cDNA cloning and amino acid sequence of human brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase

A cDNA of 1762 base pairs was obtained from a cDNA library of human brain by immunoscreening, and the nucleotide sequence of the cDNA was determined. The complete amino acid sequence of human 2',3'-cyclic-nucleotide 3'-phosphodiesterase was deduced from the nucleotide sequence of the cDNA. Human enzyme was found to contain 401 amino acids including initiation methionine and have a molecular weight of 45,098. RNA blot hybridization revealed a single mRNA band at the position of about 3000 bases. DNA blot hybridization suggested that a single-copy 2',3'-cyclic-nucleotide 3'-phosphodiesterase gene exists per haploid genome.     

Crystal structure of the catalytic fragment of human brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (CNP), a member of the 2H phosphoesterase superfamily, is firmly bound to brain white matter and found mainly in the central nervous system of vertebrates, and it catalyzes the hydrolysis of 2',3'-cyclic nucleotide to produce 2'-nucleotide. Recent studies on CNP-knockout mice have revealed that the absence of CNP causes axonal swelling and neuronal degeneration. Here, the crystal structure of the catalytic fragment (CF) of human CNP (hCNP-CF) is solved at 1.8A resolution. It is an alpha+beta type structure consisting of three alpha-helices and nine beta-strands. The structural core of the molecule is comprised of two topologically equivalent three-stranded antiparallel beta-sheets that are related by a pseudo 2-fold symmetry. Each beta-sheet contains an H-X-T-X motif, which is strictly conserved among members of the 2H phosphoesterase superfamily. The phosphate ion is bound to the side-chains of His and Thr from each of the two motifs. Structural comparison of hCNP-CF with plant 1',2'-cyclic nucleotide phosphodiesterase (CPDase) and bacterial 2'-5' RNA ligase reveals that the H-X-T-X motifs are structurally conserved among these enzymes, but the surface properties of the active site are quite different among the enzymes, reflecting the differences in their substrates. On the basis of the present crystal structure of the hCNP-CF/phosphate complex, the available structure of the CPDase/cyclic-nucleotide analogue complex, and the recent functional studies of rat CNP-CF, we propose a possible substrate-binding mode and catalytic mechanism of CNP, which employs the nucleophilic water molecule activated by His310. The proposed mechanism is basically equivalent to the second step of the well-accepted reaction mechanism of RNase A. Since the overall structure of hCNP-CF differs considerably from that of RNase A, it is likely that the similar active sites with two catalytic histidine residues in these enzymes arose through convergent evolution.     

Molecular cloning of the myelin specific enzyme 2',3'-cyclic-nucleotide 3'-phosphohydrolase

We describe the isolation of cDNA clones for bovine brain 2',3'-cyclic-nucleotide 3'-phosphohydrolase (CNPase, EC 3.1.4.37), the third most abundant protein in central nervous system myelin. The cDNA encodes the complete protein (400 amino acids) and hybridizes to a major size species of mRNA in bovine brain tissue, approx. 2.7 kb in size. CNPase mRNA levels do not appear to be affected in quaking dysmyelinating mutant mice. The sequence reveals probable sites for CNPase phosphorylation by cAMP-dependent protein kinase and a region of homology with haemocyanin.     

Cloning and characterization of zRICH, a 2',3'-cyclic-nucleotide 3'-phosphodiesterase induced during zebrafish optic nerve regeneration

We previously reported cloning of cDNAs encoding both components of a protein doublet induced during goldfish optic nerve regeneration. The predicted protein sequences showed significant homology with the mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases (CNPases). CNPases are well-established markers of mammalian myelin; hence, the cDNAs were designated gRICH68 and gRICH70 (for goldfish Regeneration-Induced CNPase Homologues of 68 and 70 kDa). Homologous cDNAs have now been isolated from zebrafish encoding a highly related protein, which we have termed zRICH. RNase protection assays show that zRICH mRNA is induced significantly (fivefold) in optic nerve regenerating zebrafish retinas 7 days following nerve crush. Western blots show a single band in zebrafish brain and retina extracts, with immunoreactivity increasing three-fold in regenerating retinas 21 days postcrush. Immunohistochemical analysis indicated that this increase in zRICH protein expression is localized to the retinal ganglion cell layer in regenerating retina. We have characterized and evaluated the relevance of a conserved beta-ketoacyl synthase motif in zRICH to CNPase activity by means of site-directed mutagenesis. Two residues within the motif, H334 and T336, are critical for enzymatic activity. A cysteine residue within the motif, which corresponds to a critical residue for beta-ketoacyl synthase, does not appear to participate in the phosphodiesterase activity.     

