Cloning and characterization of several cDNAs for UDP-glucose pyrophosphorylase from barley (Hordeum vulgare) tissues

Eleven cDNA clones encoding UDP-glucose pyrophosphorylase (UGPase) have been isolated from cDNA libraries prepared from seed embryo, seed endosperm and leaves of barley (Hordeum vulgare L.). The sequences were identical, with the exception of positioning of the poly(A) tail; at least five clones with different polyadenylation sites were found. For a putative full-length cDNA [1775 nucleotides (nt) plus polyadenylation tail], isolated from an embryo cDNA library, an open reading frame of 1419 nt encodes a protein of 473 amino acids (aa) of 51.6 kDa. An alignment of the derived aa sequence with other UGPases has revealed high identity to UGPases from eukaryotic tissues, but not from bacteria. Within the aa sequence, no homology was found to a UDP-glucose-binding motif that has been postulated for a family of glucosyl transferases. The derived aa sequence of UGPase contains three putative N-glycosylation sites and has a highly conserved positioning of five Lys residues, previously shown to be critical for catalysis and substrate binding of potato tuber UGPase. A possible role for N-glycosylation in the intracellular targeting of UGPase is discussed.     

Structure-function analysis of yeast tRNA ligase

Trl 1 is an essential 827-amino-acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two catalytic domains--an N-terminal adenylyltransferase/ligase component (amino acids 1-388) and a C-terminal 5'-kinase/cyclic phosphodiesterase component (amino acids 389-827)--that can function in tRNA splicing in vivo when expressed as separate polypeptides. Sedimentation analysis indicates that the ligase and kinase/CPD domains are monomeric proteins that do not form a stable complex in trans. To understand the structural requirements for the RNA ligase component, we performed a mutational analysis of amino acids that are conserved in Trl1 homologs from other fungi. Alanine scanning identified 23 new residues as essential for Trl1-(1-388) activity in vivo. Structure-activity relationships at these positions, and four essential residues defined previously, were clarified by introducing 50 different conservative substitutions. Lethal mutations of Lys114, Glu184, Glu266, and Lys284 abolished Trl1 adenylyltransferase activity in vitro. The essential elements embrace (1) putative equivalents of nucleotidyltransferase motifs I, Ia, III, IV, and V found in DNA ligases, T4 RNA ligase 2, and mRNA capping enzymes; (2) an N-terminal segment shared with the T4 RNA ligase 1 subfamily only; and (3) a constellation of conserved residues specific to fungal tRNA splicing enzymes. We identify yeastlike tRNA ligases in the proteomes of Leishmania and Trypanosoma. These findings recommend tRNA ligase as a target for antifungal and antiprotozoal drug discovery.     

Molecular cloning and sequence analysis of human dipeptidyl peptidase IV, a serine proteinase on the cell surface.

The cDNA coding for the human dipeptidyl peptidase IV (DPPIV) has been isolated and sequenced. The nucleotide sequence (3465 bp) of the cDNA contains an open reading frame encoding a polypeptide comprising 766 amino acids, one residue less than those of rat DPPIV. The predicted amino acid sequence exhibits 84.9% identity to that of the rat enzyme, and contains nine potential N-linked glycosylation sites, one site more than those in the rat enzyme. A putative catalytic triad for serine proteinases, serine, aspartic acid and histidine, are found in a completely conserved COOH-terminal region (positions 625-752).     

Differential mRNA expression levels and gene sequences of a putative carboxylesterase-like enzyme from two strains of the parasitoid Anisopteromalus calandrae (Hymenoptera: Pteromalidae).

