Characterization of the human prolyl 4-hydroxylase tetramer and its multifunctional protein disulfide-isomerase subunit synthesized in a baculovirus expression system.

Prolyl 4-hydroxylase (EC 1.14.11.2), an alpha 2 beta 2 tetramer, catalyzes the posttranslational formation of 4-hydroxyproline in collagens. The enzyme can easily be dissociated into its subunits, but all attempts to associate a tetramer from the dissociated subunits in vitro have been unsuccessful. Molecular cloning of the catalytically important alpha subunit has identified two types of cDNA clone due to mutually exclusive alternative splicing. The beta subunit is a highly unusual multifunctional polypeptide, being identical to the enzyme protein disulfide-isomerase (EC 5.3.4.1). We report here on expression of the alpha and beta subunits of prolyl 4-hydroxylase and a fully active enzyme tetramer in Spodoptera frugiperda insect cells by baculovirus vectors. When the beta subunit was expressed alone, the polypeptide produced was found in a 0.1% Triton X-100 extract of the cell homogenate and was a fully active protein disulfide-isomerase. When either form of the alpha subunit was expressed alone, only traces of the alpha subunit could be extracted from the cell homogenate with 0.1% Triton X-100, and 1% SDS was required to obtain efficient solubilization. These alpha subunits had no prolyl 4-hydroxylase activity. When the cells were coinfected with both alpha- and beta-subunit-producing viruses, an enzyme tetramer was formed, but significant amounts of alpha and beta subunits remained unassociated. The recombinant tetramer was indistinguishable from that isolated from vertebrate tissue in terms of its specific activity and kinetic constants for cosubstrates and the peptide substrate. The two alternatively spliced forms of the alpha subunit gave enzyme tetramers with identical catalytic properties. Baculovirus expression seems to be an excellent system for mass production of the enzyme tetramer and for detailed investigation of the mechanisms involved in the association of the monomers.     

Prolyl 4-hydroxylase: molecular cloning and the primary structure of the alpha subunit from chicken embryo.

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme required for the posttranslational hydroxylation of proline residues in collagen. The enzyme is a tetramer composed of two pairs of nonidentical subunits (alpha 2 beta 2). The beta subunit is protein disulfide-isomerase, a ubiquitous enzyme found in the endoplasmic reticulum of many cell types. We report here the amino acid sequence of the alpha subunit. One cDNA clone (alpha 1) was isolated from a chicken embryo cDNA expression library in lambda gt11 by screening with anti-alpha-subunit polyclonal immunoglobulins. This alpha 1 cDNA contains an open reading frame of 1401 base pairs. A comparison of the translation of the nucleotide sequence with protein sequences obtained from the purified chicken alpha-subunit polypeptide verified that alpha 1 cDNA encoded the alpha subunit. Polymerase chain reactions were used to extend the sequence of alpha 1 cDNA toward the 5' end of alpha-subunit mRNA. The mature alpha subunit is composed of 516 amino acids with a calculated molecular mass of 59,373 Da. The compiled amino acid sequence contains two potential glycosylation sites, an observation that agrees with a previous demonstration that the alpha subunit contains two N-linked oligosaccharide chains. Blot hybridization analysis of total chicken embryo RNA detected an mRNA of 3.5 kilobases, a size that closely resembles the size of the cloned cDNA. Since the expression of the alpha subunit is confined to cell types that synthesize and secrete collagens, the regulation of the synthesis of the alpha subunit may play a central role in determining the expression of prolyl 4-hydroxylase activity.     

Structure of an intermolecular electron-transfer complex: p-cresol methylhydroxylase at 6.0-A resolution

The structure of p-cresol methylhydroxylase [4-cresol:(acceptor) oxidoreductase (methyl-hydroxylating), EC 1.17.99.1], a flavocytochrome c, has been determined at 6.0-A resolution. The structure analysis is based on two heavy-atom derivatives with anomalous scattering and 2-fold averaging about a noncrystallographic axis. The molecule is an alpha 2 beta 2 tetramer with a cytochrome subunit of Mr approximately 8500 and a flavoprotein subunit of Mr approximately 49,000. The flavoprotein subunits are tightly packed about the molecular 2-fold axis, whereas the cytochrome subunits are located on the outside of the molecule, each in a depression on the surface of a flavoprotein subunit. The results of this study have led to the following conclusions. The alpha 2 beta 2 quaternary structure of the enzyme is different from alpha beta as originally thought. The orientation of the cytochrome subunit and the surface complementarity of the cytochrome and flavoprotein subunits are clearly defined. The cytochrome subunit is similar in size to other small bacterial cytochromes but probably forms a distinct subclass. The titration (by substrate) behavior of the enzyme and other kinetic properties are rationalized by its quaternary structure.     

