Cloning of cDNA encoding human H-protein, a constituent of the glycine cleavage system

A cDNA that encodes human H-protein, a constituent protein of the glycine cleavage system, was cloned with anti-rat H-protein antibody as a probe from a human liver cDNA library constructed with an expression vector, lambda gt11. The longest size of cDNA of the isolated clones was about 750 base long (lambda HH15B9). On the other hand, we determined the primary structure of human H-protein from the amino terminal Ser by the 12th Val, including a hexapeptide, -Glu-Lys-His-Glu-Trp-Val-. In addition to the finding that most cDNA inserts cloned hybridized with the synthetic DNA probe composed of the possible sequences for the hexapeptide, we confirmed that lambda HH15B9 encodes the partial primary structure of H-protein in an open reading frame.     

Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria.

L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration. We have isolated and characterized a cDNA from a lambda gt11 pea library encoding the complete L-protein precursor. The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues. The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA. Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved. Most of the residues located in or near the active sites remain unchanged. The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes. Confidence in this conclusion comes from the following considerations. First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected. Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues. In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves. Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome.     

Identification of the folate binding sites on the Escherichia coli T-protein of the glycine cleavage system.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. To determine the folate-binding site on the enzyme, 14C-labeled methylenetetrahydropteroyltetraglutamate (5,10-CH2-H4PteGlu4) was enzymatically synthesized from methylenetetrahydrofolate (5, 10-CH2-H4folate) and [U-14C]glutamic acid and subjected to cross-linking with the recombinant Escherichia coli T-protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker between amino and carboxyl groups. The cross-linked product was digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase high performance liquid chromatography. Amino acid sequencing of the labeled peptides revealed that three lysine residues at positions 78, 81, and 352 were involved in the cross-linking with polyglutamate moiety of 5, 10-CH2-H4PteGlu4. The comparable experiment with 5,10-CH2-H4folate revealed that Lys-81 and Lys-352 were also involved in cross-linking with the monoglutamate form. Mutants with single or multiple replacement(s) of these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis. The single mutation of Lys-352 caused similar increase (2-fold) in Km values for both folate substrates, but that of Lys-81 affected greatly the Km value for 5,10-CH2-H4PteGlu4 rather than for 5,10-CH2-H4folate. It is postulated that Lys-352 may serve as the primary binding site to alpha-carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4, whereas Lys-81 may play a key role to hold the second glutamate residue through binding to alpha-carboxyl group of the second glutamate residue.     

Isolation and sequence determination of cDNA encoding the major structural protein of human peripheral myelin.

A full length cDNA of the major structural protein of peripheral myelin (P0 protein) has been isolated from a cDNA library of human fetus spinal cord. The clone is 1948 base pairs (bp) in length and contains a 744 bp open reading frame encoding a polypeptide of 248 residues including 29 signal peptide. The deduced amino acid sequence is highly homologous to P0 protein from other species.     

Isolation and sequence determination of cDNA encoding P2 protein of human peripheral myelin.

A full length cDNA of P2 protein of peripheral myelin has been isolated from a cDNA library of human fetus spinal cord. The clone is 2150 base pairs (bp) in length and contains a 393 bp open reading frame encoding a polypeptide of 131 residues. The deduced amino acid sequence is highly homologous to P2 protein from other species.     

Molecular cloning of a cDNA encoding chicken T-protein of the glycine cleavage system and expression of the functional protein in Escherichia coli. Effect of mRNA secondary structure in the translational initiation region on expression.

DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries. Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292). The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein. A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA. Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA. Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside. Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein. The recombinant T-protein was purified to near homogeneity with a yield of about 30%. Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver. NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically. The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.     

Isolation and sequence of a cDNA encoding mouse IMP dehydrogenase.