Carboxylesterase-like enzyme cDNAs have been cloned and sequenced from malathion-resistant and susceptible strains of the parasitoid Anisopteromalus calandrae (Howard) (Hymenoptera: Pteromalidae). The cDNAs consist of 1963 nucleotides including a 35 bp untranslated 5'-end, a 1596 bp open reading frame, and a 332 bp untranslated 3'-end. The open reading frame encodes 532 amino acid residues. The predicted protein sequence from these cDNAs includes 2 potential N-glycosylation sites, a carboxylesterase type-B serine active site FGGDSENVTIFGESAG, and conserved residues Ser187, Glu317, and His432 to function as the catalytic triad. The predicted carboxylesterase-like enzyme sequence is most similar to that of the carboxylesterase from the peach-potato aphid, Myzus persicae with 45% sequence identity. Alignment of the parasitoid carboxylesterase-like enzyme cDNAs revealed that there are two nucleotide differences in the open reading frame between the parasitoid strains, including a silent mutation and a point mutation that presumably causes a gene product difference. A nucleotide thymine at position 658 in the susceptible strain cDNA is replaced by a guanine in the resistant strain cDNA. This substitution leads to an amino acid change from tryptophan (Trp220) in the susceptible strain to glycine (Gly220) in the resistant strain. This substitution is genetically linked to resistance but it is not known how or if this amino acid substitution affects detoxification of malathion. Northern blot analyses demonstrated that expression level of the carboxylesterase-like enzyme mRNA in adult A. calandrae is approximately 30-fold higher in the resistant strain relative to that in the susceptible strain. Southern analysis indicated that Pst I or Eco RI restriction sites are different in the two strains. Both a modified gene structure and an increase in expression of carboxylesterase may be responsible for the high level of resistance found in this beneficial wasp.     

In silico analysis of ORF1ab in coronavirus HKU1 genome reveals a unique putative cleavage site of coronavirus HKU1 3C-like protease

Recently we have described the discovery and complete genome sequence of a novel coronavirus associated with pneumonia, coronavirus HKU1 (CoV-HKU1). In this study, a detailed in silico analysis of the ORF1ab, encoding the 7,182-amino acid replicase polyprotein in the CoV-HKU1 genome showed that the replicase polyprotein of CoV-HKU1 is cleaved by its papain-like proteases and 3C-like protease (3CL(pro)) into 16 polypeptides homologous to the corresponding polypeptides in other coronaviruses. Surprisingly, analysis of the putative cleavage sites of the 3CL(pro) revealed a unique putative cleavage site. In all known coronaviruses, the P1 positions at the cleavage sites of the 3CL(pro) are occupied by glutamine. This is also observed in CoV-HKU1, except for one site at the junction between nsp10 (helicase) and nsp11 (member of exonuclease family), where the P1 position is occupied by histidine. This amino acid substitution is due to a single nucleotide mutation in the CoV-HKU1 genome, CAG/A to CAT. This probably represents a novel cleavage site because the same mutation was consistently observed in CoV-HKU1 sequences from multiple specimens of different patients; the P2 and P1'-P12' positions of this cleavage site are consistent between CoV-HKU1 and other coronaviruses; and as the helicase is one of the most conserved proteins in coronaviruses, cleavage between nsp10 and nsp11 should be an essential step for the generation of the mature functional helicase. Experiments, including purification and C-terminal amino acid sequencing of the CoV-HKU1 helicase and trans-cleavage assays of the CoV-HKU1 3CL(pro) will confirm the presence of this novel cleavage site.     

Characterization of new members of the pregnancy-specific β1-glycoprotein family

Three cDNAs encoding members of the pregnancy-specific 1-glycoprotein (PSG) family were isolated from human term placental cDNA library. All three cDNAs encode proteins with similar domain structure. There is a leader sequence of 34 amino acids followed by an N-domain of 109 amino acids. Immediately after the N-domain are one or two copies of a repeating A-domain of 93 amino acids, a B-domain of 85 amino acids and a C-domain of variable size. The proteins are highly hydrophilic. However, one of them has an 81-amino acid C-domain which is very hydrophobic and could potentially serve as a membrane attachment site. The putative cell-cell recognition tripeptide, Arg-Gly-Asp, is present in the N-domain of two of the proteins. Partial sequence of one of the cDNAs has been found in HeLa cells while cDNAs highly homologous to two of the cDNAs have been found in the fetal liver. Functional roles of the PSG proteins basing on their structure are proposed.Abbreviations PSG Pregnancy-Specific 1-Glycoprotein, according to nomenclature recommended at the ISOBM XVII Meeting, 1989 [31] - CEA Carcinoembryonic Antigen - bp base-pair - kb kilo-base-pair - nt nucleotide - aa amino acid - UTR Untranslated Region - RGD Arg-Gly-Asp The nucleotide sequence of two of the cDNAs presented in this article have been submitted to GenBank under the accession numbers M37102 (hPS91) and M37103 (hPS133). The nucleotide sequence of hPS176 has also been submitted. No accession number is available yet.     

