Molecular cloning of the human thyrotropin-beta subunit gene

Genomic DNA fragments that carried a gene for human thyrotropin-beta (hTSH beta) subunit were isolated. Nucleotide sequence analysis of the gene showed that the hTSH beta subunit precursor consists of 138 amino acid residues. There is an N-terminal sequence of 20 amino acids as a signal peptide, followed by 112 amino acids, whose sequence is in agreement with that known for the secretory form of hTSH beta subunit. This is followed by an additional stretch of 6 hydrophobic amino acids, which may be eliminated post-translationally. The coding region is separated by an intron of about 460 bp. Genomic Southern blot hybridization analysis suggested that the hTSH beta gene is a unique single copy gene.     

Analysis of the cleavage site specificity of the endopeptidase involved in the maturation of the large subunit of hydrogenase 3 from Escherichia coli

The maturation of [NiFe]-hydrogenases is a catalysed process in which the activities of at least seven proteins are involved. The last step consists of the endoproteolytic cleavage of the precursor of the large subunit after the [NiFe]-metal centre has been assembled. The amino acid sequence requirements for the endopeptidase HycI involved in the C-terminal processing of HycE, the large subunit of the hydrogenase 3 from Escherichia coli, were investigated. Mutational alteration of the amino acid residues neighbouring the cleavage site showed that proteolysis still occurred when chemically similar amino acids were exchanged. Processing was blocked, however, in a variant in which the methionine at the C-terminal side was replaced by a glutamate residue. Truncation of the precursor from the C-terminal end rendered variants amenable to maturation even when two-thirds of the extension were removed but abolished proteolysis upon further deletion of a cluster of six basic amino acids. A construct in which the C-terminal extension from the large subunit of the hydrogenase 2 was fused to the mature part of the large subunit of hydrogenase 3 was neither processed by HycI nor by HybD, the endopeptidase specific for the large subunit of hydrogenase 2. The maturation endopeptidase, therefore, exhibits a relaxed sequence constraint in recognition of its cleavage site and does not require the entire C-terminal extension. The results point to an interaction of the C-terminus with some domain of the large subunit, rendering a conformation amenable to recognition by the endopeptidase.     

Determination of the sidedness of the C-terminal region of the gastric H,K-ATPase alpha subunit.

It cannot be predicted from hydropathy analysis whether the C-terminal end of the alpha subunit of the gastric H,K-ATPase is cytoplasmic or extracytoplasmic. The sideness of the C-terminal amino acids was determined by taking advantage of the two C-terminal tyrosines in the primary sequence of the enzyme. Intact, cytoplasmic side out vesicles derived from hog gastric mucosa or detergent solubilized vesicles were iodinated by the lactoperoxidase method and then the C-terminal amino acids hydrolyzed by carboxypeptidase Y. The alpha and beta subunits were separated by SDS gel electrophoresis. The level of iodination of the alpha subunit following solubilization was about three fold greater than when intact vesicles were iodinated, and the beta subunit was iodinated only when solubilized enzyme was used. Carboxypeptidase Y removed 28 +/- 4% of the radioactivity from the alpha subunit iodinated in intact vesicles. These data are consistent with a cytoplasmic location of the C-terminal amino acids of the alpha subunit and with a mostly extracytoplasmic location of the amino acids of the beta subunit.     

Cloning, primary structure and properties of a novel human integrin beta subunit.

The originally described integrin beta subunits that define the three subfamilies of integrin heterodimers are beta 1, beta 2 and beta 3. In this paper, we describe the isolation of a cDNA coding for a novel human integrin beta subunit, designated as beta 5. The beta 5 cDNA was isolated from a human thymic epithelial cell library, using oligonucleotide probes that were designed from a region highly conserved among the known beta 1, beta 2 and beta 3 sequences. The beta 5 cDNA codes for 799 (or 796) amino acids, including a 23 amino acid leader sequence. There are 776 (or 773) amino acids in the mature protein, which includes a long extracellular domain of 696 amino acids, a transmembrane domain and an intracellular C-terminal domain of 57 amino acids. The beta 5 sequence resembled the known beta 3, beta 1 and beta 2 sequences by 55, 43 and 38%, respectively, including conservation of 56/56 cysteines. Rabbit antiserum was prepared against a 20 amino acid synthetic peptide predicted from the beta 5 C-terminal sequence. This serum immunoprecipitated a beta 5 protein that was 100,000 Mr (reduced) and 95,000 Mr (nonreduced). Only a single alpha subunit was detected in association with beta 5, and that alpha subunit was immunochemically indistinguishable from the alpha v subunit previously found as part of the vitronectin receptor complex. By immunoprecipitation, beta 5 was most prevalent on carcinoma cell lines, was also present on hepatoma and fibroblast cell lines, and was absent from lymphoblastoid cells and platelets.     

Functionally important conserved length of C-terminal regions of yeast and bovine ADP/ATP carriers, identified by deletion mutants studies, and water accessibility of the amino acids at the C-terminal region of the yeast carrier

Comparison of the amino acid sequence of yeast type 2 ADP/ATP carrier (yAAC2) with that of bovine type 1 AAC (bAAC1) revealed that the N- and C-terminus of yAAC2 are 15- and 6-amino acids longer, respectively, than those of bAAC1. In the present study, we focused on the difference in the C-terminal region between yAAC2 and bAAC1. Deletion of first six residues of C-terminus of yAAC did not markedly affect the function of yAAC2; however, further deletion of 1 amino acid (7th amino acid from the C-terminus) destroyed its function. On the contrary, deletion of the first amino acid residue of the C-terminus of bAAC1 caused failure of its functional expression in yeast mitochondria. Based on these results, we concluded that the 6-amino acid residue extension of the C-terminus of yAAC2 was not necessary for the function of this carrier and that the remainder of the C-terminal region of yAAC2, having a length conserved with that of bAAC1, is important for the transport function of AACs. We next prepared various single-Cys mutants in which each of 32 residues in the C-terminus of yAAC2 was replaced by a Cys residue. Since all mutants were successfully expressed in yeast mitochondria, we examined the reactivity of these cysteine residues with the membrane-impermeable sulfhydryl reagent eosin 5-maleimide (EMA). As a result, all cysteine residues that replaced the 9 continuous amino acids in Met310-Lys318 showed high reactivity with EMA regardless of the presence of carboxyatractyloside or bongkrekic acid; and so this region was concluded to be exposed to the water-accessible environment. Furthermore, based on the reactivities of cysteine residues that replaced amino acids in the sixth transmembrane segment, the probable structural features of the C-terminal region of this carrier in the presence of bongkrekic acid were discussed.     

The C-terminus of the gamma 2 chain but not of the beta 3 chain of laminin-332 is indirectly but indispensably necessary for integrin-mediated cell reactions

Using a recombinant mini-laminin-332, we showed that truncation of the three C-terminal amino acids of the gamma 2 chain, but not of the C-terminal amino acid of the beta 3 chain, completely abolished alpha 3 beta 1 integrin binding and its cellular functions, such as attachment and spreading. However, a synthetic peptide mimicking the gamma 2 chain C-terminus did not interfere with alpha 3 beta 1 integrin binding or cell adhesion and spreading on laminin-332 as measured by protein interaction assays and electric cell-substrate impedance sensing. Nor was the soluble peptide able to restore the loss of integrin-mediated cell adhesiveness to mini-laminin-332 after deletion of the gamma 2 chain C-terminus. These findings spoke against the hypothesis that the gamma 2 chain C-terminus of laminin-332 is a part of the alpha 3 beta 1 integrin interaction site. In addition, structural studies with electron microscopy showed that truncation of the gamma 2 chain C-terminus opened up the compact supradomain structure of LG1-3 domains. Thus, by inducing or stabilizing an integrin binding-competent conformation or array of the LG1-3 domains, the gamma 2 chain C-terminus plays an indirect but essential role in laminin-332 recognition by alpha 3 beta 1 integrin and, hence, its cellular functions.     

Amino acid sequence of the human fibronectin receptor

The amino acid sequence deduced from cDNA of the human placental fibronectin receptor is reported. The receptor is composed of two subunits: an alpha subunit of 1,008 amino acids which is processed into two polypeptides disulfide bonded to one another, and a beta subunit of 778 amino acids. Each subunit has near its COOH terminus a hydrophobic segment. This and other sequence features suggest a structure for the receptor in which the hydrophobic segments serve as transmembrane domains anchoring each subunit to the membrane and dividing each into a large ectodomain and a short cytoplasmic domain. The alpha subunit ectodomain has five sequence elements homologous to consensus Ca2+-binding sites of several calcium-binding proteins, and the beta subunit contains a fourfold repeat strikingly rich in cysteine. The alpha subunit sequence is 46% homologous to the alpha subunit of the vitronectin receptor. The beta subunit is 44% homologous to the human platelet adhesion receptor subunit IIIa and 47% homologous to a leukocyte adhesion receptor beta subunit. The high degree of homology (85%) of the beta subunit with one of the polypeptides of a chicken adhesion receptor complex referred to as integrin complex strongly suggests that the latter polypeptide is the chicken homologue of the fibronectin receptor beta subunit. These receptor subunit homologies define a superfamily of adhesion receptors. The availability of the entire protein sequence for the fibronectin receptor will facilitate studies on the functions of these receptors.     

Characterization of a novel synGAP isoform, synGAP-beta

We cloned a cDNA encoding a novel synGAP, synGAP-d (GenBank(TM) accession number ), from a rat brain cDNA library. The clone consisted of 4801 nucleotides with a coding sequence of 3501 nucleotides, encoded a protein consisting of 1166 amino acids with >99% homology with 1092 amino acid overlaps to synGAP, and contained a 13-nucleotide insertion to the previously reported synGAP mRNAs, which suggested that the clone was a splice variant of synGAP. We also found that there are at least seven variants in the 3' portion of the synGAP mRNA and that they encoded five different protein isoforms. The coding sequence of these C-terminal variants were classified into alpha1, alpha2, beta1, beta2, beta3, beta4, and gamma, and synGAP-d was classified as the beta1 form. The previously reported synGAPs (synGAP-a, -b, and -c and p135synGAP) can be classified as the alpha1 isoform. All isoforms were expressed specifically in the brain. Unexpectedly, the beta isoform, which lacks a C-terminal PSD-95-binding motif ((S/T)XV), was more restricted to the postsynaptic density fraction than the motif-containing alpha1 isoform. The beta isoform did not interact with PSD-95 but specifically interacted with a nonphosphorylated alpha subunit of Ca(2+)/calmodulin-dependent protein kinase II through its unique C-terminal tail.     

Topology of subunits of the mammalian cytochrome c oxidase: relationship to the assembly of the enzyme complex.

The arrangement of three subunits of beef heart cytochrome c oxidase, subunits Va, VIa, and VIII, has been explored by chemical labeling and protease digestion studies. Subunit Va is an extrinsic protein located on the C side of the mitochondrial inner membrane. This subunit was found to label with N-(4-azido-2-nitrophenyl)-2-aminoethane[35S]sulfonate and sodium methyl 4-[3H]formylphenyl phosphate in reconstituted vesicles in which 90% of cytochrome c oxidase complexes were oriented with the C domain outermost. Subunit VIa was cleaved by trypsin both in these reconstituted vesicles and in submitochondrial particles, indicating a transmembrane orientation. The epitope for a monoclonal antibody (mAb) to subunit VIa was lost or destroyed when cleavage occurred in reconstituted vesicles. This epitope was localized to the C-terminal part of the subunit by antibody binding to a fusion protein consisting of glutathione S-transferase (G-ST) and the C-terminal amino acids 55-85 of subunit VIa. No antibody binding was obtained with a fusion protein containing G-ST and the N-terminal amino acids 1-55. The mAb reaction orients subunit VIa with its C-terminus in the C domain. Subunit VIII was cleaved by trypsin in submitochondrial particles but not in reconstituted vesicles. N-Terminal sequencing of the subunit VIII cleavage product from submitochondrial particles gave the same sequence as the untreated subunit, i.e., ITA, indicating that it is the C-terminus which is cleaved from the M side. Subunits Va and VIII each contain N-terminal extensions or leader sequences in the precursor polypeptides; subunit VIa is made without an N-terminal extension.     

The intracellular segment of the sodium channel beta 1 subunit is required for its efficient association with the channel alpha subunit

Sodium channels consist of a pore-forming alpha subunit and auxiliary beta 1 and beta 2 subunits. The subunit beta 1 alters the kinetics and voltage-dependence of sodium channels expressed in Xenopus oocytes or mammalian cells. Functional modulation in oocytes depends on specific regions in the N-terminal extracellular domain of beta 1, but does not require the intracellular C-terminal domain. Functional modulation is qualitatively different in mammalian cells, and thus could involve different molecular mechanisms. As a first step toward testing this hypothesis, we examined modulation of brain Na(V)1.2a sodium channel alpha subunits expressed in Chinese hamster lung cells by a mutant beta1 construct with 34 amino acids deleted from the C-terminus. This deletion mutation did not modulate sodium channel function in this cell system. Co-immunoprecipitation data suggest that this loss of functional modulation was caused by inefficient association of the mutant beta 1 with alpha, despite high levels of expression of the mutant protein. In Xenopus oocytes, injection of approximately 10,000 times more mutant beta 1 RNA was required to achieve the level of functional modulation observed with injection of full-length beta 1. Together, these findings suggest that the C-terminal cytoplasmic domain of beta 1 is an important determinant of beta1 binding to the sodium channel alpha subunit in both mammalian cells and Xenopus oocytes.     

The primary structure of the VLA-2/collagen receptor alpha 2 subunit (platelet GPIa): homology to other integrins and the presence of a possible collagen-binding domain

VLA-2 (also called gpIa/IIa on platelets) is a collagen receptor with a unique alpha subunit and a beta subunit common to other adhesion receptors in the VLA/integrin family. Multiple cDNA clones for the human VLA-2 alpha 2 subunit have been selected from a lambda gtll library by specific antibody screening. The 5,374-bp nucleotide sequence encoded for 1,181 amino acids, including a signal peptide of 29 amino acids followed by a long extracellular domain (1,103 amino acids), a transmembrane domain, and a short cytoplasmic segment (22 amino acids). Direct sequencing of purified alpha 2 protein confirmed the identity of the 15 NH2-terminal amino acids. Overall, the alpha 2 amino acid sequence was 18-25% similar to the sequences known for other integrin alpha subunits. In particular, the alpha 2 sequence matched other integrin alpha chains in (a) the positions of 17 of its 20 cysteine residues; (b) the presence of three metal-binding domains of the general structure DXDXDGXXD; and (c) the transmembrane domain sequence. In addition, the alpha 2 sequence has a 191-amino acid insert (called the I-domain), previously found only in leukocyte integrins of the beta 2 integrin family. The alpha 2 I-domain was 23-41% similar to domains in cartilage matrix protein and von Willebrand factor, which are perhaps associated with collagen binding. The NH2-terminal sequence reported here for alpha 2 does not match the previously reported alpha 2 NH2-terminal sequence (Takada, Y., J. L. Strominger, and M. E. Hemler. 1987. Proc. Natl. Acad. Sci. USA. 84:3239-3243). Resolution of this discrepancy suggests that there may be another VLA heterodimer that resembles VLA-2 in size but has a different amino acid sequence.     

Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus

A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.     

Characterization of the bilin attachment sites in R-phycoerythrin

The amino acid sequence around the sites of attachment of all the bilin prosthetic groups of Gastroclonium coulteri R-phycoerythrin, (alpha beta)6 gamma, have been determined. The sequences of tryptic peptides derived from the alpha and beta subunits are (Formula: see text) where the designations alpha and beta refer to the subunits from which the peptides derived. Cysteinyl residues involved in bilin attachment are indicated with an asterisk. Each peptide carries a single bilin, either phycoerythrobilin (PEB) or phycourobilin (PUB). Spectroscopic studies on the gamma subunit indicate the presence of one PEB and three PUB groups. However, five unique tryptic peptides, gamma-A through gamma-E, were characterized, indicating that Gastroclonium R-phycoerythrin is a mixture of at least two species, (alpha beta)6 gamma and (alpha beta)6 gamma', with gamma subunits differing in amino acid sequence. The sequences of the gamma subunit bilin peptides (see below) were not homologous to those from alpha and beta subunits of any biliprotein. (Formula: see text) The bilins in all these peptides are attached through single linkages to a cysteinyl residue, except for the phycourobilin on peptide beta-3 which is attached through two thioether linkages to cysteinyl residues 10 amino acids apart. The availability of small bilin peptides was exploited to obtain more accurate molar extinction coefficients for peptide-linked PEB and PUB groups. Application of these extinction coefficients in the calculation of the bilin content of R-, B-, and C-phycoerythrins shows that there are 5 bilins/alpha beta in each of these three biliprotein types.     