Inosinic acid (IMP) dehydrogenase (IMPD) catalyzes the conversion of IMP to XMP as the first committed step in GMP biosynthesis de novo. We have isolated a cDNA containing the complete coding region of mouse IMPD by its ability to complement a bacterial mutant lacking IMPD activity. Two independent cDNA clones were isolated by complementation, of which the longest was 1.7 kb in length. Northern analyses, using the IMPD cDNA as a probe, indicated that mature IMPD mRNA was a single species approx. 2.0 kb in size. Mouse IMPD is almost identical to Chinese hamster and human IMPDs and is highly conserved between Escherichia coli and mouse, with a direct amino acid (aa) identity of 39%, which increases to 60% if conserved aa are considered. The leader region of our longest cDNA clone is G + C-rich and contains two tandem copies of a G + C-rich direct repeat.     

Isolation and sequence of cDNA clones encoding rat phosphatidylinositol transfer protein

Phosphatidylinositol (PtdIns) transfer protein is a cytosolic protein that catalyzes the transfer of PtdIns between membranes. It is expressed in organisms from yeast to man, and activity has been found in all animal tissues examined. Using antibodies prepared against bovine brain PtdIns transfer protein, lambda gt11 rat brain cDNA libraries were screened and several clones isolated. DNA sequence analysis showed that the cDNAs encoded a polypeptide of 271 amino acids with a mass of 31,911 Da. Comparison of the deduced amino acid sequence with N-terminal sequence data obtained for the intact purified bovine brain protein and rat lung phospholipid transfer protein verified that the cDNAs were PtdIns transfer protein clones. The predicted protein shows no significant sequence similarity to other known (phospholipid)-binding proteins. DNA blot hybridization suggests that the rat genome may contain more than one gene encoding PtdIns transfer protein. RNA blot hybridization reveals that the PtdIns transfer protein gene is expressed at low levels in a wide variety of rat tissues; all tissues examined showed a major mRNA component of 1.9 kilobases and a minor component of 3.4 kilobases. The isolation of clones encoding rat PtdIns transfer protein will greatly facilitate studies of the structure and function of PtdIns transfer proteins and their role in lipid metabolism.     

Mechanism of the glycine cleavage reaction. Properties of the reverse reaction catalyzed by T-protein

T-protein, one of the components of the glycine cleavage system, catalyzes the synthesis of the H-protein-bound intermediate from methylenetetrahydrofolate, ammonia, and H-protein having a reduced lipoyl prosthetic group (Okamura-Ikeda, K., Fujiwara, K., and Motokawa, Y. (1982) J. Biol. Chem. 257, 135-139). Spectroscopic studies indicated that the utilization of methylenetetrahydrofolate occurred only in the presence of the three substrates, indicating the formation of a quaternary complex. The amount of methylenetetrahydrofolate consumed was equal to that of methylene carbon attached to H-protein. Steady-state kinetic studies show that the reaction proceeds through an Ordered Ter Bi mechanism. Reduced H-protein is the first substrate that binds T-protein followed by methylenetetrahydrofolate and ammonia. The order of release of products is tetrahydrofolate and the H-protein-bound intermediate. Km values for H-protein, methylenetetrahydrofolate, and ammonia are 0.55 microM, 0.32 mM, and 22 mM, respectively.     

Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.     

Isolation and sequence analysis of a cDNA encoding rat liver alpha-L-fucosidase.

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].     