Bovine parathymosin: amino acid sequence and comparison with rat parathymosin

Parathymosin has been purified from calf liver and its primary sequence established, except for a segment containing approximately 11 amino acid residues in the central part of the polypeptide chain. Bovine parathymosin contains approximately 101 amino acid residues and shows 90% identity with rat parathymosin, with substitution of Glu for Asp at positions 21, 57, and 58, Asp for Glu at positions 60 and 63, and Ala for Val at position 77. Three non-conservative substitutions were Ala for Thr at position 81, Leu for Arg at position 78, and Val for Lys at position 79. The replacement at the last two positions of a pair of basic by hydrophobic amino acid residues may account for differences in chromatographic behavior observed for the bovine and rat polypeptides. Analysis of the NH2-terminus employed a new deblocking procedure which was also employed to analyze rat parathymosin, requiring correction of the previously published NH2-terminal sequence for that polypeptide.     

Molecular abnormality of a Japanese glucose-6-phosphate dehydrogenase variant (G6PD Tokyo) associated with hereditary non-spherocytic hemolytic anemia

Summary The entire coding sequence of a Japanese class 1 variant (G6PD Tokyo) was amplified by the polymerase chain reaction from genomic DNA. Nucleotide analysis by a direct sequencing technique revealed a unique nucleotide substitution (1246 G to A) in exon 10, which predicts a Glu to Lys substitution at the 416th amino acid. This is another member of a conspicuous mutation cluster surrounding the putative NADP-binding domain.     

Mutational analysis of the octapeptide sequence motif at the NS1-NS2A cleavage junction of dengue type 4 virus.

We have previously shown that proper processing of dengue type 4 virus NS1 from the NS1-NS2A region of the viral polyprotein requires a hydrophobic N-terminal signal and the downstream NS2A. Results from deletion analysis indicate that a minimum length of eight amino acids at the C terminus of NS1 is required for cleavage at the NS1-NS2A junction. Comparison of this eight-amino-acid sequence with the corresponding sequences of other flaviviruses suggests a consensus cleavage sequence of Met/Leu-Val-Xaa-Ser-Xaa-Val-Xaa-Ala. Site-directed mutagenesis was performed to construct mutants of NS1-NS2A that contained a single amino acid substitution at different positions of the consensus cleavage sequence or at the immediate downstream position. Three to eight different substitutions were made at each position. A total of 50 NS1-NS2A mutants were analyzed for their cleavage efficiency relative to that of the wild-type dengue type 4 virus sequence. As predicted, nearly all substitutions at positions P1, P3, P5, P7, and P8, occupied by conserved amino acids, yielded low levels of cleavage, with the exception that Pro or Ala substituting for Ser (P5) was tolerated. Substitutions of an amino acid at the remaining positions occupied by nonconserved amino acids generally yielded high levels of cleavage. However, some substitutions at nonconserved positions were not tolerated. For example, substitution of Gly or Glu for Gln (P4) and substitution of Val or Glu for Lys (P6) each yielded a low level of cleavage. Overall, these data support the proposed cleavage sequence motif deduced by comparison of sequences among the flaviviruses. This study also showed that in addition to the eight-amino-acid sequence, the amino acid immediately following the NS1-NS2A cleavage site plays a role in cleavage.     

