Molecular cloning of human thiamin pyrophosphokinase

Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) catalyses phosphorylation of thiamin to thiamin pyrophosphate, an active enzyme cofactor. Here we describe the cloning of complete human TPK1 cDNA from an adult liver library. Human TPK1 is 89% identical to murine TPK1 at the protein level. The gene maps to chromosome 7q34-36, consists of at least eight exons, and spans a distance at least of 420 kb. The mRNA of human TPK1 is highly expressed in testis, small intestine and kidney with lesser but detectable expression in brain, liver, placenta and spleen. The availability of the human TPK1 gene will provide another useful tool for studying the role of this enzyme in human thiamin metabolism and deficiency state.     

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.     

A new crystal form of mouse thiamin pyrophosphokinase

Thiamin pyrophosphokinase (TPK) transfers a pyrophosphate group from ATP to the hydroxyl group of thiamin and produces thiamin pyrophosphate (TPP). TPP is the cofactor of metabolically important enzymes such as pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-keto acid dehydrogenase, transketolase and 2-hydroxyphytanoyl-CoA lyase. Thiamin deficiency results in Wernike-Korsakof Syndrome (WKS) due to neurological disorder and wet beriberi, a potentially fatal cardiovascular disease. Mouse TPK associates as a dimer revealed by previous solved crystallographic structures. In this study, we report mouse TPK complexed with TPP-Mg²⁺ and thiamin -Mg²⁺, respectively, in a new crystal form. In these two structures, four mouse TPK molecules were found in each asymmetric unit. Although we cannot rule out this tetramer form can be an artifact from crystal packing, mouse TPK tetramer has a more closed ATP binding pocket and has the potential to provide specific interactions between mouse TPK and ATP compared with the previous dimeric structure and is likely to be an active form.     

Molecular cloning of mouse tumour necrosis factor cDNA and its eukaryotic expression.

Tumour necrosis factor (TNF), released by induced macrophages, causes tumour necrosis in animals and kills preferentially transformed cells in vitro. mRNA induced in the established mouse monocytic PU 5.1.8 cell line by lipopolysaccharide, was converted into double-stranded cDNA and cloned in the pAT153 vector. Recombinant plasmids were screened by plus-minus hybridization and TNF-specific oligonucleotide probes constructed on the basis of partial amino acid sequences of rabbit TNF. A series of TNF specific clones were identified and confirmed by hybrid selection of mouse TNF-specific mRNA. The sequence codes for a 235 amino acids long polypeptide, of which 156 amino acids presumably correspond to the mature product. It can be concluded that mature mouse TNF is a glycosylated dimer. Biologically active TNF was secreted by both Cos-I and CHO-cells transfected with the chimaeric expression vector pSV2d2-mTNF containing the coding region of the mouse TNF cDNA gene.     

Molecular cloning and expression of rat betacellulin cDNA

The cDNA encoding an entire open reading frame of rat betacellulin has been cloned from rat kidney. Expression of this cDNA in COS7 cells showed a significant amount of mitogenic activity in the culture media. Western blotting of the cell lysates suggested that the membrane-anchored precursor was cleaved to release its ectodomain very efficiently.     

Molecular cloning and expression of salmon prolactin cDNA

Prolactin was purified from chum salmon pituitaries. It was resolved into two variants by reverse-phase high-performance liquid chromatography. A cDNA library was prepared from Pacific chinook salmon pituitaries. Salmon prolactin gene was screened using a synthetic oligonucleotide based on partial protein sequence. A positive clone (PRL-10) was identified and sequenced. It is a full-size clone containing 1.1 kb and coding for a preprolactin of 211 amino acids. A modified prolactin plasmid (PRL-10A), in which the 5' untranslated sequence and the nucleotide sequence coding for the signal peptide of prolactin were deleted, was reconstructed into an expression vector using the heat-inducible lambda pL promotor. Mature prolactin, a single polypeptide of 22 kDa, was efficiently expressed in the bacteria at an elevated temperature.     

Crystal structure of thiamin pyrophosphokinase.

