Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells.

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required. We report here the partial cloning of the CMP-sialic acid:Galbeta1,3GalNAcalpha2, 3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1, 3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1, 3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.

Synthesis of bisected glycoforms of recombinant IFN-beta by overexpression of beta-1,4-N-acetylglucosaminyltransferase III in Chinese hamster ovary cells.

Genetic engineering of oligosaccharide biosynthesis pathways in mammalian cells makes possible generation of new recombinant glycoproteins of potential importance in the biopharmaceutical industry. Most prior investigations of glycosylation engineering of secreted heterologous glycoproteins involve terminal steps of oligosaccharide biosynthesis. In particular, increasing the frequency of bisected structures within the glycoform distribution has not before been considered. A Chinese hamster ovary (CHO) cell line capable of producing bisected oligosaccharides on glycoproteins was created by overexpression of a recombinant N-acetylglucosaminyltransferase III (GnT-III). Interferon beta (IFN-beta) was chosen as a model and potential therapeutic secreted heterologous protein to demonstrate the effect of recombinant GnT-III-expression on product glycosylation. IFN-beta with bisected oligosaccharides was produced by the GnT-III-engineered CHO cells but not by the unmodified parental cell line.

Antisense strategies for glycosylation engineering of Chinese hamster ovary (CHO) cells.

Novel glycoproteins, inaccessible by other techniques, can be obtained by metabolic engineering of the oligosaccharide biosynthesis pathway. Furthermore, alteration of cell-surface oligosaccharides can change the properties of receptors involved in cell-cell adhesion. Sialyl Lewis X (sLex) is a cell-surface oligosaccharide determinant which is specifically expressed on granulocytes and monocytes and which interacts with selectins to influence leukocyte trafficking, thrombosis, inflammation, and cancer. Antisense technology targeting fucosyltransferase VI (Fuc-TVI), an enzyme necessary for the synthesis of the sLex in engineered Chinese hamster ovary (CHO) cells, has reduced Fuc-TVI activity, sLex synthesis, and adhesion to endothelial cells. Antisense methodology to reduce targeted activity in oligosaccharide biosynthesis or other pathways is an important addition to CHO cell metabolic engineering capabilities.

Inverse metabolic engineering: A strategy for directed genetic engineering of useful phenotypes.

The classical method of metabolic engineering, identifying a rate-determining step in a pathway and alleviating the bottleneck by enzyme overexpression, has motivated much research but has enjoyed only limited practical success. Intervention of other limiting steps, of counterbalancing regulation, and of unknown coupled pathways often confounds this direct approach. Here the concept of inverse metabolic engineering is codified and its application is illustrated with several examples. Inverse metabolic engineering means the elucidation of a metabolic engineering strategy by: first, identifying, constructing, or calculating a desired phenotype; second, determining the genetic or the particular environmental factors conferring that phenotype; and third, endowing that phenotype on another strain or organism by directed genetic or environmental manipulation. This paradigm has been successfully applied in several contexts, including elimination of growth factor requirements in mammalian cell culture and increasing the energetic efficiency of microaerobic bacterial respiration. (c) 1996 John Wiley & Sons, Inc.

Deregulated expression of cloned transcription factor E2F-1 in Chinese hamster ovary cells shifts protein patterns and activates growth in protein-free medium.

Engineering of the cell cycle can be an effective means for bypassing growth factor requirements of animal cells. Cloned human E2F-1 from Nalm 6 cells was subcloned into pRc/CMV and transfected into Chinese hamster ovary (CHO) cells. Ten stable transfectant clones isolated from cells cultured under neomycin-resistance selection pressure all expressed significantly higher amounts of E2F-1 than control cells as determined by Western analysis. Confocal immunofluorescent microscopy and Southern analysis of several clones also provided evidence for the expression of cloned E2F-1 in these cells. CHO K1:E2F-1 cells are able to proliferate on well-defined serum- and protein-free basal medium and exhibit an S-phase extended by 65% compared to CHO K1 cells mitogenically stimulated by basic fibroblast growth factor (bFGF). Two-dimensional electrophoresis of the intracellular proteins of E2F-1 clones shows an increase in 236 gene products compared to CHO K1 control cells, further verifying a functional regulatory role of cloned E2F-1 in CHO cells. Among these upregulated species is the cell cycle regulatory protein, cyclin A, which has already been shown to be regulated by E2F-1 in human fibroblasts. Overexpression of cloned E2F-1 in CHO cells is a potentially useful new strategy for bypassing serum requirements in mammalian cell culture. Furthermore, such cell cycle control stimulus-protein pattern response data can contribute to a clearer understanding of complex multigene networks involved in mammalian cell cycle regulation.

Chitin synthase 3 from yeast has zymogenic properties that depend on both the CAL1 and the CAL3 genes.