Identification of 2',3'-cyclic nucleotide 3'-phosphodiesterase in bovine adrenal medulla

The adrenal medulla contains an enzyme which catalyzes the hydrolysis of 2',3'-cAMP to 2'-AMP. For the parameters which have been examined, the adrenal medulla 2',3'-cAMP phosphodiesterase appears to be similar to brain 2',3'-cyclic nucleotide 3'-phosphodiesterase (also commonly referred to as CNPase). The apparent Km of the adrenal medulla CNPase for 2',3'-cAMP is 0.88 mM. The enzyme activity is unaltered by either EDTA, MgCl2 or CaCl2 in the presence or absence of calmodulin. The apparent molecular weight is 102,500 daltons. The function of the enzyme in either the brain or the adrenal medulla is, at the present time, unknown.     

cDNA cloning and amino acid sequence for human myelin-associated glycoprotein

cDNA clones of human myelin-associated glycoprotein were isolated and analyzed. The combination of the two overlapping cDNA clones covered the full coding region and the complete amino acid sequence was deduced. In rat and mouse, expression of the two forms of mRNA is developmentally regulated; the mRNA without exon 12 portion is expressed mainly in the actively myelinating stage of development. Although the cDNA library used here was prepared from adult human brain poly(A)+ RNA, all five clones obtained corresponded to the mRNA without exon 12 portion.     

cDNA sequence and deduced amino acid sequence of bovine oviductal fluid catalase

A bovine oviductal fluid catalase (OFC) which preferentially binds to the acrosome surface of some mammalian spermatozoa has recently been purified. The objectives of this study were to clone the OFC, obtain the full-length cDNA and protein sequence and determine which characteristics of the proteins are associated with the binding of the enzyme to sperm surface. Northern blot analysis revealed low levels of catalase mRNA in bovine oviducts and uterus compared to the liver and kidney. Screening of a cDNA library from the cow oviduct permit to obtain a full-length cDNA of 2282 bp, with an open reading frame of 1581 bp coding for a deduced protein of 526 amino acids (59 789 Da). The deduced protein contained four potential N-glycosylation sites and many potential O-glycosylation sites. The OFC protein exhibited high identity with catalase from other bovine tissues, likewise with catalases from human fibroblast and kidney, and with rat liver catalase. The homology of amino acid sequence of OFC with bovine liver catalase was about 99%. However the OFC posses an extended carboxyl terminus of 20 amino acids not present on the liver catalase. This result is supported by a lower mobility of the OFC compared to the liver catalase when both proteins are submitted on SDS-PAGE. Mol. Reprod. Dev. 51:265–273, 1998. © 1998 Wiley-Liss, Inc.     

Transketolase and 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I isoforms are specifically recognized by IgG autoantibodies in multiple sclerosis patients

The presence of autoantibodies in multiple sclerosis (MuS) is well known, but their target antigens have not been clearly identified. In the present study, IgG autoreactivity to neural antigens of normal human white matter separated by bidimensional electrophoresis was assessed in serum and cerebrospinal fluid of 18 MuS and 20 control patients. Broad IgG autoreactivity was detected by two-dimensional immunoblotting in all cases to neural antigens, most of which were identified by mass spectrometry. The comparative analysis of MuS and non-MuS reactive spots showed that a restricted number of neural protein isoforms were specifically recognized by MuS IgG. Almost all MuS patients had cerebrospinal fluid IgG directed to isoforms of one of the oligodendroglial molecules, transketolase, 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I, collapsin response mediator protein 2, and tubulin beta 4. Interestingly 50% of MuS IgG recognized transketolase, which was mostly localized on oligodendrocytes in human white matter from normal and MuS samples. IgG autoreactivity to cytoskeletal proteins (radixin, sirtuin 2, and actin-interacting protein 1) was prevalent in secondary progressive MuS patients. Among the proteins recognized by serum IgG, almost all MuS patients specifically recognized a restricted number of neuronal/cytoskeletal proteins, whereas 2',3'-cyclic-nucleotide 3'-phosphodiesterase type I was the oligodendroglial antigen most frequently recognized (44%) by MuS seric IgG. Our immunomics approach shed new light on the autoimmune repertoire present in MuS patients revealing novel oligodendroglial and/or neuronal putative autoantigens with potential important pathogenic and diagnostic implications.     

Molecular cloning, cDNA structure and predicted amino acid sequence of bovine 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4 isomerase

We have used our recently characterized human 3 beta-hydroxy-5-ene steroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNA as probe to isolate cDNAs encoding bovine 3 beta-HSD from a bovine ovary lambda gtll cDNA library. Nucleotide sequence analysis of two overlapping cDNA clones of 1362 bp and 1536 bp in length predicts a protein of 372 amino acids with a calculated molecular mass of 42,093 (excluding the first Met). The deduced amino acid sequence of bovine 3 beta-HSD displays 79% homology with human 3 beta-HSD while the nucleotide sequence of the coding region shares 82% interspecies similarity. Hybridization of cloned cDNAs to bovine ovary poly(A)+ RNA shows the presence of an approximately 1.7 kb mRNA species.     

