amino acid sequences of peptide fragments derived from the purified AST-RB1 showed no significant homology with previously reported STs, but those from the purified AST-RB2 shared a high similarity with those of the ST2 family. Both enzymes were expressed specifically in the liver. The present results strongly suggest that the purified AST-RB1 is a novel enzyme in terms of structure and catalytic properties showing high selectivity for amine substrates, and AST-RB2 is a quite unique from among ST2A enzymes of other species in its substrate specificity.     

Molecular cloning and characterization of rat lin-10

In Caenorhabditis elegans, the vulval induction is mediated by tyrosine kinase receptor/Ras signal transduction pathway composed of the lin-3, let-23, and let-60 products. In addition to these gene products, the lin-2, lin-7, and lin-10 products are also implicated in this pathway. Lin-2 encodes a MAGUK and lin-7 encodes a small protein with one PDZ domain. The lin-10 product has no homology to known proteins. Here, we have cloned a rat homologue of the lin-10 product and characterized it. Rat lin-10 is ubiquitously expressed in various rat tissues and distributed in both the cytosol and membrane fractions. In brain, however, rat lin-10 is distributed only in the membrane fraction and enriched in the synaptic plasma membrane and postsynaptic density fractions. These results suggest that rat lin-10 is involved at least in synaptic functions in brain.     

Molecular cloning and characterization of Borrelia burgdorferi rpoB

To examine the association of vascular endothelial growth factor (VEGF) expression with tumor angiogenesis, survival and thymidine phosphorylase/platelet-derived endothelial cell growth factor (dThdPase/PD-ECGF) expression in human colorectal cancer, immunohistochemical studies were performed on 136 cases of resected colorectal cancer specimens using antibodies for VEGF, KDR, CD34 and dThdPase/PD-ECGF. Fifty-nine cases (43%) were evaluated as positive for VEGF staining and 71 cases (52%) were evaluated as positive for dThdPase/PD-ECGF staining. The expression of VEGF correlated significantly with vessel counts and the expression of dThdPase/PD-ECGF (P = 0.01 and 0.01, respectively). Cox proportional hazards model analysis showed that vessel counts and VEGF expression were significant and independent prognostic factors, but that KDR expression was not.