Active site geometry of glucose-1-phosphate uridylyltransferase

Glucose-1-phosphate uridylyltransferase, or UGPase, catalyzes the production of UDP-glucose from glucose-1-phosphate and UTP. Because of the biological role of UDP-glucose in glycogen synthesis and in the formation of glycolipids, glycoproteins, and proteoglycans, the enzyme is widespread in nature. Recently this laboratory reported the three-dimensional structure of UGPase from Escherichia coli. While the initial X-ray analysis revealed the overall fold of the enzyme, details concerning its active site geometry were limited because crystals of the protein complexed with either substrates or products could never be obtained. In an effort to more fully investigate the active site geometry of the enzyme, UGPase from Corynebacterium glutamicum was subsequently cloned and purified. Here we report the X-ray structure of UGPase crystallized in the presence of both magnesium and UDP-glucose. Residues involved in anchoring the ligand to the active site include the polypeptide chain backbone atoms of Ala 20, Gly 21, Gly 117, Gly 180, and Ala 214, and the side chains of Glu 36, Gln 112, Asp 143, Glu 201, and Lys 202. Two magnesium ions are observed coordinated to the UDP-glucose. An alpha- and a beta-phosphoryl oxygen, three waters, and the side chain of Asp 142 ligate the first magnesium, whereas the second ion is coordinated by an alpha-phosphoryl oxygen and five waters. The position of the first magnesium is conserved in both the glucose-1-phosphate thymidylyltransferases and the cytidylyltransferases. The structure presented here provides further support for the role of the conserved magnesium ion in the catalytic mechanisms of the sugar-1-phosphate nucleotidylyltransferases.     

Mutational analysis of the human immunodeficiency virus type 1 env gene product proteolytic cleavage site.

The structural requirements for proteolytic cleavage of the human immunodeficiency virus type 1 env gene product, gp160, to gp120 and gp41 have been assessed by specific mutagenesis of the sequence Lys Ala Lys Arg Arg Val Val Glu Arg Glu Lys Arg located between amino acids 500 and 511, i.e., at the putative C terminus of gp120. The basic amino acids underlined have been mutated, individually and in combination, to neutral amino acids, and the cleavability of the mutated env gene products was examined after expression in CV-1 cells. The results show that the replacement of Arg-511 (cleavage presumably occurs C terminal to this amino acid) with Ser completely abolishes recognition and cleavage by the cellular protease(s), i.e., the remaining basic amino acids in the vicinity do not serve as alternative substrates. However, Arg-508 and Lys-510 are important features of the recognition site since, when they are individually changed to neutral amino acids, cleavage is severely impaired. The basic amino acids 500, 502, and 504 are, individually, not important for cleavage, since their individual replacement by neutral amino acids does not impair cleavage. However, when all four basic amino acids 500, 502, 503, and 504 are changed to neutral amino acids, cleavage is almost completely abolished. This shows that the sequence Arg Glu Lys Arg at the cleavage site is alone not sufficient for cleavage but that a contribution of other amino acids is required, whether the other amino acids provide a basic character or a certain structure in the vicinity of the cleavage site. When noncleavable or poorly cleavable mutant env genes are expressed from the infectious plasmid pNL4-3 in CD4+ human lymphoblastoid cells, noninfectious virus, incapable of spread throughout the culture, is produced.     

Sequence characterization and expression patterns of defensin and lysozyme encoding genes from the gut of the reduviid bug Triatoma brasiliensis

The cDNAs encoding an intestinal defensin (def1) and lysozyme (lys1) of the reduviid bug Triatoma brasiliensis have been amplified by PCR using specific oligonucleotide primers and 5'- and 3'-RACE, cloned and sequenced. The 576 bp clone has an open reading frame of 282 bp and encodes a pre-prodefensin with 94 amino acid residues, containing a putative signal and activation peptide cleavage site at Ser19 and Arg51, respectively. The genomic DNA contains a second defensin gene with similar characteristics, 88.3% identity and also one intron of 107 nucleotides. The 538 bp clone has an open reading frame of 417 bp, encoding a pre-lysozyme with 139 amino acid residues. The putative signal peptide is cleaved at alanine 18. Using whole mount in situ hybridization, high expression of both genes has been found, distributed uniformly throughout the entire cardia and the blood-storing stomach and to a much lower extent in the digesting small intestine. Using quantitative real-time PCR, the expression level of def1 was also shown to be very low in small intestine, rectum and salivary glands; in the stomach, expression was 500-2500 times higher than in the cardia and fat body. No expression of lys1 could be detected in the salivary glands and rarely a very low expression in the small intestine, rectum and fat body. Lys1 expression in the stomach was 60-300 times higher than in the cardia. Comparing the levels in unfed fifth instars and up to 15 days after feeding, a strong def1 induction was evident in the fat body at 15 days after feeding and in the stomach a maximum level of def1 and lys1 at 5 days after feeding.     

