Molecular cloning and characterization of LR3, a novel LDL receptor family protein with mitogenic activity

Mice with hemophilia B have been engineered using gene targeting techniques. These animals exhibit severe factor IX deficiency and a clinical phenotype that mirrors the human disease. We have bred the founder animals onto two different strains of mice, C57B1/6 and CD-1, and have sought to determine whether adenoviral vectors expressing human factor IX could correct the bleeding diathesis of mice with hemophilia B. Initial experiments showed that purified plasma-derived human factor IX added to murine factor IX-deficient plasma resulted in complete correction of the activated partial thromboplastin time (aPTT), and that injection of 10(11) particles of an adenoviral vector expressing human factor IX resulted in normalization of a modified aPTT in mouse plasma. As an additional method of assessing the function of human factor IX in the murine coagulation system, bleeding times were performed in normal, hemophilic, and adenoviral-treated hemophilic mice. By two different bleeding-time techniques, the treated hemophilic mice gave values identical to normal littermate controls, whereas the untreated hemophilic mice exhibited heavy blood loss and prolonged bleeding. There was a marked difference in antibody formation in the two strains of mice; 100% of the hemophilic CD-1 mice formed antibodies to human factor IX, but none of the C57B1/6 mice did. These data suggest that the C57B1/6 hemophilic mice will be more useful for gene transfer studies, while the CD-1 hemophilic mice may be of greater utility in studying the development of inhibitors.     

Molecular cloning and characterization of a highly conserved chicken cellular nucleic acid binding protein cDNA

Three variants of the Candida antarctica B lipase have been constructed and characterized. The variant containing the T103G mutation, which introduces the consensus sequence G-X-S-X-G found in most other known lipases, shows an increased thermostability but retains only half the specific activity of the native enzyme. Also in ester synthesis the activity is lowered but the specificity and enantioselectivity remains unchanged. The W104H mutant, in which more space is introduced into the active site, has more dramatically changed properties. Both the thermostability and the specific activity are slightly reduced but the activity and specificity in ester synthesis is highly different from the native enzyme. In general, the activity is very low and the enantioselectivity is, furthermore, highly reduced. Finally, the mutation M72L was introduced to increase the oxidation stability of the enzyme. This variant did exhibit an increased resistance towards oxidation but the thermostability was, unfortunately, also reduced.     

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.     

Molecular cloning and biochemical characterization of bovine spleen myristoyl CoA:protein N-myristoyltransferase

Myristoyl-CoA:protein N-myristoyltransferase (NMT) is an essential eukaryotic enzyme that catalyzes the cotranslational transfer of myristate to the NH2-terminal glycine residue of a number of important proteins of diverse function. We have isolated full-length cDNA encoding bovine spleen NMT (sNMT). The single long open reading frame of 1248 bp of sNMT specifies a protein of 416 amino acids with a predicted mass of 46,686 Da. The protein coding sequence was expressed in Escherichia coli resulting in the production of functionally active 50-kDa NMT. Deletion mutagenesis showed that the C-terminus is essential for activity whereas up to 52 amino acids can be deleted from the N-terminus without affecting the function. One of the N-terminal deletions resulted in threefold higher NMT activity. Genomic Southern analysis indicated the presence of two strong hybridizing bands with three different restriction enzyme digests suggesting the possibility of two copies of the NMT gene in the bovine genome. RNA blot hybridization analysis of total cellular RNA prepared from bovine brain, heart, spleen, lung, liver, kidney, and skeletal muscle probed with bovine sNMT cDNA revealed a single 1.7-kb mRNA. Western blot analysis of various bovine tissues with human NMT peptide antibody indicated a common prominent immunoreactive band with an apparent molecular mass of 48.5-50 kDa in all tissues. Additional immunoreactive bands were observed in brain (84 and 50 kDa), lung (58 kDa), and skeletal muscle (58 kDa). Activity measurements demonstrated that brain contained the highest NMT activity followed by spleen, lung, kidney, heart, skeletal muscle, pancreas, and liver. It appears therefore that mRNA and protein expression do not correlate with NMT activity, suggesting the presence of regulators of the enzyme activity.     