Expression cloning of a CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1''Cer alpha 2,8-sialyltransferase (GD3 synthase) from human melanoma cells.

Using an expression cloning approach, we have isolated a cDNA encoding GD3 synthase (CMP-NeuAc:NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer alpha 2,8-sialyltransferase, EC 2.4.99.8), which is a key regulatory enzyme determining the prominence of the ganglioside biosynthesis pathway. The cloned cDNA encodes a 341-amino acid protein containing a single transmembrane domain at its N-terminal region, suggesting that the protein has a type II transmembrane topology. The sequence of alpha 2,8-sialyltransferase showed a high level of similarity with other sialyltransferases at two conserved regions typical in the sialyltransferase family. Transfected cells containing the cloned cDNA expressed GD3 ganglioside on the cell surface, which was detectable with specific anti-GD3 antibody by immunofluorescence and immunostaining after separation of isolated glycolipids on thin-layer chromatography. The cDNA hybridized to a single mRNA species of 2.4 kb in melanoma cells. This sialyltransferase is distinctive in catalyzing the formation of the alpha 2-8 linkage of sialic acids.     

Methionine adenosyltransferase II beta subunit gene expression provides a proliferative advantage in human hepatoma

BACKGROUND & AIMS: Of the 2 genes (MAT1A, MAT2A) encoding methionine adenosyltransferase, the enzyme that synthesizes S-adenosylmethionine, MAT1A, is expressed in liver, whereas MAT2A is expressed in extrahepatic tissues. In liver, MAT2A expression associates with growth, dedifferentiation, and cancer. Here, we identified the beta subunit as a regulator of proliferation in human hepatoma cell lines. The beta subunit has been cloned and shown to lower the K(m) of methionine adenosyltransferase II alpha2 (the MAT2A product) for methionine and to render the enzyme more susceptible to S-adenosylmethionine inhibition. METHODS: Methionine adenosyltransferase II alpha2 and beta subunit expression was analyzed in human and rat liver and hepatoma cell lines and their interaction studied in HuH7 cells. beta Subunit expression was up- and down-regulated in human hepatoma cell lines and the effect on DNA synthesis determined. RESULTS: We found that beta subunit is expressed in rat extrahepatic tissues but not in normal liver. In human liver, beta subunit expression associates with cirrhosis and hepatoma. beta Subunit is expressed in most (HepG2, PLC, and Hep3B) but not all (HuH7) hepatoma cell lines. Transfection of beta subunit reduced S-adenosylmethionine content and stimulated DNA synthesis in HuH7 cells, whereas down-regulation of beta subunit expression diminished DNA synthesis in HepG2. The interaction between methionine adenosyltransferase II alpha2 and beta subunit was demonstrated in HuH7 cells. CONCLUSIONS: Our findings indicate that beta subunit associates with cirrhosis and cancer providing a proliferative advantage in hepatoma cells through its interaction with methionine adenosyltransferase II alpha2 and down-regulation of S-adenosylmethionine levels.     

The alpha and beta subunits of phosphorylase kinase are homologous: cDNA cloning and primary structure of the beta subunit.

We have cloned cDNA molecules encoding the beta subunit of phosphorylase kinase (ATP:phosphorylase-b phosphotransferase; EC 2.7.1.38) from rabbit fast-twitch skeletal muscle and have determined the complete primary structure of the polypeptide by a combination of peptide and DNA sequencing. In the mature beta subunit, the initial methionine is replaced by an acetyl group. The subunit is composed of 1092 amino acids and has a calculated molecular mass of 125,205 Da. Alignment of its sequence with the alpha subunit of phosphorylase kinase reveals extensive regions of homology, but each molecule also possesses unique sequences. Two of the three phosphorylation sites known for the beta subunit and all seven phosphorylation sites known for the alpha subunit are located in these unique domains.     

Ouabain-binding-site photoaffinity probes that label both subunits of Na+,K+-ATPase.

4"'-Diazomalonyldigitoxin and its isomer, 3"'-diazomalonyldigitoxin, have been synthesized at high specific radioactivity and used as photolabels for the Na,K-ATPase (ATP phosphohydrolase, EC 3.6.1.3) purified from Electrophorus electricus. Photoaffinity labeling experiments using both type I and type II complexes of enzyme with both photolabels showed ouabain-protectable labeling of the alpha as well as the beta subunit. These data suggest that, in the purified eel enzyme, the alpha and beta subunits are in intimate contact, at least in the region of the third digitoxose of the "sugar-specific" binding site.     

Pertussis toxin substrate, the putative Ni component of adenylyl cyclases, is an alpha beta heterodimer regulated by guanine nucleotide and magnesium.

The final step in a scheme for the purification of the guanine nucleotide- and Mg2+-binding stimulatory regulatory component (Ns) of adenylyl cyclase [adenylate cyclase; ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from human erythrocyte membranes involves chromatography over hydroxylapatite (HAP) which yields two fractions. The first fraction (HAP I) contains predominantly two peptides that, upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis, migrate with Mr values of 39,000 and 35,000. The second fraction (HAP II) contains predominantly Ns formed of two peptides of Mr 42,000 and 35,000. The HAP I, Mr 39,000 peptide is shown to be a substrate for the ADP-ribosylating toxin of Bordetella pertussis (pertussis toxin). Upon sucrose density gradient centrifugation, both the Mr 39,000 and the Mr 35,000 peptides of HAP I migrate at about 4 S. Treatment of HAP I with guanine nucleotide and Mg2+ prior to centrifugation results in a coordinated change in the migration of both peptides to 2 S. It is postulated that HAP I contains an alpha beta heterodimeric protein composed of an alpha subunit of Mr 39,000 and a beta subunit of Mr 35,000. Further, this protein dissociates under the influence of guanine nucleotides and Mg2+ into its individual alpha and beta subunits. Because previous studies have shown that treatment of cells and cell membranes with pertussis toxin results in attenuation of the effects of hormones that inhibit adenylyl cyclase activity, and because this effect correlates with the ADP-ribosylation of a Mr approximately equal to 40,000 peptide, we believe that we have purified a guanine nucleotide- and Mg2+-binding inhibitory regulatory component of adenylyl cyclases--i.e., the Ni.     

Prolyl 4-hydroxylase is required for viability and morphogenesis in Caenorhabditis elegans

The genome of Caenorhabditis elegans possesses two genes, dpy-18 and phy-2, that encode alpha subunits of the enzyme prolyl 4-hydroxylase. We have generated deletions within each gene to eliminate prolyl 4-hydroxylase activity from the animal. The dpy-18 mutant has an aberrant body morphology, consistent with a role of prolyl 4-hydroxylase in formation of the body cuticle. The phy-2 mutant is phenotypically wild type. However, the dpy-18; phy-2 double mutant is not viable, suggesting an essential role for prolyl 4-hydroxylase that is normally accomplished by either dpy-18 or phy-2. The effects of the double mutation were mimicked by small-molecule inhibitors of prolyl 4-hydroxylase, validating the genetic results and suggesting that C. elegans can serve as a model system for the discovery of new inhibitors.     

Evidence for two dissimilar polypeptide chains in the beta 2 subunit of hexosaminidase.

The major isoenzymes of human hexosaminidase have the structures alpha beta 2 (hex A) and 2 beta 2 (hex B). In this study, we present evidence that the beta 2 subunit of hex B and hex BA (the form of hex B derived from hex A) is composed of two nonidentical polypeptide chains. We have called these chains beta a and beta b. They have similar molecular weights (25,000) but have pI values that differ by 1 unit. We have used a two-dimensional analytical gel electrophoresis method in combination with peptide mapping to compare the primary sequence structure of the two beta chains. In this method, the polypeptide chains of hex B or hex BA were first separated by isoelectric focusing in 8.5 M urea. The separated chains were subjected to partial proleolytic digestion in the stacking gel of a second NaDodSO4/polyacrylamide gel with subsequent separation of peptides by electrophoresis into the second gel. Partial digestion by protease V8 or papain showed that the beta a and beta b species have distinct primary structures, neither of which was similar to that of the alpha chain. On the basis of these results, we suggest that the beta 2 subunit of hexosaminidase has the structure of beta a beta b. The possibility that the distinct beta chains are encoded by a single gene is discussed in the light of genetic and other data.     

Three-dimensional structure of holo 3 alpha,20 beta-hydroxysteroid dehydrogenase: a member of a short-chain dehydrogenase family.

The x-ray structure of a short-chain dehydrogenase, the bacterial holo 3 alpha,20 beta-hydroxysteroid dehydrogenase (EC 1.1.1.53), is described at 2.6 A resolution. This enzyme is active as a tetramer and crystallizes with four identical subunits in the asymmetric unit. It has the alpha/beta fold characteristic of the dinucleotide binding region. The fold of the rest of the subunit, the quaternary structure, and the nature of the cofactor-enzyme interactions are, however, significantly different from those observed in the long-chain dehydrogenases. The architecture of the postulated active site is consistent with the observed stereospecificity of the enzyme and the fact that the tetramer is the active form. There is only one cofactor and one substrate-binding site per subunit; the specificity for both 3 alpha- and 20 beta-ends of the steroid results from the binding of the steroid in two orientations near the same cofactor at the same catalytic site.     

Genes encoding alpha and beta subunits of Na,K-ATPase are located on three different chromosomes in the mouse.

We have made use of a panel of mouse-hamster somatic cell hybrids and restriction fragment length polymorphisms between two mouse species (Mus musculus and Mus spretus) to determine the chromosomal localization of genes encoding the alpha and beta subunits of the Na,K-ATPase (Na+,K+-activated ATP phosphohydrolase, EC 3.6.1.3). DNA probes for three distinct isoforms of the Na,K-ATPase alpha subunit mapped to three different mouse chromosomes: the alpha 1 gene (Atpa-1) cosegregated with the Egf gene on chromosome 3; alpha 2 (Atpa-2) with the cytochrome P-450PB gene family/coumarin hydroxylase locus on chromosome 7; alpha 3 (Atpa-3) with the alpha-spectrin gene on chromosome 1. The Na,K-ATPase beta-subunit gene (Atpb) mapped to the same region of chromosome 1, but it was not tightly linked to the Atpa-3 gene. These results indicate that three isoforms of the Na,K-ATPase alpha subunit are encoded by three distinct genes. The dispersion of Na,K-ATPase genes suggests that their expression is not likely to be controlled by a common cis-acting regulatory element.     

Assignment of the genes for the alpha and beta subunits of thyrotropin to different mouse chromosomes.

A series of mouse-hamster somatic cell hybrids, containing reduced numbers of mouse chromosomes and a complete set of hamster chromosomes, was used to determine the chromosomal locations of the genes for the alpha and beta subunits of mouse thyrotropin. Cloned cDNA probes for each subunit, in conjunction with Southern blot analysis of DNA treated with the restriction enzyme BamHI, allowed for assignment of the alpha-subunit gene to mouse chromosome 4 and of the beta-subunit gene to chromosome 3. Mouse alpha-subunit gene sequences always segregated with chromosome 4 (concordant in 14 hybrids) and the enzyme markers phosphoglucomutase 2 and 6-phosphogluconate dehydrogenase. Mouse beta-subunit gene sequences always segregated with chromosome 3 (concordant in 15 hybrids). Thus, the genes for at least one of the glycoprotein hormones, thyrotropin, are on different chromosomes.     

Role of interferon alpha/beta receptor chain 1 in the structure and transmembrane signaling of the interferon alpha/beta receptor complex.

A previously cloned cDNA encodes one subunit of the human interferon alpha/beta receptor (IFN alpha R), denoted IFN alpha R1. To study the expression and signaling of IFN alpha R1, we used monoclonal antibodies (mAbs) generated against the baculovirus-expressed ectodomain of IFN alpha R1. Immunoprecipitation and immunoblotting of lysates from a variety of human cell lines showed that IFN alpha R1 has an apparent molecular mass of 135 kDa. Binding analysis with 125I-labeled mAb demonstrated high levels of cell surface expression of IFN alpha R1 in human cells and in mouse cells transfected with IFN alpha R1 cDNA, whereas no cross-reactivity was observed in control mouse L929 cells expressing only the endogenous mouse receptor. The subunit was rapidly down-regulated by IFN alpha (80% decrease within 2 hr) and degraded upon internalization. The IFN alpha R1 chain appeared to be constitutively associated with the 115-kDa subunit of the IFN alpha/beta receptor, since the mAbs coprecipitated this protein. IFN alpha/beta treatment induced tyrosine phosphorylation of IFN alpha R1 within 1 min, with kinetics paralleling that of the IFN-activated protein-tyrosine kinases Jak1 and Tyk2. Ligand-induced tyrosine phosphorylation of IFN alpha R1 was blocked by the kinase inhibitors genistein or staurosporine. Although IFN alpha R1 cDNA-transfected mouse cells expressed high levels of this subunit when compared with empty vector-transfected cells the number of binding sites for human IFN alpha (50-75 sites per cell) was not increased. Human IFN alpha induced the expression of a mouse IFN alpha/beta-responsive gene (the 204 gene) in mouse L929 cells transfected with the IFN alpha R1 cDNA, but not in mock-transfected cells. These results suggest that the IFN alpha R1 subunit acts as a species-specific signal transduction component of the IFN alpha/beta receptor complex.     