Thiamin pyrophosphate (TPP) is a coenzyme derived from vitamin B1 (thiamin). TPP synthesis in eukaryotes requires thiamin pyrophosphokinase (TPK), which catalyzes the transfer of a pyrophosphate group from ATP to thiamin. TPP is essential for central metabolic processes, including the formation of acetyl CoA from glucose and the Krebs cycle. Deficiencies in human thiamin metabolism result in beriberi and Wernicke encephalopathy. The crystal structure of mouse TPK was determined by multiwavelength anomalous diffraction at 2.4 A resolution, and the structure of TPK complexed with thiamin has been refined at 1.9 A resolution. The TPK polypeptide folds as an alpha/beta-domain and a beta-sandwich domain, which share a central ten-stranded mixed beta-sheet. TPK subunits associate as a dimer, and thiamin is bound in the dimer interface. Despite lacking apparent sequence homology with other proteins, the alpha/beta-domain resembles the Rossman fold and is similar to other kinase structures, including another pyrophosphokinase and a thiamin biosynthetic enzyme. Comparison of mouse and yeast TPK structures reveals differences that could be exploited in developing species-specific inhibitors of potential use as antimicrobial agents.     

Molecular cloning and expression of cDNA encoding a galactose/N-acetylgalactosamine-specific lectin on mouse tumoricidal macrophages.

We previously reported that the mouse macrophage galacose and N-acetylgalactosamine-specific lectin (MMGL) may participate in the binding of the macrophages to tumor cells [Oda, S., Sato, M., Toyoshima, S., & Osawa, T. (1989) J. Biochem. 105, 1040-1043]. We now report the cloning and characterization of a cDNA encoding MMGL. The MMGL gene encoded a protein consisting of 304 amino acid residues with a molecular weight of 34,595. The deduced amino acid sequence indicated that MMGL had a single membrane-spanning region, three leucine zipper-like domains, and a carbohydrate recognition domain. Two N-glycosylation sites were found in the extracellular region of MMGL, corresponding to the heavy N-glycosylation in the native MMGL. Comparison of the amino acid sequence of MMGL with those of rat hepatic lectins revealed a high overall sequence homology. The sequence homology was especially high in the putative membrane-spanning region and carbohydrate recognition domain. There was, however, a region of 25 amino acids which did not exist on hepatic lectins. The MMGL cDNA without the region encoding the putative membrane-spanning region and intracellular region was expressed in Escherichia coli. The expressed protein had galactose-binding activity and its sugar-binding specificity was same as that of the native lectin.     

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.     

Molecular cloning and expression of rat interleukin-1 alpha cDNA

A cDNA sequence coding for rat interleukin-1 alpha (IL-1 alpha) has been isolated from a cDNA library that was prepared with mRNA derived from LPS-stimulated rat peritoneal macrophages by using human IL-1 alpha cDNA as a probe. The rat cDNA encodes a 270 amino acid residue protein which is homologous (65%) to human IL-1 alpha. The rat cDNA sequence under SV40 early promoter directed the synthesis of biologically active IL-1 in monkey COS-1 cells. Rat IL-1 alpha mRNA is not expressed in spleen, lung, liver or brain, and is also not expressed in these organs of LPS-treated rat except spleen. This suggests that IL-1 alpha is not produced constitutively in various tissues and LPS is not sufficient to induce IL-1 alpha in most tissues. Our data indicate that the IL-1 activities which have been reported to be produced in the brain are not of alpha type. We have constructed a plasmid expressing the carboxy terminal 156 amino acids in Escherichia coli. Recombinant rat IL-1 alpha produced in COS cells or E. coli has cytotoxic activity against the human melanoma cell line A375S1 (GIF activity), which has been reported to be sensitive to human IL-1 alpha and IL-1 beta. This suggests that GIF activity is common to IL-1s derived from various sources.     

Molecular cloning and functional expression of cDNA encoding a mammalian inorganic pyrophosphatase.

