Amino acid sequence of beta-galactoside-binding bovine heart lectin. Member of a novel class of vertebrate proteins

A variety of animal tissues contain beta-galactoside-binding lectins with molecular masses in the range 13-17 kDa. There is evidence that these lectins may constitute a new protein family although their function in vivo is not yet clear. In this work the major part of the amino acid sequence of the 13 kDa lectin from bovine heart muscle has been determined. Comparison of this sequence with the cDNA-deduced sequence published for the chick embryo skin lectin showed 58% homology. Comparison of the bovine lectin sequence with partial sequences from two cDNA clones from a human hepatoma library and partial amino acid sequences of human lung lectin showed 70, 40 and 85% homology, respectively. The sequences of these vertebrate lectins are thus clearly related, supporting earlier results of immunological cross-reactivity within this group of proteins. Computer searching of protein sequence databases did not detect significant homologies between the bovine lectin sequence and other known proteins.     

Covalent linkage of phospholipid to myelin basic protein: identification of serine-54 as the site of attachment

The peptide portion of the lipopeptide isolated from bovine myelin basic protein contained glycine, lysine, and serine in a 2:1:1 molar ratio as determined by amino acid analysis. The N-terminus of the peptide was determined to be glycine. The tetrapeptide Gly53-Ser-Gly-Lys56 was the only segment of myelin basic protein that matched the above two characteristics. This tetrapeptide is highly conserved among the myelin basic proteins sequenced so far. After the selective degradation of the lipopeptide, phosphoserine was identified in the acid hydrolysate, thus indicating that Ser-54 of myelin basic protein in bovine brain is the site of attachment of polyphosphoinositide. Interestingly, serine-54 of myelin basic protein can be phosphorylated by the endogenous protein kinase myelin. However, myelin basic protein phosphorylated by the catalytic subunit of an exogenous soluble protein kinase failed to produce radioactively labeled lipopeptide. Hence the endogenous enzymes of myelin are thought to be involved in the formation of the covalent linkage between polyphosphoinositide and myelin basic protein. The conservation in sequence suggests a possible important structural role for the "phospholipidation" of myelin basic protein.     

The sequence of the mouse 14 kDa beta-galactoside-binding lectin and evidence for its synthesis on free cytoplasmic ribosomes.

The partial amino acid sequence of the mouse 14 kDa beta-galactoside-binding lectin has been deduced from cDNA clones corresponding to 86% of the coding sequence and extending to the polyadenylation signal. The deduced amino acid sequence for the murine lectin shows 94% identity with the rat, 89% with human, 86% with bovine and 46% with the chicken 14 kDa lectins. A cDNA probe has been used to analyse genomic DNA and identify a single mRNA of approx. 570 bp in 3T3 fibroblasts, murine erythroleukaemia cells and the murine basement-membrane-secreting Engelbreth-Holm-Swarm tumour. Analysis of free and bound polyribosomes has shown that the lectin message is translated on free cytoplasmic ribosomes.     

Isolation of bovine liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase cDNA: bovine liver and heart forms of the enzyme are separate gene products.

In order to ascertain whether the heart and liver forms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase were products of two different genes or arose via alternative splicing of a single gene, the bovine liver cDNA of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was isolated from a lambda gt10 phage library and its sequence compared with that of bovine heart cDNA. The deduced amino acid sequence of the bovine liver cDNA was also compared with the amino acid sequence of the human and rat liver phosphofructo-2-kinase/fructose-2,6-bisphosphatase enzyme. The bovine liver cDNA codes for a protein that has 81.6% amino acid identity with the bovine heart form and 97.0 and 98.3% identity with the rat and human liver forms of the enzyme, respectively. Comparison of the nucleotide sequences of the two bovine cDNAs and their deduced amino acid sequences demonstrates that while there is conservation of the active sites of liver/muscle and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases they are encoded by different genes.     

The cDNA for the heart/muscle isoform of bovine cytochrome c oxidase subunit VIa encodes a presequence.

We have used mixed oligonucleotide probes to isolate a cDNA for the heart/muscle isoform of cytochrome c oxidase (COX) subunit VIa (COX VIa-H) from a bovine heart cDNA library in lambda gt10. This cDNA, and a second one isolated upon rescreening, predict a 97 amino acid COX VIa precursor protein comprised of a 12 amino acid, basic presequence plus an 85 residue mature VIa protein. The presence of a presequence contrasts with the rat heart COX VIa cDNA.     