3':5'-cyclic-nucleotide phosphodiesterase in the bovine pituitary gland

Cyclic-AMP phosphodiesterase activity in the homogenate of the anterior pituitary gland was 2-fold higher than that in the homogenate of the posterior pituitary, whereas cyclic-GMP phosphodiesterase activity was dominant in the posterior homogenate. There were two peaks of cyclic-AMP phosphodiesterase activity with different isoelectric points of 4.3 and 5.2. Fraction I had a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S; fraction II had a molecular weight of 180 000 and a sedimentation coefficient of 3.1 S. Cyclic AMP hydrolytic activity in the supernatant of the posterior lobe corresponded to fraction I in the anterior lobe. Cyclic GMP hydrolytic activity in both the anterior and posterior lobes (activated by Ca2+/calmodulin) had an isoelectric point of 5.2, a molecular weight of 240 000 and a sedimentation coefficient of 6.2 S. Cyclic AMP and GMP hydrolytic activities in both the anterior and posterior lobes appeared in fraction I and did not separate when the preparations were mixed before electric focusing or sucrose density gradient procedures. Cyclic AMP hydrolytic activity in fraction II could be separated from cyclic GMP hydrolytic activity.     

Complete amino acid sequence of canine cardiac calsequestrin deduced by cDNA cloning

cDNA cloning was used to deduce the complete amino acid sequence of canine cardiac calsequestrin, the principal Ca2+-binding protein of cardiac junctional sarcoplasmic reticulum. Cardiac calsequestrin contains 391 amino acid residues plus a 19-residue amino-terminal signal sequence. The molecular weight of the mature protein, excluding carbohydrate, is 45,269. Cardiac calsequestrin is highly acidic, and a striking feature is the enrichment of acidic residues (60%) within the 63 carboxyl-terminal residues. No part of the sequence contains EF hand Ca2+-binding structures. The photo-affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine was used to localize the Ca2+-regulated hydrophobic site to amino acid residues 192-223. The cardiac and skeletal muscle isoforms of calsequestrin (Fliegel, L., Ohnishi, M., Carpenter, M. R., Khanna, V. K., Reithmeier, R. A. F., and MacLennan, D. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1167-1171), although the products of different genes, are 65% identical, are acidic, and share one glycosylation site. However, cardiac calsequestrin has several unique features. First, it has a 31-amino acid extension at its carboxyl terminus (residues 361-391), which contains 71% acidic residues and a second glycosylation site. Second, its mRNA contains a second open reading frame with the capacity to code for a 111-amino acid protein. Third, contrary to the restricted expression of the fast skeletal isoform, cardiac calsequestrin mRNA is present in both cardiac and slow skeletal muscle, but not in fast skeletal muscle. We conclude that the deduced amino acid sequence of cardiac calsequestrin is consistent with its ability to bind large amounts of Ca2+ (40 mol of Ca2+/mol of calsequestrin). The protein probably binds Ca2+ by acting as a charged surface rather than by presenting multiple discrete Ca2+-binding sites.     

cDNA cloning and predicted amino acid sequence of Glycera dibranchiata monomer hemoglobin IV

The three major monomer hemoglobins from Glycera dibranchiata erythrocytes isolated in this laboratory were sequenced from their N-termini. A stretch of amino acid sequence identity was used to determine the sequence of a mixed oligodeoxynucleotide that would be complementary to all 12 possible mRNA sequences coding for the amino acids. A cDNA library was constructed by using poly(A+) RNA from G. dibranchiata erythrocytes, the library was probed with the oligonucleotide, and the longest positive inserts found were subcloned into a sequencing plasmid and then sequenced. The first one was 745 bases long, containing 85 bases of 5'-untranslated RNA, an open reading frame of 444 bases coding for 148 amino acids, and a 3'-untranslated region of 216 bases. The predicted amino acid sequence matches the first 25 amino acids of G. dibranchiata monomer globin component IV. The sequence contains an N-terminal methionine plus 18 other mostly conservative sequence changes compared to the published sequence of Imamura et al. (1972), which appears from our partial sequencing to be monomer globin component II. We confirm the presence of leucine in the E7 position, which is histidine in most myoglobins and hemoglobins.     