Extracts of soluble proteins from bovine retina contain multiple species of inorganic pyrophosphatase (PPase) that can be resolved by hydroxylapatite or ion exchange chromatography. We have purified one of these isoforms by a combination of chromatography and electrophoresis under denaturing conditions and have partially sequenced four peptides generated from it by CNBr digestion. This sequence information was used to clone PPase cDNA from a retinal cDNA library. Of five cDNA inserts, three were 1.3 kilobase pairs in length and two of these contained a complete open reading frame that was 867 base pairs long and encoded a 289-amino acid protein of 33 kDa. The deduced amino acid sequence is 49.5% identical to that of PPase from Saccharomyces cerevisiae, and contains identical amino acid residues at all of the positions previously identified as essential for catalytic activity in that enzyme. When the bovine PPase cDNA was expressed in Escherichia coli, catalytically active PPase was produced that comigrated with bovine retinal PPase in a nondenaturing gel and was clearly distinguishable from the host PPase. Northern analysis of poly(A)+ RNA from human, canine, and bovine retinas revealed that each contained a single major band of 1.4 kilobases that hybridized strongly with a pyrophosphatase cDNA probe. Southern analysis of bovine genomic DNA was consistent with the existence of one PPase gene. Thus, the multiple forms separated by chromatography may be derived from a common precursor or from mRNAs of very similar size.     

Molecular cloning and expression of mouse brain sialidase.

Sialidase (EC 3.2.1.18) catalyzes the release of sialic acid from sialo-oligosaccharides, gangliosides, or sialo-glycoproteins. In this investigation, we cloned a novel cDNA for mouse brain sialidase and expressed the cDNA in COS-7 cells. This 1,699 bp cDNA codes for a 41.6 kDa protein consisting of 372 deduced amino acid residues. In COS-7 cells transiently transfected with the cDNA, a 250-fold increase was observed in specific activity toward 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid. Similarity searches of the nonredundant GenBank peptide sequence database by the PSI-BLAST program identified rat, hamster, human, and bacterial sialidases homologous to this mouse brain sialidase. Amino acid sequence identities to rat and hamster sialidases (84% and 77%, respectively) suggest that this form of sialidase is conserved in rodents. Sequence identities to human and mouse lysosomal sialidases (30% and 28%, respectively) indicate that the mouse brain sialidase is distinct from the lysosomal enzyme. Mouse brain sialidase has two amino acid sequence motifs common to bacterial sialidases: the 'F/YRIP' motif and the 'Asp-box' motif. The 'F/YRIP' motif is present near the N terminus while two 'Asp-box' motifs are present downstream.     

The crystal structure of yeast thiamin pyrophosphokinase.

BACKGROUND: Thiamin pyrophosphokinase (TPK) catalyzes the transfer of a pyrophosphate group from ATP to vitamin B1 (thiamin) to form the coenzyme thiamin pyrophosphate (TPP). Thus, TPK is important for the formation of a coenzyme required for central metabolic functions. TPK has no sequence homologs in the PDB and functions by an unknown mechanism. The TPK structure has been determined as a significant step toward elucidating its catalytic action. RESULTS: The crystal structure of Saccharomyces cerevisiae TPK complexed with thiamin has been determined at 1.8 A resolution. TPK is a homodimer, and each subunit consists of two domains. One domain resembles a Rossman fold with four alpha helices on each side of a 6 strand parallel beta sheet. The other domain has one 4 strand and one 6 strand antiparallel beta sheet, which form a flattened sandwich structure containing a jelly-roll topology. The active site is located in a cleft at the dimer interface and is formed from residues from domains of both subunits. The TPK dimer contains two compound active sites at the subunit interface. CONCLUSIONS: The structure of TPK with one substrate bound identifies the location of the thiamin binding site and probable catalytic residues. The structure also suggests a likely binding site for ATP. These findings are further supported by TPK sequence homologies. Although possessing no significant sequence homology with other pyrophospokinases, thiamin pyrophosphokinase may operate by a mechanism of pyrophosphoryl transfer similar to those described for pyrophosphokinases functioning in nucleotide biosynthesis.