Complementary DNA cloning and sequencing of rat ovarian basic fibroblast growth factor and tissue distribution study of its mRNA

Three cDNA clones encoding rat basic fibroblast growth factor (FGF) were isolated from 10(6) independent clones prepared from a pregnant mare serum gonadotropin (PMSG)-stimulated rat ovarian cDNA library. One of the cDNA clones contained the entire coding sequence for basic FGF. The other two possessed the sequence coding the carboxy terminal 61 amino acids of rat basic FGF, the putative upstream intron sequence, and a 3'-noncoding region. The cDNAs encoding rat basic FGF predict a molecule consisting of 154 amino acid residues, which is one amino acid shorter than the human and bovine basic FGF. Otherwise, there are only 5 conservative amino acid substitutions between the rat and the human/bovine sequences. Poly A+ RNA from brain cortex and hypothalamus show a single 6.0 kb band that hybridizes to the cloned cDNA probe by Northern analyses. The observation that basic FGF mRNA is below the limits of detection in adrenal, spleen, heart, lung, kidney, liver, stomach, small intestine, large intestine, testis, and ovary support the notion that the that the high levels of the protein found in these tissues is due to storage of the mitogen in the extracellular matrix and not continuous gene expression. The significance of the abundance of mRNA in tissues which are not undergoing either active angiogenesis or cell proliferation (hypothalamus and brain cortex) is unclear but emphasizes the potential neuronotrophic function of basic FGF.     

Cloning of a full-length complementary DNA for fatty-acid-binding protein from bovine heart

A full-length cDNA for bovine heart fatty-acid-binding protein (H-FABP) was cloned from a lambda gt11 cDNA library established from bovine heart muscle. The cDNA sequence shows an open reading frame coding for a protein with 133 amino acids. Colinearity with the amino acid sequences of four tryptic peptides was asserted. H-FABP isolated from bovine heart begins with an N-acetylated valine residue, however, as derived from analysis of the tryptic, amino-terminal-blocked peptide and the molecular mass of the peptide obtained via secondary-ion mass spectrometry. The molecular mass of the total protein is 14673 Da. Bovine H-FABP is 89% homologous to rat H-FABP and 97% homologous to the bovine mammary-derived growth-inhibition factor described recently by B?hmer et al. [J. Biol. Chem. 262, 15137-15143 (1987)]. Significant homologies were also found with bovine myelin protein P2 and murine adipocyte protein p422. Secondary-structure predictions were proposed for these proteins, based on computer analysis, which reveal striking similarities.     

Isolation and characterization of a complementary DNA for the nuclear-coded precursor of the beta-subunit of bovine mitochondrial F1-ATPase

We have isolated a cDNA clone encoding the precursor of the beta-subunit of the bovine heart mitochondrial F1-ATPase. Two probes were used to isolate this precursor from a bovine heart cDNA library. One probe was a mixed-sequence oligonucleotide directed against a portion of the amino acid sequence of the mature protein, and the other probe was the F1-ATPase beta-subunit gene from Saccharomyces cerevisiae. Determination of the nucleotide sequence of this cDNA reveals that it contains a 1584-nucleotide-long open reading frame that encodes the complete mature beta-subunit protein and a 48 amino acid long NH2-terminal extension. This amino-terminal presequence contains four basic arginine residues, one acidic glutamic acid residue, four polar uncharged serine residues, and five proline residues. Southern blot hybridization analyses suggest that the bovine F1-ATPase beta-subunit precursor is encoded by a single genetic locus. RNA blot hybridization analyses reveal a single mRNA species of approximately 1.9 kilobases from both bovine liver and heart.     

Cloning of the C alpha catalytic subunit of the bovine cAMP-dependent protein kinase.

The bovine C alpha type catalytic subunit of the cAMP-dependent protein kinase was cloned. A partial cDNA was isolated from a bovine heart cDNA library. This clone contained 120 bp of the coding sequence and the entire 3' untranslated region of 1431 bp. The complete coding region was cloned by PCR amplification from total bovine heart and skeletal muscle RNA. The sequence of the 3' oligonucleotide was taken from the partial cDNA clone whereas the 5' oligonucleotide was chosen by comparison of sequences of published C alpha subunits from other species. In the deduced amino acid sequence there is one deviation from the published bovine C alpha protein sequence, aspartic acid 286 is exchanged by an asparagine. The C alpha mRNA was found to be expressed differentially in various bovine tissues.     

The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bc1 complex deduced from cDNA analysis

We isolated a cDNA encoding human Rieske Fe-S protein of mitochondrial cytochrome bc1 complex from a fibroblast cDNA library by colony hybridization. The cDNA contains the nucleotide sequence encoding all of the amino acids (274 residues) comprising the putative precursor to the protein. Based on the known amino acid sequence of bovine Rieske Fe-S protein, the N-terminal extension sequence is presumed to be composed of 78 amino acids with a molecular weight of 8053. The mature protein consists of the same number of amino acid residues as that of its rat and bovine counterparts, having a homology of about 92% with the latter.     

Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli. Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library. A full-length clone was obtained consisting of a 27-bp 5' noncoding region, an open reading frame encoding the expected 157 amino acid protein, and an extensive 3' nontranslated sequence. The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly(A)+ mRNA from bovine heart. The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E. coli. The expressed enzyme was soluble, and catalytically active and was readily isolated and purified. The recombinant protein had the expected Mr of 18,000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes. The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from the bovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated. A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction. Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate. By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.     