The amino-terminal region of the Escherichia coli T-protein of the glycine cleavage system is essential for proper association with H-protein.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. Our previous work on Escherichia coli T-protein (ET) showed that the lack of the N-terminal 16 residues caused a loss of catalytic activity [Okamura-Ikeda, K., Ohmura, Y., Fujiwara, K. and Motokawa, Y. (1993) Eur. J. Biochem. 216, 539-548]. To define the role of the N-terminal region of ET, a series of deletion mutants were constructed by site-directed mutagenesis and expressed in E. coli. Deletions of the N-terminal 4, 7 and 11 residues led to reduction in the activity to 42, 9 and 4%, respectively, relative to the wild-type enzyme (wtET). The mutant with 7-residue deletion (ETDelta7) was purified and analyzed. ETDelta7 exhibited a marked increase in Km (25-fold) for E. coli H-protein (EH) accompanied by a 10-fold decrease in kcat compared with wtET, indicating the importance of the N-terminal region in the interaction with EH. The role of this region in the ET-EH interaction was investigated by cross-linking of wtET-EH or ETDelta7-EH complex with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker, in the presence of folate substrates. The resulting tripartite cross-linked products were cleaved with lysylendopeptidase and V8 protease. After purification by reversed-phase HPLC, the cross-linked peptides were subjected to Edman sequencing. An intramolecular cross-linking between Asp34 and Lys216 of wtET which was not observed in wtET alone and an intermolecular cross-linking between Lys288 of wtET and Asp-43 of EH were identified. In contrast, no such cross-linking was detected from the cross-linked product of ETDelta7. These results suggest that EH, when it interacts with ET, causes a change in conformation of ET and that the N-terminal region of ET is essential for the conformational change leading to the proper interaction with EH.     

Isolation and sequence of a cDNA encoding the major structural protein of peripheral myelin

The myelin sheath is a multilayered membrane, unique to the nervous system, which functions as an insulator to increase greatly the velocity of axonal impulse conduction. We have used the techniques of differential screening and hybrid selection to identify a cDNA clone encoding the Schwann cell glycoprotein P0, the major structural protein of the peripheral myelin sheath. The sequence of this protein, deduced from the nucleotide sequence of the cloned cDNA, indicates that P0 is an integral membrane protein containing a single membrane-spanning region, a large hydrophobic extracellular domain, and a smaller basic intracellular domain. The structure of the protein suggests that each of these domains plays an essential role in generating the highly ordered structure of the myelin sheath. Furthermore, we find that the induction of P0 mRNA coincides with the initiation of myelin formation, and we propose a model in which the glycoprotein serves as a molecular guidepost for this process.     

Isolation and sequence of cDNA encoding the soybean protease inhibitors PI IV and C-II

Two full-length (or nearly so) cDNA clones containing information for the protease inhibitors PI IV and C-II from soybean seeds were identified by means of a synthetic probe. DNA sequencing revealed that the two protease inhibitors are synthesized as precursors with a short peptide leader. The coding regions of the two clones show 80% homology, wheraes the 5 non-coding regions are 90% homologous. Homology of 75% is found in the region extending beyond the stop codons.     

Crystal structure of human T-protein of glycine cleavage system at 2.0 A resolution and its implication for understanding non-ketotic hyperglycinemia

T-protein, a component of the glycine cleavage system, catalyzes the formation of ammonia and 5,10-methylenetetrahydrofolate from the aminomethyl moiety of glycine attached to the lipoate cofactor of H-protein. Several mutations in the human T-protein gene cause non-ketotic hyperglycinemia. To gain insights into the effect of disease-causing mutations and the catalytic mechanism at the molecular level, crystal structures of human T-protein in free form and that bound to 5-methyltetrahydrofolate (5-CH3-H4folate) have been determined at 2.0 A and 2.6 A resolution, respectively. The overall structure consists of three domains arranged in a cloverleaf-like structure with the central cavity, where 5-CH3-H4folate is bound in a kinked shape with the pteridine group deeply buried into the hydrophobic pocket and the glutamyl group pointed to the C-terminal side surface. Most of the disease-related residues cluster around the cavity, forming extensive hydrogen bonding networks. These hydrogen bonding networks are employed in holding not only the folate-binding space but also the positions and the orientations of alpha-helix G and the following loop in the middle region, which seems to play a pivotal role in the T-protein catalysis. Structural and mutational analyses demonstrated that Arg292 interacts through water molecules with the folate polyglutamate tail, and that the invariant Asp101, located close to the N10 group of 5-CH3-H4folate, might play a key role in the initiation of the catalysis by increasing the nucleophilic character of the N10 atom of the folate substrate for the nucleophilic attack on the aminomethyl lipoate intermediate. A clever mechanism of recruiting the aminomethyl lipoate arm to the reaction site seems to function as a way of avoiding the release of toxic formaldehyde.