Cloning of a human liver microsomal UDP-glucuronosyltransferase cDNA.

A cDNA clone (HLUG 25) encoding the complete sequence of a human liver UDP-glucuronosyltransferase was isolated from a lambda gt11 human liver cDNA library. The library was screened by hybridization to a partial-length human UDP-glucuronosyltransferase cDNA (pHUDPGT1) identified from a human liver pEX cDNA expression library by using anti-UDP-glucuronosyltransferase antibodies. The authenticity of the cDNA clone was confirmed by hybrid-select translation and extensive sequence homology to rat liver UDP-glucuronosyltransferase cDNAs. The sequence of HLUG 25 cDNA was determined to be 2104 base-pairs long, including a poly(A) tail, and contains a long open reading frame. The possible site of translation initiation of this sequence is discussed with reference to a rat UDP-glucuronosyltransferase cDNA clone (RLUG 38).     

Cloning and characterization of DNA complementary to rat liver UDP-glucuronosyltransferase mRNA

UDP-glucuronosyltransferase (transferase) clones were isolated from a cDNA bank constructed in pBR322 using transferase-enriched mRNA from the livers of phenobarbital-treated rats. The enrichment of mRNA was accomplished by polysome immunoadsorption with antibody to purified mouse liver transferase. This antibody was shown to bind specifically to rat transferase by Ouchterlony double diffusion analysis, immunoadsorption of glucuronidating activities, and selective inhibition of the immunoadsorption of in vitro synthesized enzyme by purified rat liver transferase. The isolated clones were verified to contain DNA complementary to transferase mRNA by hybrid translation-selection. Three classes of transferase cDNAs were characterized by restriction endonuclease mapping, and the largest insert-containing clone of each class was designated pUDPGTr-1, pUDPGTr-2, and pUDPGTr-3. Their insert sizes were approximately 2,400, 2,000, and 2,000 bp, respectively. All three cDNAs hybridized with a 2,300 +/- 150 bp mRNA, and each selected the translation of a 52,000-dalton polypeptide. Immunoadsorption of the 35S-labeled translation product could be competitively inhibited in each case by the addition of purified rat liver transferase. pUDPGTr-1 and pUDPGTr-3 inserts shared extensive sequence homology. This was demonstrated by Southern blot analysis using purified inserts and electron microscopic heteroduplex analysis. Southern blot analysis revealed that these cDNAs hybridized to overlapping genomic fragments. pUDPGTr-2 shared less sequence homology with the other two classes of cDNAs, based on the above criteria. In addition, mRNA corresponding to pUDPGTr-2 was elevated 5-fold by phenobarbital treatment, whereas the other mRNAs levels were unaffected. These studies demonstrate that in rat liver there are a minimum of three distinct transferase mRNAs, two of which may be associated with a common gene or gene family.     

Cloning and characterization of cDNA encoding 3-methylcholanthrene inducible rat mRNA for UDP-glucuronosyltransferase

We have isolated cDNA clones of the mRNA for rat UDP-glucuronosyltransferase that catalyzes the glucuronidation of 4-nitrophenol, by using synthetic oligonucleotides as hybridization probes. The complete nucleotide sequence of the 1,927-base pairs cDNA insert has been determined. With untranslated sequences of 124 and 216 base pairs in the 5'- and 3'-terminal regions, respectively, the cDNA insert contained 1,587 base pairs that encode a complete primary structure of a putative precursor form of 4-nitrophenol UDP-glucuronosyltransferase with a calculated molecular weight of 60,114. The cDNA sequence also indicates the presence of 25 amino acids preceding the sequence determined by microsequence of the isolated protein. This extrapeptide, for the most part, consists of hydrophobic amino acids which are characteristic of the signal peptides as found for secretory proteins and most transmembrane proteins. Furthermore, the deduced amino acid sequence contains a putative halt transfer signal of a hydrophobic segment (residues 487-510), which is flanked on both sides by the peptide segments of highly charged amino acid residues (residues 463-486 and 511-529). These features are consistent with the properties of transmembrane proteins. Specific cDNA probes were used to analyze the induction of the enzyme in rat tissues by treatment with 3-methylcholanthrene. RNA blot analysis showed that 3-methylcholanthrene increased 10- to 15-fold the amount of hybridizable mRNA in liver. The livers and kidneys from 3-methylcholanthrene-treated rats were found to contain almost the same amount of hybridizable mRNA, although the basal level in the kidney was much higher than that of the liver, and the amounts in the lung were much lower than that of the liver and kidney.     