In previous studies, chitin synthase 3 (Chs3), the enzyme responsible for synthesis of most of the chitin present in the yeast cell, was found to be inactivated by incubation with trypsin, in contrast to other yeast chitin synthases (Chs1 and Chs2), which are stimulated by this treatment (chitin synthase; UDP-N-acetyl-D-glucosamine:chitin 4-beta-N-acetylglucosaminyl-transferase, EC 2.4.1.16). It has now been found that the substrate UDPGlcNAc protects Chs3 against proteolytic inactivation. Treatment of Chs3-containing membranes with detergents drastically reduced the enzymatic activity. Activity could, however, be restored by subsequent incubation with trypsin or other proteases in the presence of UDPGlcNAc. Under such conditions, protease treatment stimulated activity as much as 10-fold. A change in divalent cation specificity after trypsin treatment suggests that the protease directly affects the enzyme molecule. Experiments with mutants in the three genes involved in Chs3 activity--CAL1, CAL2, and CAL3--showed that only CAL1 and CAL3 are required for the protease-elicited (zymogenic) activity. It is concluded that Chs3 is a zymogen and that the CAL2 product functions as its activator. The differences and possible similarities between Chs3 and the other chitin synthases are discussed.

Phosphorylation of human and bovine prothymosin alpha in vivo.

Prothymosin alpha is post-translationally modified. When human myeloma cells were metabolically labeled with [32P]orthophosphoric acid, they synthesized [32P]prothymosin alpha. The incorporated radioactivity was resistant to DNase and RNases A, T1, and T2, but could be completely removed by alkaline phosphatase. No evidence was found for an RNA adduct as postulated by Vartapetian et al. [Vartapetian, A., Makarova, T., Koonin, E. V., Agol, V. I., & Bogdanov, A. (1988) FEBS Lett. 232, 35-38]. Thin-layer electrophoresis of partially hydrolyzed [32P]prothymosin alpha indicated that serine residues were phosphorylated. Analysis of peptides derived from bovine prothymosin alpha and human [32P]prothymosin alpha by treatment with endoproteinase Lys-C revealed that the amino-terminal 14-mer, with serine residues at positions 1, 8, and 9, was phosphorylated at a single position. Approximately 2% of the peptide in each case contained phosphate. Further digestion of the phosphopeptide with Asp-N followed by C18 reversed-phase column chromatography produced two peptides: a phosphate-free 9-mer containing amino acids 6-14 and a labeled peptide migrating slightly faster than the N-terminal 5-mer derived from the unmodified 14-mer. Positive identification of the phosphorylated amino acid was obtained by colliding the 14-residue phosphopeptide with helium in the mass spectrometer and finding phosphate only in a nested set of phosphorylated fragments composed of the first three, four, and five amino acids. The results prove that prothymosin alpha contains N-terminal acetylserine phosphate. In a synchronized population of human myeloma cells, phosphorylation occurred throughout the cell cycle. Furthermore, prothymosin alpha appeared to be stable, with a half-life slightly shorter than the generation time. Although prothymosin alpha is known to be essential for cell division, the constancy of both the amount of the protein and the degree of its phosphorylation suggests that prothymosin alpha does not directly govern mitosis.

Nuclear targeting of prothymosin alpha.

Prothymosin alpha is a highly acidic protein which lacks an amino-terminal signal peptide, yet was once thought to be a precursor for thymosin alpha 1, a putative peptide hormone secreted by the thymus. Here, two lines of evidence are presented that strongly implicate prothymosin alpha as a nuclear protein: 1) in COS cells transfected with the human prothymosin alpha gene copious amounts of prothymosin alpha were present in sealed nuclei obtained by treating these cells with cytochalasin B and enucleating them centrifugally. 2) Constructs in which human prothymosin alpha nucleic acid sequences were fused in-frame either near the amino terminus of the beta-galactosidase gene in pCH110 or at the carboxyl terminus, when expressed in COS cells, resulted in nuclear localization of the fusion protein; indirect immunofluorescence in situ was used as the assay. The basic cluster of amino acids at the carboxyl terminus of prothymosin alpha, TKKQKT, has been identified as part of the nuclear targeting signal, whereas the basic cluster of amino acids situated within the thymosin alpha 1 sequence at the amino terminus failed to effect nuclear transport.

Prothymosin alpha antisense oligomers inhibit myeloma cell division.

The function of prothymosin alpha has been investigated by using four different antisense oligodeoxyribonucleotides directed at selected regions of its mRNA. In every case, when synchronized human myeloma cells were released from stationary phase by incubation in fresh medium containing antisense oligomers, cell division was prevented or inhibited; sense oligomers and random antisense oligomers had no effect. A detailed analysis of synchronized cell populations indicated that sense-treated and untreated cells divided approximately 17 hr after growth initiation, whereas cells incubated with antisense oligomer 183, a 16-mer targeted 5 bases downstream of the initiation codon, entered mitosis approximately one cell division late. The failure to divide correlated directly with a deficit in prothymosin alpha and with the continued presence of intact intracellular antisense oligomers over a period of at least 24 hr. Because antisense oligomers had no effect either on the timing of the induction of prothymosin alpha mRNA upon growth stimulation or on mRNA levels seen throughout the cell cycle, we concluded that antisense DNA caused specific hybrid arrest of translation. Our data suggest that prothymosin alpha is required for cell division. However, there is no evidence that prothymosin alpha directly regulates mitosis.