2',3'-cyclic nucleotide 2'-phosphodiesterase from Fusarium culmorum

We describe the properties of a 2',3'-cyclic nucleotide 2'-phosphodiesterase (EC 3.1.4.16), found in Fusarium culmorum, which hydrolyzes nucleoside 2',3'-cyclic monophosphates to nucleoside 3'-phosphates. In contrast with a similar enzyme found in bacteria, the Fusarium enzyme does not exhibit nucleotidase activity and does not show a requirement for metal ions, but is inhibited by micromolar concentrations of Cu++ and Zn++, and is very stable to heat. This cyclic phosphodiesterase hydrolyzes the four major nucleoside 2',3'-cyclic monophosphates and has greater affinity for purine (Kms for Ado-2',3'-P = 0.3 mM and for Guo-2',3'-P = 0.1 mM) than for pyrimidine nucleotides (Kms for Cyd-2',3'-P = 0.6 mM and for Urd-2',3'-P = 2 mM). The respective Vmax for Urd-2',3'-P; Cyd-2',3'-P; Ado-2',3'-P; and Guo-2',3' are 100:45:16:5. The efficacy of the phosphodiesterase to hydrolyze the four major 2',3' cyclic nucleotides (based on the relative values of Vmax/Km) is not significantly different. The Fusarium enzyme differs from a previously described 2',3' cyclic phosphodiesterase from Neurospora, in that it is inactive on 3',5'-nucleoside monophosphates and nucleoside 2' or 3' phosphates.     

Diastereomeric specificity of 2',3'-cyclic nucleotide 3'-phosphodiesterase.

The diastereomers of adenosine and uridine 2',3'-cyclic phosphorothioates were tested as substrates for 2',3'-cyclic nucleotide 3'-phosphodiesterase from bovine brain. The enzyme cleaves the Sp (or exo) diastereomers efficiently, whereas the Rp (or endo) diastereomers are resistant to hydrolysis, even after long incubation. As the enzyme exhibits strong substrate inhibition the precise determination of kinetic parameters posed problems, particularly with phosphorothioates. The stereoselectivity of this enzyme is opposite to that of RNase T1 and RNase A and thus could be a useful complement in determination of the configuration of nucleoside 2',3'-cyclic phosphorothioates resulting from hydrolysis reactions of unknown stereochemical course.     

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.     

Purification and characterization of wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase

The 2',3'-cyclic nucleotide 3'-phosphodiesterase which hydrolyzes nucleoside 2',3'-cyclic phosphates (N greater than p) to nucleoside 2'-phosphates has been purified 16,000-fold to near homogeneity from wheat germ. The purified enzyme is a single polypeptide with a molecular weight of 23,000-24,000. It has a pH optimum of 7.0. The apparent Km values for A greater than p, G greater than p, C greater than p, and U greater than p are 13.1, 9.2, 25.2, and 25.3 mM, respectively. Vmax values for A greater than p, G greater than p, C greater than p, and U greater than p are 2090, 280, 2140, and 600 mumol/min/mg of purified protein, respectively. Wheat germ 2',3'-cyclic nucleotide 3'-phosphodiesterase does not hydrolyze 2',3'-cyclic esters in cyclic phosphate-terminated oligoribonucleotides or in nucleoside 5'-phosphate, 2',3'-cyclic phosphate (pN greater than p). This is in contrast to the 3'-phosphodiesterase activity associated with a wheat germ RNA ligase which hydrolyzes cyclic phosphate-terminated oligonucleotides and pN greater than p substrates much more efficiently than nucleoside 2',3'-cyclic phosphates. The enzyme characterized in this work appears to be the only known 2',3'-cyclic nucleotide 3'-phosphodiesterase specific for 2',3'-cyclic mononucleotides.     

Human hepatic triglyceride lipase: cDNA cloning, amino acid sequence and expression in a cultured cell line

By immunoscreening of a human cDNA expression library and hybridization of colonies, four partially overlapping cDNA clones of human hepatic triglyceride lipase (HTGL) mRNA were isolated. The clones included the complete coding sequence, the 3'- and at least part of the 5'-untranslated region. The length of the composite HTGL cDNA segment (1.7 kb) was consistent with the size of the mRNA identified in an established human hepatoma cell line. DNA-sequence analysis of cDNAs of partially unspliced mRNAs, and of cloned genomic DNA indicated that the HTGL coding sequence comprises at least six exons. As predicted from the cDNA, the unprocessed HTGL protein has a molecular weight of 56, three potential glycosylation sites, and a signal peptide of 23 amino acids. Sequence comparison with cDNA of other lipases, including rat hepatic lipase, revealed 30%-75% protein-sequence homology. The data establish that HTGL is a secretory protein produced in the hepatocyte, and that its synthesis can be continued in permanent cell lines of hepatoma origin. Our studies also showed that HTGL is another member of a lipase gene family which has interfacial binding sites and possibly other functional domains in common.