A cloned bovine cDNA encodes an alternate form of the catalytic subunit of cAMP-dependent protein kinase

While attempting to isolate a cDNA clone for the catalytic subunit of the bovine cAMP-dependent protein kinase, we have isolated cDNAs which code for a protein slightly different than the known amino acid sequence. The alternate cDNA was identified by screening a bovine pituitary cDNA library using synthetic oligonucleotides predicted from the known amino acid sequence of the catalytic subunit. The cDNA which we identified, encodes a protein which is 93% identical to the known amino acid sequence of the bovine catalytic subunit. It seems likely that this cDNA represents a previously undiscovered catalytic subunit of the cAMP-dependent protein kinase. The mRNA for the alternate catalytic subunit is different in size from the mRNA coding for the previously known catalytic subunit and also has a different tissue distribution. These findings suggest that there are at least two different genes for the catalytic subunit. The differences in amino acid sequence and tissue distribution suggest the possibility of important functional differences in the two enzymes.     

Complete amino acid sequence of a beta-galactoside-binding lectin from human placenta

The complete amino acid sequence of a beta-galactoside-binding lectin from human placenta was determined at protein level. The lectin consists of 134 amino acids and its N-terminal alanine is blocked with acetate. The lectin shows about 50% similarity with chick 14K lectin, which was the first vertebrate beta-galactoside-binding lectin completely sequenced. Only 14 residues proved to be different from those of rat lung lectin, the sole mammalian lectin of which the complete sequence has been reported.     

Bovine dopamine beta-hydroxylase cDNA. Complete coding sequence and expression in mammalian cells with vaccinia virus vector.

We have isolated cDNA clones for bovine dopamine beta-hydroxylase from an adrenal medulla cDNA library and have determined the complete coding sequence. The largest cDNA clone isolated from the library is 2.4 kilobase pairs (kb) and contains an open reading frame of 1788 bases, coding for a protein of 597 amino acids and Mr = 66,803. The predicted amino acid sequence of the bovine cDNA contains 85% identity with human dopamine beta-hydroxylase (Lamouroux, A., Vingny, A., Faucon Biquet, N., Darmon, M. C., Franck, R., Henry, J.P., and Mallet, J. (1987) EMBO J. 6, 3931-3937; Kobayashi, K., Kurosawa, Y., Fujita, K., and Nagatsu, T. (1989) Nucleic Acids Res. 17, 1089-1102). Northern blot analysis reveals that the cDNA hybridizes to an mRNA of 2.4 kb present in bovine adrenal medulla, but not in kidney, heart, or liver. In addition, the cDNA hybridizes to a second RNA species of 5.5 kb, which is 4-fold less abundant than the 2.4-kb RNA. In vitro translation of a synthetic RNA transcribed from the 2.4-kb cDNA produces a 68-kDa protein, which is specifically immunoprecipitated by antiserum to bovine dopamine beta-hydroxylase. The 2.4-kb cDNA was cloned into a vaccinia virus vector, and the recombinant virus was used to infect the rat pheochromocytoma PC12 and monkey BSC-40 fibroblast cell lines. In both cell lines, infection with recombinant virus produces a protein of Mr = 75,000, which reacts with antiserum to bovine dopamine beta-hydroxylase. These results indicate that the 2.4-kb cDNA contains the genetic information necessary to code for the bovine dopamine beta-hydroxylase subunit.     

Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Study of the expression of myelin proteolipid protein (lipophilin) using a cloned complementary DNA.

We have prepared a lambda gt10 cDNA library with the mRNA isolated from fetal calf brains which were actively myelinating. Using two oligonucleotides made according to the known amino acid sequence of myelin proteolipid protein (PLP or lipophilin), we have isolated several cDNA clones for this major intrinsic membrane protein of myelin. One of these clones, designated as pLP1, is found to contain 444 bp of coding sequence for the C-terminal half of PLP and 486 bp of 3' untranslated sequence. Using pLP1 as a hybridization probe, we have studied the regulation of PLP at the mRNA level during rat brain development. Our results show that the relative amounts of mRNA for PLP and that for the major extrinsic protein of the myelin membrane, myelin basic protein, increase coordinately during the course of myelination in the brain.     

Cloning and sequencing of a cDNA encoding the Rieske iron-sulfur protein of bovine heart mitochondrial ubiquinol-cytochrome c reductase

Two cDNA clones encoding bovine heart mitochondrial Rieske iron-sulfur protein were obtained by immunological screening of a bovine heart cDNA expression library in lambda gt11 with antiserum directed against Rieske iron-sulfur protein isolated from bovine heart mitochondrial ubiquinol-cytochrome c reductase. The cDNA inserts were 1005 and 1100 base pairs with an open reading frame of 807 base pairs which encoded a 196-amino acid mature Rieske iron-sulfur protein and a 73-amino acid presequence. The amino acid sequence of Rieske iron-sulfur protein deduced from nucleotide sequencing is the same as that obtained from protein sequencing except at residues #73 and #191 which are Ser and Asp instead of Ala and Gly, respectively.     

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%.