Molecular cloning of a human co-beta-glucosidase cDNA: evidence that four sphingolipid hydrolase activator proteins are encoded by single genes in humans and rats

Authentic cDNAs encoding the activator protein for acid beta-glucosidase (EC3.2.1.45), co-beta-glucosidase, were cloned from the pCD and lambda gt11 human cDNA libraries. Initial screening with oligonucleotide mixtures encoding amino acid sequences of co-beta-glucosidase identified partial cDNAs which were used to obtain a potentially full-length cDNA from the lambda gt11 library. This clone (2767 bp), EGTISI, contained 5' (38 bp) and 3' (1157 bp) noncoding sequences, a translation initiation site, and an open reading frame encoding 524 amino acids which included a typical hydrophobic signal sequence (16 amino acids). Computer analyses identified three regions of high similarity to co-beta-glucosidase encoded by tandem sequences in EGTISI. Searches revealed that two of these regions encoded peptides of known function; SAP1 (sphingolipid activator protein 1) and protein C (a new sphingolipid activator protein) were encoded by EGTISI sequences 5' and 3', respectively, to those for co-beta-glucosidase. The third region of similarity, encoding a theoretical peptide (undefined function), was located most 5' in the cDNA. EGTISI and its encoded polypeptide had high similarity (77% nucleotide identity and about 80% amino acid similarity) to a rat Sertoli cell cDNA and its encoded sulfated glycoprotein-1. These results indicate that a single highly conserved gene encodes the precursor for four potential sphingolipid activator proteins in rat and man.     

A Single Amino Acid Substitution in One of the Lipases of Aspergillus nidulans Confers Resistance to the Antimycotic Drug Undecanoic Acid

A plausible approach to evaluate the inhibitory action of antifungals is through the investigation of the fungal resistance to these drugs. We describe here the molecular cloning and initial characterization of the A. nidulans lipA gene, where mutation (lipA1) conferred resistance to undecanoic acid, the most fungitoxic fatty acid in the C(7:0)-C(18:0) series. The lipA gene codes for a putative lipase with the sequence consensus GVSIS and WIFGGG as the catalytic signature. Comparison of the wild-type and LIP1 mutant strain nucleotide sequences showed a G --> A change in lipA1 allele, which results in a Glu(214) --> Lys substitution in LipA protein. This ionic charge change in a conserved LipA region, next to its catalytic site, may have altered the catalytic properties of this enzyme resulting in resistance to undecanoic acid.     

Role of Glu318 at the putative distal site in the catalytic function of cytochrome P450d.

Most microsomal P450s have a conserved "threonine cluster" composed of three Thrs (Thr319, Thr321, Thr322 for P450d) at a putative distal site. An ionic amino acid at 318 is also well conserved as Glu or Asp for most P450s. To understand the role of these conserved polar amino acids at the putative distal site in the catalytic function of microsomal P450, we studied how mutations at this site of P450d influence the activation of molecular oxygen in the reconstituted system. Catalytic activity (0.02 min-1) toward 7-ethoxycoumarin of the Glu318Ala mutant of P450d was just 6% of that (0.33 min-1) of the wild type, while those of Glu318Asp, Thr319Ala, and Thr322Ala were comparable to or even higher than that of the wild type. Consumption rates of O2 and formation rates of H2O2 of those mutants varied in accord with the catalytic activities. Especially, the efficiency (0.5%) of incorporated oxygen atom to the substrate versus produced H2O2 for the Glu318Ala mutant was much lower than that (3.7%) of the wild type, while that (58.8%) for the mutant Glu318Asp was 16-fold higher than that of the wild type. In addition, the autoxidation [Fe(II)---- Fe(III)] rate (0.074 s-1) of the Glu318Ala mutant was much lower than those (0.374-0.803 s-1) of the wild type and other mutants. Thus, we strongly suggest that Glu318 plays an important role in the catalytic function toward 7-ethoxycoumarin of microsomal P450d.