Molecular cloning of the dnaK locus, and purification and characterization of a DnaK protein from Bacillus brevis HPD31

Using part of the dnaK gene from Bacillus subtilis as a probe, a 4. 4-kbp SacI-BglII fragment of chromosomal DNA of Bacillus brevis, a protein-hypersecreting bacterium, was cloned. Nucleotide sequencing revealed 3 open reading frames in the order of grpE-dnaK-dnaJ homologues. We purified DnaK protein to homogeneity from B. brevis HPD31 harboring a multi-copy dnaK expression plasmid. Purified DnaK showed ATPase activity which was synergistically stimulated 14-fold by the addition of glutathione S-transferase-DnaJ and glutathione S-transferase-GrpE fusion proteins. DnaK hydrolyzed not only ATP but also CTP, UTP, and GTP at about 40% of the efficiency of ATP. The specific activity of DnaK-ATPase was 7.25x10-3 unit/mg protein (the turnover number against ATP was 0.47 min-1) under our assay conditions. The DnaK dimers dissociated into monomers on addition of ATP, GTP, CTP, UTP and ATPgammaS, but not ADP or AMP. DnaK formed a stable complex with permanently unfolded carboxymethylated alpha-lactalbumin but not with native alpha-lactalbumin, and this complex was dissociated by addition of ATP/Mg. Formation of this complex was inhibited in the presence of inorganic phosphate.     

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 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.     

Purification, cloning, and preliminary characterization of a Spiroplasma citri ribosomal protein with DNA binding capacity

The rpsB-tsf-x operon of Spiroplasma citri encodes ribosomal protein S2 and elongation factor Ts, two components of the translational apparatus, and an unidentified X protein. A potential DNA-binding site (a 20-base pair (bp) inverted repeat sequence) is located at the 3' end of rpsB. Southwestern analysis of S. citri proteins, with a 30-bp double-stranded oligonucleotide probe (IRS), containing the 20-bp inverted repeat sequence and the genomic flanking sequences, detected an IRS-binding protein of 46 kDa (P46). P46 protein, which displays preferential affinity for the IRS, was purified from S. citri by a combination of affinity and gel filtration chromatographies. The native form of P46 seems to be homomultimeric as estimated by SDS-polyacrylamide gel electrophoresis analysis and gel filtration. A 3.5-kilobase pair S. citri DNA fragment comprising the P46 gene and flanking sequences was cloned and sequenced. Sequence analysis of this DNA fragment indicated that the P46 gene is located within the S10-spc operon of S. citri at the position of the gene coding for ribosomal protein L29 in the known S10-spc operons. The similarity between the N-terminal domain of P46 and the L29 ribosomal protein family and the presence of a 46-kDa IRS-binding protein in S. citri ribosomes indicated that P46 is the L29 ribosomal protein of S. citri. We suggest that P46 is a bifunctional protein with an L29 N-terminal domain and a C-terminal domain involved in IRS binding.     

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.     

Molecular cloning and characterization of the noncatalytic subunit of the Rab3 subfamily-specific GTPase-activating protein

We recently purified and characterized from rat brain a GTPase-activating protein (GAP) specific for the Rab3 small G protein subfamily implicated in Ca2+-dependent exocytosis. Rab3 GAP showed two bands with Mr of about 130,000 (p130) and 150,000 (p150) on SDS-polyacrylamide gel electrophoresis. p130, but not p150, showed the catalytic activity. Because p150 was likely the subunit of Rab3 GAP, here we cloned the cDNA of p150, determined its primary structure, and characterized it. The tissue and subcellular distribution patterns of p150 and p130 were similar, and both the proteins were enriched in the synaptic soluble fraction. p150 was co-immunoprecipitated with p130 from this fraction. Recombinant p150 formed a heterodimer with recombinant p130 as estimated by sucrose density gradient ultracentrifugation. Recombinant p150 neither showed the Rab3A GAP activity nor affected the activity of recombinant p130. When p150 and p130 were co-expressed in the cells, the subcellular localization of each protein did not change. These results indicate that p150 is the noncatalytic subunit of Rab3 GAP.     

Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta

Ca2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain VIII for maximal activity. Two distinct CaMKI/IV kinases, CaMKKalpha and CaMKKbeta, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996) J. Biol. Chem. 271, 10806-10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKKbeta, tissue and regional brain localization of CaMKKbeta protein, and mRNA and functional characterization of recombinant CaMKKbeta in vitro and in Jurkat T cells. The sequences of human and rat CaMKKbeta demonstrate 65% identity and 80% similarity with CaMKKalpha and 30-40% identity with CaMKI and CaMKIV themselves. CaMKKbeta is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKKbeta tracks more closely with CaMKIV than does CaMKKalpha. Bacterially expressed CaMKKbeta undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKKbeta activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKKbeta activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.     

Molecular cloning and characterization of a novel human classic cadherin homologous with mouse muscle cadherin

We used a novel cDNA cloning method based on the cadherin-beta-catenin protein interaction and identified a new human classic-type cadherin, which we named cadherin-15, from adult brain and skeletal muscle cDNA libraries. Sequence analysis revealed that this cadherin was closely related to mouse muscle cadherin and seemed to be its human counterpart. However, its deduced amino acid sequence differed from that of mouse muscle cadherin in that it had an extra 31-amino acid sequence at its C terminus that has been found neither in mouse muscle cadherin nor in any other known classic cadherin. Analysis of cadherin-15 protein expressed in L fibroblasts showed that it was cleaved proteolytically, expressed on the cell surfaces as a mature form of about 124-kDa, and functioned as a cell-cell adhesion molecule in a homophilic and specific manner, but Ca2+ did not protect it against degradation by trypsin. Our findings also suggest that cadherin-15 mediates cell-cell adhesion with a binding strength comparable to that of E-cadherin.     

Molecular cloning and characterization of a plasma membrane-associated sialidase specific for gangliosides

Gangliosides are plasma membrane components thought to play important roles in cell surface interactions, cell differentiation, and transmembrane signaling. A mammalian sialidase located in plasma membranes is unique in specifically hydrolyzing gangliosides, suggesting crucial roles in regulation of cell surface functions. Here we describe the cloning and expression of a cDNA for the ganglioside sialidase, isolated from a bovine brain cDNA library based on the amino acid sequence of the purified enzyme from bovine brain. This cDNA encodes a 428-amino acid protein containing a putative transmembrane domain and the three Asp boxes characteristic of sialidases and sharing 19-38% sequence identity with other sialidases. Northern blot and polymerase chain reaction analyses revealed a general distribution of the gene in mammalian species, including man, and the mouse. In COS-7 cells transiently expressing the sialidase, the activity was found to be 40-fold that of the control level with ganglioside substrates in the presence of Triton X-100, and the hydrolysis was almost specific to gangliosides other than GM1 and GM2, both alpha2-->3 and alpha2-->8 sialyl linkages being susceptible. The major subcellular localization of the expressed sialidase was assessed to be plasma membrane by Percoll density gradient centrifugation of cell homogenates and by immunofluorescence staining of the transfected COS-7 cells. Analysis of the membrane topology by protease protection assay suggested that this sialidase has a type I membrane orientation with its amino terminus facing to the extracytoplasmic side and lacking a signal sequence.     

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.     

Molecular cloning and functional characterization of murine sphingosine kinase

Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison with Saccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylated D-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide, D,L-threo-dihydrosphingosine or N, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger.