Molecular cloning and functional expression of bovine deoxyhypusine hydroxylase cDNA and homologs

Deoxyhypusine hydroxylase is the second of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here, we describe the cloning and characterization of bovine deoxyhypusine hydroxylase cDNA and its homologs. The deduced bovine deoxyhypusine hydroxylase protein is 87% identical to human enzyme and 45% identical to yeast enzyme. The overexpressed enzyme showed activity in catalyzing the hydroxylation of the deoxyhypusine residue in the eIF5A intermediate. An amino acid substitution from Glu 57 to Gly located at one of the four conserved His-Glu (HE) pairs, the potential metal coordination sites, resulted in severe reduction of deoxyhypusine hydroxylase activity. A deletion at the HEAT-repeats 1-3 resulted in complete losses of deoxyhypusine hydroxylase activity.     

Molecular evolution by change of function. Alkaloid-specific homospermidine synthase retained all properties of deoxyhypusine synthase except binding the eIF5A precursor protein

Deoxyhypusine synthase participates in the post-translational activation of the eukaryotic initiation factor 5A (eIF5A). The enzyme transfers the aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein, i.e. eIF5A(lys). Homospermidine synthase catalyzes an analogous reaction but uses putrescine instead of eIF5A(lys) as substrate yielding the rare polyamine homospermidine as product. Homospermidine is an essential precursor in the biosynthesis of pyrrolizidine alkaloids, an important class of plant defense compounds against herbivores. Sequence comparisons of the two enzymes indicate an evolutionary origin of homospermidine synthase from ubiquitous deoxyhypusine synthase. The two recombinant enzymes from Senecio vernalis were purified, and their properties were compared. Protein-protein binding and kinetic substrate competition studies confirmed that homospermidine synthase, in comparison to deoxyhypusine synthase, lost the ability to bind the eIF5A(lys) to its surface. The two enzymes show the same unique substrate specificities, catalyze the aminobutylation of putrescine with the same specific activities, and exhibit almost identical Michaelis kinetics. In conclusion, homospermidine synthase behaves like a deoxyhypusine synthase that lost its major function (aminobutylation of eIF5A precursor protein) but retained unaltered its side activity (aminobutylation of putrescine). It is suggested as having evolved from deoxyhypusine synthase by gene duplication and being recruited for a new function.     

Essential role of eIF5A-1 and deoxyhypusine synthase in mouse embryonic development

The eukaryotic initiation factor 5A (eIF5A) contains a polyamine-derived amino acid, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed post-translationally by the addition of the 4-aminobutyl moiety from the polyamine spermidine to a specific lysine residue, catalyzed by deoxyhypusine synthase (DHPS), and subsequent hydroxylation by deoxyhypusine hydroxylase (DOHH). The eIF5A precursor protein and both of its modifying enzymes are highly conserved, suggesting a vital cellular function for eIF5A and its hypusine modification. To address the functions of eIF5A and the first modification enzyme, DHPS, in mammalian development, we knocked out the Eif5a or the Dhps gene in mice. Eif5a heterozygous knockout mice and Dhps heterozygous knockout mice were viable and fertile. However, homozygous Eif5a1 (gt/gt) embryos and Dhps (gt/gt) embryos died early in embryonic development, between E3.5 and E7.5. Upon transfer to in vitro culture, homozygous Eif5a (gt/gt) or Dhps (gt/gt) blastocysts at E3.5 showed growth defects when compared to heterozygous or wild type blastocysts. Thus, the knockout of either the eIF5A-1 gene (Eif5a) or of the deoxyhypusine synthase gene (Dhps) caused early embryonic lethality in mice, indicating the essential nature of both eIF5A-1 and deoxyhypusine synthase in mammalian development.     

Molecular cloning of bovine beta-lactoglobulin cDNA

A cDNA library from bovine mammary gland mRNA was constructed in pBR322 and screened by hybrid-selected translation and immunoscreening. Several beta-lactoglobulin clones were identified and sequenced. All clones contained cDNA fragments corresponding to the 3' region of beta-lactoglobulin mRNA. The 3' non-translated region of beta-lactoglobulin mRNA consists of 187 nucleotides; the polyadenylation signal AATAAA occurs 17 nucleotides before the poly(A) tail. The amino-acid sequence predicted from the 3' coding region corresponds completely to the previously determined amino-acid sequence of beta-lactoglobulin.     

Molecular cloning of bovine class I MHC cDNA

Two cDNA cloned from a Hereford cow B cell line (BL-3) have allowed the determination of the complete coding region for two class I molecules encoded by the bovine MHC (BoLA). The predicted protein sequences have all the features expected of expressed class I molecules that present peptide Ag to cytotoxic T cells. Comparison with class I molecules from other species strongly suggests these cDNA are derived from different genes and provides evidence for the existence of a second expressed class I BoLA locus. The BoLA proteins show greater similarity to HLA than to H-2 molecules, correlating with the cross-reactions of W6/32 and other murine anti-HLA-A,B,C mAb with BoLA molecules. The basis for the W6/32 epitope and the preferential association of H-2 class I H chains with bovine beta 2-m is examined.     

Molecular cloning of a bovine immunoglobulin lambda chain cDNA

A cDNA library of the bovine mammary gland constructed in pBR322 was screened by mRNA hybrid-selected translation and by differential hybridization. Several immunoglobulin (Ig) lambda light-chain clones were identified and sequenced. Nucleotide sequence comparison of bovine and human Ig lambda chains showed a high degree of homology for constant regions and for J regions. The amino acid (aa) sequence encoded by the constant region of the bovine Ig lambda chain cDNA contains 107 aa with differences at 24 aa positions from the human Ig lambda chain. Three complementarity-determining regions (CDR1,2,3) characteristic of the variable region of bovine Ig lambda chain cDNA can be distinguished. The bovine and human sequences display good homology in the framework region 3 (FR3) but only patches of homology throughout the FR2 region. The 5' end of the bovine Ig lambda chain cDNA fragment of clone 1-14E contains five stop codons: two in CDR1, one in FR1 and two in the hydrophobic prepeptide region. These data suggest that the Ig lambda mRNA of clone 1-14E is transcribed from the V lambda pseudogene.     

Molecular cloning and nucleotide sequence of a bovine alpha-lactalbumin cDNA

A cDNA clone for the bovine milk protein, alpha-lactalbumin (alpha LA), has been identified using a rat cDNA probe. The bovine cDNA clone is 703 nucleotides (nt) long, contains 8 nt of 5'-untranslated sequence and 269 nt of 3'-untranslated sequence. When compared with previously reported sequences, the bovine alpha LA mRNA sequence has 74% similarity with rat alpha LA mRNA, 79% similarity with human mRNA and 74% similarity with guinea pig mRNA.     

Molecular cloning and expression of a cDNA encoding endothelial cell nitric oxide synthase.

Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is derived from a guanidino nitrogen of L-arginine via its metabolism by nitric oxide synthase (NOS). Herein, we report the molecular cloning of a cDNA encoding the constitutive calcium-calmodulin (Ca2+/CaM)-regulated nitric oxide synthase (ECNOS). A full-length ECNOS clone was isolated by screening a bovine aortic endothelial cell cDNA library using a fragment of rat brain NOS (bNOS) cDNA. This cDNA has an open reading frame of 3615 nucleotides encoding a 1205-amino acid protein. Membranes prepared from COS cells transfected with the ECNOS cDNA demonstrated NADPH- and Ca2+/CaM- dependent conversion of L-, but not D-, arginine to NO and citrulline that was inhibited by NG-nitro-L-arginine methyl ester. Comparison of the deduced amino acid sequence of ECNOS to the bNOS and macrophage NOS (Mac-NOS) sequences revealed 57 and 50% identity, respectively. In addition, ECNOS contains a unique N-myristylation consensus sequence (not shared by bNOS or Mac-NOS) that may explain its membrane localization.     

Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the M_r-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.     

cDNA cloning and expression of human lactosylceramide synthase.

Lactosylceramide synthase is an enzyme that catalyzes the transfer of galactose from UDP-Gal to glucosylceramide, and thus participates in the biosynthesis of most glycolipids in mammals. We have isolated and sequenced the cDNA clone encoding human lactosylceramide synthase. The deduced amino acid sequence of the human lactosylceramide synthase showed 94.2% identity with rat lactosylceramide synthase. Northern blotting analysis revealed that lactosylceramide synthase mRNA was expressed in various tissues, with the highest level in brain and adrenal gland.     

Molecular cloning and sequence analysis of a novel chalcone synthase cDNA from Ginkgo biloba.

A chalcone synthase (CHS) gene was cloned from Ginkgo biloba for the first time and it was also the first cloned gene involved in flavonoids metabolic pathway in G. biloba. The full-length cDNA of G. biloba CHS (designated as Gbchs) was 1608bp with poly(A) tailing and it contained a 1173bp open reading frame (ORF) encoding a 391 amino acid protein. Gbchs was found to have extensive homology with those of other plant chs genes via multiple alignments. The active sites of the CoA binding, coumaroyl pocket and cyclization pocket in CHS protein of Medicago sativa were also found in GbCHS. Molecular modeling of GbCHS indicated that the three-dimensional structure of GbCHS strongly resembled that of M. sativa (MsCHS2), implying GbCHS may have similar functions with MsCHS2. Phylogenetic tree analysis revealed that GbCHS had closer relationship with CHSs from gymnosperm plants than from other plants. Gbchs is a useful tool to study the regulation of flavonoids metabolism in G. biloba.     

Human geranylgeranyl diphosphate synthase. cDNA cloning and expression

Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR.RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed in Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA.     

Molecular cloning and partial sequencing of hepatitis A viral cDNA.

Hepatitis A virus was purified from infected monkey kidney cell cultures, and the viral RNA was used to synthesize double-stranded cDNA. This cDNA was cloned either after insertion into a plasmid-primed synthesis system or after insertion into the PstI site of pBR322. The resulting clones were mapped by restriction endonuclease analysis and by cross hybridization of the viral inserts to generate a composite map which represented at least 97% of the viral genome, lacking ca. 220 bases from the 5' end of the genome. The clones were verified to be hepatitis A virus specific based on their positive hybridization to viral RNA and to total hepatitis A virus-infected cellular RNA from a heterologous marmoset host system. The nucleotide sequence of 3,054 base pairs of cDNA homologous to the 5' half of the viral genome was determined, and an open reading frame of 854 consecutive coding triplets was identified. In addition, sequences which encode the VP-1 and VP-3 viral structural proteins were located in the nucleotide sequence.     

Reversal of the deoxyhypusine synthesis reaction. Generation of spermidine or homospermidine from deoxyhypusine by deoxyhypusine synthase

Deoxyhypusine synthase catalyzes the first step in hypusine (N epsilon-(4-amino-2-hydroxybutyl)lysine) synthesis in a single cellular protein, eIF5A precursor. The synthesis of deoxyhypusine catalyzed by this enzyme involves transfer of the 4-aminobutyl moiety of spermidine to a specific lysine residue in the eIF5A precursor protein to form a deoxyhypusine-containing eIF5A intermediate, eIF5A(Dhp). We recently discovered the efficient reversal of deoxyhypusine synthesis. When eIF5A([3H]Dhp), radiolabeled in the 4-aminobutyl portion of its deoxyhypusine residue, was incubated with human deoxyhypusine synthase, NAD, and 1,3-diaminopropane, [3H]spermidine was formed by a rapid transfer of the radiolabeled 4-aminobutyl side chain of the [3H]deoxyhypusine residue to 1,3-diaminopropane. No reversal was observed with [3H]hypusine protein, suggesting that hydroxylation at the 4-aminobutyl side chain of the deoxyhypusine residue prevents deoxyhypusine synthase-mediated reversal of the modification. Purified human deoxyhypusine synthase also exhibited homospermidine synthesis activity when incubated with spermidine, NAD, and putrescine. Thus it was found that [14C]putrescine can replace eIF5A precursor protein as an acceptor of the 4-aminobutyl moiety of spermidine to form radiolabeled homospermidine. The Km value for putrescine (1.12 mM) as a 4-aminobutyl acceptor, however, is much higher than that for eIF5A precursor (1.5 microM). Using [14C]putrescine as an acceptor, various spermidine analogs were evaluated as donor substrates for human deoxyhypusine synthase. Comparison of spermidine analogs as inhibitors of deoxyhypusine synthesis, as donor substrates for synthesis of deoxyhypusine (or its analog), and for synthesis of homospermidine (or its analog) provides new insights into the intricate specificity of this enzyme and versatility of the deoxyhypusine synthase reaction.     

Neuron-specific ablation of eIF5A or deoxyhypusine synthase leads to impairments in growth,viability, neurodevelopment,and cognitive functions in mice

Eukaryotic initiation factor 5A (eIF5A)^†, ^‡ is an essential protein that requires a unique amino acid, hypusine, for its activity. Hypusine is formed exclusively in eIF5A post-translationally via two enzymes, deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase. Each of the genes encoding these proteins, Eif5a, Dhps, and Dohh, is required for mouse embryonic development. Variants in EIF5A or DHPS were recently identified as the genetic basis underlying certain rare neurodevelopmental disorders in humans. To investigate the roles of eIF5A and DHPS in brain development, we generated four conditional KO mouse strains using the Emx1-Cre or Camk2a-Cre strains and examined the effects of temporal- and region-specific deletion of Eif5a or Dhps. The conditional deletion of Dhps or Eif5a by Emx1 promotor–driven Cre expression (E9.5, in the cortex and hippocampus) led to gross defects in forebrain development, reduced growth, and premature death. On the other hand, the conditional deletion of Dhps or Eif5a by Camk2a promoter–driven Cre expression (postnatal, mainly in the CA1 region of the hippocampus) did not lead to global developmental defects; rather, these KO animals exhibited severe impairment in spatial learning, contextual learning, and memory when subjected to the Morris water maze and a contextual learning test. In both models, the Dhps-KO mice displayed more severe impairment than their Eif5a-KO counterparts. The observed defects in the brain, global development, or cognitive functions most likely result from translation errors due to a deficiency in active, hypusinated eIF5A. Our study underscores the important roles of eIF5A and DHPS in neurodevelopment.     

Molecular cloning and sequence analysis of the cDNA for small proteoglycan II of bovine bone.

The cDNA for the full-length core protein of the small chondroitin sulphate proteoglycan II of bovine bone was cloned and sequenced. A 1.3 kb clone (lambda Pg28) was identified by plaque hybridization with a previously isolated 1.0 kb proteoglycan cDNA clone (lambda Pg20), positively identified previously by polyclonal and monoclonal antibody reactivity and by hybrid-selected translation in vitro [Day, Ramis, Fisher, Gehron Robey, Termine & Young (1986) Nucleic Acids Res. 14, 9861-9876]. The cDNA sequences of both clones were identical in areas of overlap. The 360-amino-acid-residue protein contains a 30-residue propeptide of which the first 15 residues are highly hydrophobic. The mature protein consists of 330 amino acid residues corresponding to an Mr of 36,383. The core protein contains three potential glycosaminoglycan-attachment sites (Ser-Gly), only one of which is within a ten-amino-acid-residue homologous sequence seen at the known attachment sites of related small proteoglycans. Comparisons of the published 24-residue N-terminal protein sequence of bovine skin proteoglycan II core protein with the corresponding region in the deduced sequence of the bovine core protein reveals complete homology. Comparison of the cDNA-derived sequences of bovine bone and human embryonic fibroblast proteoglycans shows a hypervariable region near the N-terminus. Nucleotide homology between bone and fibroblast core proteins was 87% and amino acid homology was 90%.