Cloning, characterization and site-directed mutagenesis of canine renin

Inhibition of renin has been shown to be successful in managing hypertension and maintaining cardiac health. Canine models have played a key role in preclinical assessment of renin inhibitors. Here we report the cloning of canine prorenin gene. The amino acid sequence of mature canine renin was approximately 70% identical to that of human renin. The full-length prorenin was expressed in HEK 293 cells, purified and converted to its active form by trypsin-mediated cleavage of the 43 residue propeptide. The mature enzyme was characterized by steady-state kinetics using a peptide corresponding to the canine angiotensinogen sequence, Ac-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser-OH (cleavage between Leu(10)-Leu(11)). The reaction followed Michaelis-Menten kinetics with a K(M) of 120 microM and a second-order rate constant (k(cat)/K(M)) of 1.7 x 10(5) M(-)(1)s(-)(1). The enzyme was inhibited by various human renin inhibitors, but at reduced potency compared to the human renin. The basis of the species specificity was investigated by mutagenesis. Based on primary sequence and structural alignments, three mutants were prepared (G149S-S150T, V286L, G149S-S150T-V286L). Each mutant yielded catalytically active enzymes with lower specific activities than native canine renin. V286L had the greatest effect on substrate specificity, while G149S, S150T mutations produced enzymes with inhibitor profiles similar to human renin.     

Cloning and characterization of cDNA encoding canine alpha-L-iduronidase. mRNA deficiency in mucopolysaccharidosis I dog.

alpha-L-Iduronidase is a lysosomal enzyme, the deficiency of which causes mucopolysaccharidosis I (MPS I); a canine MPS I colony has been bred to test therapeutic intervention. The enzyme was purified to apparent homogeneity from canine testis and found to consist of two electrophoretically separable proteins that had common internal peptides but differed at their amino termini. A 57-base oligonucleotide, corresponding to the most probable codons of the longest peptide, was used to screen a canine testis cDNA library. Three cDNAs were isolated, two of which lacked the 5'-end whereas the third was full-length except for a small internal deletion. The composite sequence encodes an open reading frame of 655 amino acids that includes all sequenced peptides. The amino terminus of the larger protein, glutamic acid 26, is at the predicted signal peptide cleavage site, whereas the amino terminus of the smaller protein is leucine 106. There are six potential N-glycosylation sites and a non-canonical polyadenylation signal, CTTAAA. A search of GenBank showed that the amino acid sequence of alpha-L-iduronidase has similarity to that of a bacterial beta-xylosidase. A full-length cDNA corresponding to the composite sequence was constructed (pcIdu) and inserted into the pSVL expression vector (pSVcIdu). Two days after Cos-1 cells were transfected with pSVcIdu, their intracellular and secreted level of alpha-L-iduronidase activity has increased 8- and 22-fold, respectively, over the endogenous activity. Fibroblasts of MPS I dogs, which have no alpha-L-iduronidase activity, lacked the normal alpha-L-iduronidase mRNA of 2.2 kilobases and contained instead a trace amount of a 2.8-kilobase species. Isolation and characterization of an expressible alpha-L-iduronidase cDNA represents the first step toward mutation analysis and replacement therapy.     

Purification and characterization of an ethanol-induced UDP-glucuronosyltransferase

A UDP-glucuronosyltransferase (GT) enzyme was isolated from ethanol-induced male New Zealand white rabbit hepatic protein. The animals were pretreated for 2 weeks with 10% ethanol in their drinking water. The GT enzyme was purified by anion-exchange and affinity chromatography and was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The molecular mass of the ethanol-induced UDP-glucuronosyltransferase was determined to be 57,000 Da. Tryptic digests of the ethanol-induced GT and a similarly purified GT from control rabbit liver appeared to be different by HPLC analysis, even though the molecular masses of the enzymes were indistinguishable. Amino acid compositions of the two proteins were different for six amino acids. The apparent Km values for the ethanol-induced GT enzyme for 1-naphthol and morphine as substrates were 43 and 109 microM, respectively. The apparent Vmax values for the ethanol-induced GT enzyme for these substrates were 83 and 4.6 nmol/min/mg protein. The increases in catalytic efficiencies, apparent Vmax/Km for 1-naphthol and for morphine, for the ethanol-induced isozyme compared to the control isozyme activities were 2.0- and 2.4-fold. A polyclonal antibody raised in sheep to the rabbit ethanol-induced GT demonstrated a 520-fold selectivity for precipitation of the ethanol-induced protein rather than the control protein. These results demonstrate the production of an unique isozyme of UDP-glucuronosyltransferase that is produced in rabbits as a result of chronic ethanol exposure.     

Cloning and characterization of opticin cDNA: evaluation as a candidate for canine oculo-skeletal dysplasia

Opticin, a novel member of the leucine-rich repeat (LRR) family, has been reported to bind to collagen fibrils. Many members of the LRR family of extracellular matrix proteins have been reported to bind to fibrillar collagen and regulate the diameter of collagen fibrils and lateral fusion between fibrils. Collagen fibrils are important for the maintenance of the vitreous body in the eye and growth plate cartilage of joints. Oculo-skeletal dysplasia (OSD) is a heterogeneous group of heritable genetic disorders affecting humans and a few breeds of dogs. Labrador retrievers and Samoyeds affected with non-allelic forms of OSD exhibit vitreous dysplasia and dwarfism, and could serve as an animal model for the disorder. To test the opticin gene as a candidate for OSD, canine opticin cDNA has been cloned and characterized. The predicted 327 amino acid sequence is 77% homologous to human opticin, and maintains characteristic structural domains including seven LRR domains, two cysteine clusters and potential O-linked glycosylation sites. It shows highest protein sequence identity to epiphycan (37%) and osteoglycin (31%) and belongs to the Class III family of LRR extracellular matrix proteins. In addition to ocular tissues and cartilage, opticin mRNA and protein have been identified in ligament, skin, muscle, and testes. No alteration of opticin expression at the protein level was observed in OSD affected dogs relative to normal controls. Based on linkage analysis using a newly identified intragenic single nucleotide polymorphism opticin has been excluded from having any causal association with the OSD loci in both Samoyeds and Labrador retrievers.     

Cloning and characterization of a Drosophila tyramine receptor.

Receptors for biogenic amines such as dopamine, serotonin and epinephrine belong to the family of receptors that interact with G proteins and share a putative seven transmembrane domain structure. Using a strategy based on nucleotide sequence homology between the corresponding genes, we have isolated Drosophila cDNA clones encoding a new member of the G protein-coupled receptor family. This protein exhibits highest homology to the human alpha 2 adrenergic receptors, the human 5HT1A receptor and a recently cloned Drosophila serotonin receptor. The corresponding mRNA is found predominantly in adult Drosophila heads. Membranes from mammalian cells expressing this receptor displayed high affinity binding sites for [3H]yohimbine, an alpha 2 adrenergic receptor antagonist (Kd = 4.45 x 10(-9) M). Tyramine was the most efficient of the putative Drosophila neurotransmitters at displacing [3H]yohimbine binding (EC50 = 1.25 x 10(-6) M). Furthermore tyramine induced an inhibition of adenylate cyclase activity in NIH 3T3 cells expressing this receptor. The Drosophila tyramine receptor that we have isolated might therefore be an invertebrate equivalent of the mammalian alpha 2 adrenergic receptors.     

Cloning and functional characterization of a cocaine-sensitive dopamine transporter.