Studies of the functional topography of Escherichia coli RNA polymerase. A method for localization of the sites of affinity labelling

A method is proposed for localization of the sites of affinity labelling of the beta subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids. The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short-term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the beta subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-terminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed. The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the beta subunit of E. coli RNA polymerase.     

The DNA bend angle and binding affinity of an HMG box increased by the presence of short terminal arms.

The HMG box of human LEF-1 (hLEF-1, formerly TCF1alpha) has been expressed in four forms: a parent box of 81 amino acids and constructs having either a 10 amino acid C-terminal extension, a 9 amino acid N-terminal extension, or both. These four species have been compared for DNA binding and bending ability using a 28 bp recognition sequence from the TCR alpha-chain enhancer. In the bending assay, whereas the parent box and that with the N-terminal extension bent the DNA by 57/58 degrees, the box extended at the C-terminus bent the DNA by 77/78 degrees, irrespective of the presence or absence of the N-terminal extension. A 6- fold increase in DNA affinity also resulted from addition of both terminal extensions. These observations redefine the functional boundaries of the HMG box. The structure of a mouse LEF-1/DNA complex recently published [Love et al. (1995) Nature 376, 791-795] implies that the higher DNA affinity and in particular the increased bend angle observed are consequences, at least in part, of the C-terminal extension spanning the major groove on the inside of the DNA bend.     

The asparagine-linked oligosaccharides at individual glycosylation sites in human thyrotrophin.

The asparagine-linked carbohydrate structures at each of the three glycosylation sites of human thyrotrophin were investigated by 400 MHz 1H-NMR spectroscopy. Highly purified, biologically active human thyrotrophin (hTSH) was dissociated into its subunits hTSH alpha (glycosylated at Asn 52 and Asn 78) and hTSH beta (glycosylated at Asn 23). The alpha-subunit was further treated with trypsin which gave two glycopeptides that were subsequently separated by reverse-phase HPLC and identified by amino acid sequence analysis. The oligosaccharides were liberated from hTSH alpha glycopeptides and from intact hTSH beta by hydrazinolysis, and were fractionated as alditols by anion-exchange and ion-suppression amine-adsorption HPLC preparatory to structural analysis. The N-glycans present on hTSH were mainly diantennary complex-type structures with a common Man alpha 1-3 branch that terminated with 4-O-sulphated GalNAc. The Man alpha 1-6 branch displayed structural heterogeneity in the terminal sequence, with chiefly alpha 2-3-sialylated Gal and/or 4-O-sulphated GalNAc. The relative amounts of the two major complete diantennary oligosaccharides and their core fucosylation differed according to glycosylation site; the sulphated/sialylated diantennary oligosaccharide was most abundant at the two sites on the alpha-subunit, whereas the disulphated, core-fucosylated oligosaccharide was more plentiful on the beta-subunit. Some interesting structural features, not previously reported for the N-glycans of hTSH, included 3-O-sulphated galactose (SO4-3Gal) and peripheral fucose (Fuc alpha 1-3GlcNAc) in the Man alpha 1-6 branch of some diantennary structures; the former suggests the presence of a hitherto uncharacterized galactose-3-O-sulphotransferase in thyrotroph cells of the human anterior pituitary gland.     

Sequence conservation in the alpha and beta subunits of pyruvate dehydrogenase and its similarity to branched-chain alpha-keto acid dehydrogenase

Amino acid sequence comparison of 8 alpha and 6 beta subunits of the alpha-keto acid dehydrogenase (E1) component of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex form multiple species was performed by computer analysis. In addition to 2 previously recognized regions of homology in the alpha subunit, a 3rd region of extensive homology was identified in E1 alpha, and may be one of the sites involved in subunit interaction. E1 beta contains 4 regions of extensive homology. Region 1 contains 10 amino acids that are homologous to a 10-amino acid stretch in Escherichia coli E1. Regions 2 and 3 have sequence homologies with other dehydrogenases suggesting that these regions may be involved in catalysis.