Protein kinase CK2 as an ectokinase: the role of the regulatory CK2beta subunit

Protein kinase CK2 (also known as casein kinase 2) is present in the cytoplasm, nuclei, and several other organelles. In addition, this enzyme has been found bound to the external side of the cell membrane where it acts as an ectokinase phosphorylating several extracellular proteins. Previous experiments with transfection of HEK-293T cells demonstrated that expression of both subunits, CK2alpha (catalytic) and CK2beta (regulatory), was necessary for the appearance of the ectopic enzyme as an ectokinase. In this work, using deletion and point mutations of CK2beta, it was possible to demonstrate that the region between amino acids 20 and 33 was necessary for the export of the enzyme as an ectokinase. Phenylalanines 21 and 22 and acidic residues in positions 26-28 are involved in the structural aspects that are required for export. However, the region encompassing amino acids 20-33 of CK2beta is not sufficient to make the carboxyl half of this subunit functional in bringing CK2 to the ectokinase locus. In cells transfected with only CK2beta, it was demonstrated that 3-4% of the subunit is exported to the cell medium, but the subunit is not bound to the external membrane.     

Subunits of purified calcium channels: a 212-kDa form of alpha 1 and partial amino acid sequence of a phosphorylation site of an independent beta subunit.

Antibodies prepared against peptides CP2, CP4, and CP5, which occur within the first 1522 amino acid residues of the alpha 1 subunit of dihydropyridine-sensitive skeletal muscle calcium channels, specifically recognized a 175-kDa form of the alpha 1 subunit in immunoblots and immunoprecipitation experiments. In contrast, antibodies prepared against peptide CP1, which represents the C-terminal 18 amino acid residues predicted by cloning and sequence analysis of the alpha 1 subunit, recognized a minor, previously undescribed 212-kDa protein, which is the size predicted for the full length of the alpha 1 subunit from cDNA cloning [Tanabe, T., Takeshima, H., Mikami, A., Flockerzi, V., Takahashi, H., Kangawa, K., Kojima, M., Matsuo, H., Hirose, T. & Numa, S. (1987) Nature (London) 328, 313-318]. Both the 175-kDa and 212-kDa forms were phosphorylated by cAMP-dependent protein kinase and both were present in isolated transverse tubule membranes. The 175-kDa form may arise from posttranslational proteolytic cleavage of the C terminus of the 212-kDa form of the alpha 1 subunit predicted by cDNA cloning and sequence analysis. Partial amino acid sequencing of the 54-kDa beta subunit of the calcium channel indicated this protein was not derived from the proteolytically cleaved C terminus of the alpha 1 subunit. This analysis identified a threonine residue in the sequence (Lys/Arg)-Arg-Pro-Thr-Pro of the beta subunit that was phosphorylated by cAMP-dependent protein kinase. Phosphorylation of this residue in the beta subunit may play a role in modulation of calcium channel function. Separate functional roles of the 175-kDa form of the alpha 1 subunit in excitation-contraction coupling and of the 212-kDa form in ion conductance are proposed.     

Integrins alpha1beta1 and alpha2beta1 are receptors for the rotavirus enterotoxin

Rotavirus NSP4 is a viral enterotoxin capable of causing diarrhea in neonatal mice. This process is initiated by the binding of extracellular NSP4 to target molecule(s) on the cell surface that triggers a signaling cascade leading to diarrhea. We now report that the integrins alpha1beta1 and alpha2beta1 are receptors for NSP4. NSP4 specifically binds to the alpha1 and alpha2 I domains with apparent K(d) = 1-2.7 muM. Binding is mediated by the I domain metal ion-dependent adhesion site motif, requires Mg(2+) or Mn(2+), is abolished with EDTA, and an NSP4 point mutant, E(120)A, fails to bind alpha2 integrin I domain. NSP4 has two distinct integrin interaction domains. NSP4 amino acids 114-130 are essential for binding to the I domain, and NSP4 peptide 114-135 blocks binding of the natural ligand, collagen I, to integrin alpha2. NSP4 amino acids 131-140 are not associated with the initial binding to the I domain, but elicit signaling that leads to the spreading of attached C2C12-alpha2 cells, mouse myoblast cells stably expressing the human alpha2 integrin. NSP4 colocalizes with integrin alpha2 on the basolateral surface of rotavirus-infected polarized intestinal epithelial (Caco-2) cells as well as surrounding noninfected cells. NSP4 mutants that fail to bind or signal through integrin alpha2 were attenuated in diarrhea induction in neonatal mice. These results indicate that NSP4 interaction with integrin alpha1 and alpha2 is an important component of enterotoxin function and rotavirus pathogenesis, further distinguishing this viral virulence factor from other microbial enterotoxins.