We report the cloning of a rat cDNA encoding a functional dopamine transporter. This cDNA, derived from an intron-containing gene, encodes a protein of 620 amino acids. Hydropathicity analysis of the protein sequence suggests the presence of 12 putative transmembrane domains. The protein displays considerable identity with transporters for noradrenaline and GABA (64 and 30%, respectively). Transient expression of the cDNA in COS7 cells directs the expression of dopamine uptake activity with appropriate pharmacology and in a sodium-dependent fashion. In situ hybridization reveals that the mRNA for this transporter is expressed in the substantia nigra and ventral tegmental area, regions that contain dopaminergic cell bodies.     

Cloning and characterization of a cysteine proteinase from Saccharomyces cerevisiae.

We have isolated a gene from Saccharomyces cerevisiae that encodes a protein homologous to the mammalian cysteine proteinase bleomycin hydrolase. Sequence comparison between the yeast and rabbit proteins indicates an amino acid identity of 41.5% over 277 residues and a similarity of 78.3% when conservative substitutions are included. The apparent mass of the yeast protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 47 kDa, although sequence analysis indicates two potential initiator methionines that suggest calculated masses of either 51 or 55 kDa. The protein is nonessential in yeast as haploid mutants disrupted at several positions along the open reading frame remain viable. Furthermore, these mutants do not exhibit any readily observable growth defects under varying conditions of temperature, nutrients, osmotic strength, or exogenous bleomycin. However, the purified protein does exhibit marked hydrolytic activity toward the substrate arginine 4-methyl-7-coumarylamide (Km = 12.8 microM, Vmax = 2.56 mumol mg-1 h-1), and yeast cells engineered to express this protein at higher levels maintain increased resistance to bleomycin compared to wild-type cells. Because this protein represents the first example of a cysteine proteinase identified in yeast, we have named it Ycp1 (yeast cysteine proteinase).     

Isolation and characterization of hyodeoxycholic-acid: UDP-glucuronosyltransferase from human liver

The enzyme hyodeoxycholic-acid: UDP-glucuronosyltransferase was purified about 230-fold from a solubilized human liver microsomal preparation utilizing anion-exchange chromatography, ampholyte-displacement chromatography and UDP-hexanolamine--Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of Mr 52000 in SDS/PAGE; the elution of a single peak in reversed-phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6 alpha-hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13:1:2.7. UDP-glucuronosyltransferase activities toward the 3 alpha-hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and p-nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte-displacement chromatography and/or UDP-hexanolamine--Sepharose affinity chromatography. Two-substrate kinetic analysis of hyodeoxycholic-acid-conjugating activity gave a sequential mechanism with apparent Km values of 12 microM and 4 microM for hyodeoxycholic acid and UDP-glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.     

Cloning and expression of human UDP-glucuronosyltransferase 1A4 in Bac-to-Bac system

UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from uridine diphosphate-glucuronic acid (UDP-GA) to compounds with amine, hydroxyl, and carboxylic acid moieties. N-glucuronidation is an important pathway for elimination of many tertiary amine therapeutic agents used in humans. UGT1A4 has been reported to be specific for glucuronidating primary, secondary, and tertiary amines, forming N-glucuronides. To further investigate the drugs metabolized by UGT1A4, the Bac-to-Bac expression system was used to express the recombinant UGT1A4 with His-tag on the C-terminal. The His-tagged recombinant UGT1A4 expressed in Spodoptera frugiperda (Sf9) cells were detected using anti-His antibody and the molecular weight of the recombinant protein was approximately 55kDa. The enzyme activity towards imipramine in cell homogenate protein was found to be 83.14+/-15pmol/min/mg protein (n=3) with 0.5mM imipramine by HPLC, but was not detectable in blank Sf9 cells. It paved the way for the further studies for drug glucuronidation by UGT1A4. The purification of the UGT1A4 can be done by Ni-resin. This is helpful to do research on the structure of the UFT1A4.     

Isolation and characterization of canine secretory immunoglobulin M.

Canine secretory immunoglobulin M, isolated from both colostrum and bronchial secretions, contained the unique glycoprotein bound secretory component. The presence of this extra subunit accounted for the differences in size, quaternary structure, and antigenicity observed upon comparison of secretory immunoglobulin M with its serum counterpart. Approximately 90% of the isolated secretory immunoglobulin M contained covalently bound secretory component while, in the remainder of the population, secretory component was loosely attached and easily dissociated from the immunoglobulin. Following peptide bond cleavage with cyanogen bromide, the release of bound secretory component and J chain from secretory immunoglobulin M was not detected. Because cyanogen bromide cleavage of secretory immunoglobulin A results in the release of these subunits, differences in the primary structure of secretory immunoglobulin M and secretory immunoglobulin A must exist around the binding sites for secretory component and J chain.     

Cloning and characterization of a family of proteins associated with Mpl.

Thrombopoietin (TPO) controls the formation of megakaryocytes and platelets from hematopoietic stem cells via activation of the c-Mpl receptor and multiple downstream signal transduction pathways. We used two-hybrid screening to identify new proteins that interacted with the cytoplasmic domain of Mpl, and we found a new family of proteins designated A2D (for Ataxin-2 Domain protein). The A2D are 130-kDa proteins that have three regions similar to those of Ataxin-2, the gene product causing familial type 2 spinocerebellar ataxia. A2D has several isoforms with different C-terminal domains, all produced from a single gene by alternative splicing. Northern blotting indicated that the A2D gene is widely expressed in immortalized cell lines and hematopoietic and fetal tissues. A2D proteins were constitutively associated with Mpl in vivo in human hematopoietic UT7 cells. TPO also caused the release of A2D from the activated receptor, and the phosphorylation of A2D on tyrosines residues was dependent on the Mpl C-terminal domain. Finally, A2D bound to the unstimulated erythropoietin receptor, whereas erythropoietin caused dissociation from the erythropoietin receptor, suggesting that A2D proteins are new components of the cytokine signaling system.     

Cloning and characterization of human syndecan-3.

Syndecans are cell-surface heparan sulfate proteoglycans, which perform a variety of functions in the cell. Most important, they are co-receptors for growth factors and mediate cell-cell and cell-matrix interactions. Four syndecans (syndecan 1-4) have been described in different species. The aim of this work was the cloning and characterization of human syndecan-3. The human syndecan-3 sequence has high homology to the rat and mouse sequences, with the exception of the 5'-region. Syndecan-3 mRNA is mostly expressed in the nervous system, the adrenal gland, and the spleen. When different cell lines were transiently transfected with full-length syndecan-3 cDNA, it was localized to the membrane and induced the formation of long filopodia-like structures, microspikes, and varicosities. Consequently, the actin cytoskeleton was re-organized, since actin staining was mostly found in the cellular extensions and at the cell periphery, co-localizing with the syndecan-3 staining. The development of the phenotype depended on the presence of sugar chains, as transfected glycosaminoglycan-deficient Chinese hamster ovary (CHO) 745 cells did not show these structural changes, nor did transfected CHO K1 cells in the presence of heparin. The similarity of the cloned DNA sequence with that of other mammalian species and the high expression in the nervous system led us to the assumption that human syndecan-3 could perform comparable functions to those described for syndecan-3 in rat and mouse. Additionally, transient transfection experiments suggest a role of human syndecan-3 in the organization of cell shape by affecting the actin cytoskeleton, possibly by transferring signals from the cell surface in a sugar-dependent mechanism.     

Purification and characterization of canine alpha-1-antiproteinase.

The principal canine plasma protease inhibitor, alpha-1-antiproteinase, has been purified 90-fold with a 25% yield to apparent homogeneity. The purification scheme includes anion-exchange chromatography, to separate away the bulk of the serum albumin; affinity chromatography by insolubilized concanavalin A, to remove most of the other serum proteins as well as traces of albumin; and, finally, sizing on Sephacryl-S-200. Unique to this purification scheme is the batch use of insolubilized hemoglobin--Sepharose beads to remove the ubiquitous contaminant haptoglobin. The purified material has an apparent molecular weight of 58 000, 11.2% carbohydrate, and an E280nm1% = 5.82, and can be separated by isoelectric focusing into at least two distinct forms with pI values of 4.40 and 4.52. In addition, canine alpha-1-antiproteinase is immunologically distinct from human alpha-1-antiproteinase.