Human prothymosin alpha: purification of a highly acidic nuclear protein by means of a phenol extraction.

Human prothymosin alpha, virtually alone among proteins, is recovered from the aqueous phase of phenol-extracted cell lysates prepared from human myeloma cells or COS cells that were transfected with the human prothymosin alpha gene. This observation forms the basis for purification of the protein to homogeneity in two steps--phenol extraction followed by electrophoresis in sodium dodecyl sulfate polyacrylamide gels to remove residual contaminants consisting chiefly of carbohydrate and RNA.

Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Fungal cell wall synthesis: the construction of a biological structure.

The cell wall is of vital importance for the protection of the fungal cell. It consists of structural components, mostly beta-linked polysaccharides, and of interstitial components, usually glycoproteins. Cell shape is determined by the wall and is attained by localized growth. Regulatory mechanisms that act on chitin and beta(1----3) glucan synthetase indicate how localized biosynthesis can be achieved. Whereas structural polysaccharides appear to be exported by vectorial synthesis through the plasma membrane, glycoproteins are manufactured in an assembly line that takes them through several cellular compartments before exocytosis to the periplasmic space. In this space, the last phase of cell wall construction takes place, i.e. branching of structural polysaccharides and linkage between different components.

Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae.

Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast. Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that the gene product is essential for both septum formation and cell division.

Spectroscopic evidence for a porphobilinogen deaminase-tetrapyrrole complex that is an intermediate in the biosynthesis of uroporphyrinogen III.

Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer (pH 9.2) to total PBG consumption resulted in low yields of uroporphyrinogen I (uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane (tetrapyrrylmethane) but was different from that of a 2-(hydroxymethyl)bilane. The 13C NMR spectra of incubations carried out with [11-13C]PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. The tetrapyrrole could only be partially displaced from the enzyme by ammonium ions, although a small amount of 2-(aminomethyl)bilane was always formed together with the tetrapyrrole intermediate. A protonated uro'gen I structure for this intermediate was ruled out by incubations using [2,11-13C]PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time.

Chitin synthetase 2, a presumptive participant in septum formation in Saccharomyces cerevisiae.

Strains containing a disrupted structural gene for chitin synthetase (chs1::URA3) are defective in chitin synthetase 1 (Chs1) activity but contain normal amounts of chitin (Bulawa, C.E., Slater, M., Cabib, E., Au-Young, J., Sburlati, A., Adair, L., and Robbins, P. W. (1986) Cell 46, 213-225). We have now detected in such strains a new chitin synthetase activity (Chs2), at levels about 5% of those of Chs1 in wild-type cells. Thus, Chs2 is presumably the physiological agent for chitin deposition in strains with a disrupted CHS1 gene and probably also in wild-type strains. Chs1 and Chs2 share certain properties, such as stimulation by N-acetylglucosamine and by partial proteolysis. They differ sharply, however, in divalent cation specificity and in pH optimum. Chs2 also shows less sensitivity than Chs1 to inhibition by polyoxin D or sodium chloride, a property that was used to demonstrate the presence of Chs2 in wild-type extracts. As in the case of Chs1, most of the Chs2 activity was found to be associated with the plasma membranes. This finding, together with the apparent zymogenic nature of Chs2, is consistent with the hypothesis, previously put forward for Chs1, that localized deposition of chitin is attained by activation of the zymogen form at a specific time and place. Function and significance of the two chitin synthetases are discussed in connection with fungal morphogenesis and evolution.

The S. cerevisiae structural gene for chitin synthase is not required for chitin synthesis in vivo.

The chitin synthase of Saccharomyces is a plasma membrane-bound zymogen. Following proteolytic activation, the enzyme synthesizes insoluble chitin that has chain length and other physical properties similar to chitin found in bud scars. We isolated mutants lacking chitin synthase activity (chs1) and used these to clone CHS1. The gene has an open reading frame of 3400 bases and encodes a protein of 130 kd. The fission yeast S. pombe lacks chitin synthase and chitin. When a plasmid encoding a CHS1-lacZ fusion protein is introduced into S. pombe, both enzymatic activities are expressed in the same ratio as in S. cerevisiae, demonstrating that CHS1 encodes the structural gene of chitin synthase. Three CHS1 gene disruption experiments were performed. In all cases, strains with the disrupted gene have a recognizable phenotype, lack measurable chitin synthase activity in vitro but are viable, contain normal levels of chitin in vivo, and mate and sporulate efficiently.

The regulation of porphobilinogen oxygenase and porphobilinogen deaminase activities in rat bone marrow under conditions of erythropoietic stress.

Porphorbilinogen oxygenase (EC 4.2.1.24) was associated with the microsomal fraction of bone marrow in normal rats and in rats submitted to erythropoietic stress, while porphobilinogen deaminase (EC 4.3.1.8) of the same origin was present in the cytosol. An NADPH-dependent electron-donor system for the oxygenase was also present in the microsomes of the bone marrow. Under conditions of erythropoietic stress caused by hypoxia, the activities of both enzymes were found to be inversely correlated. While the oxygenase showed minimum activity between the 4th and 8th day of hypoxia, porphobilinogen deaminase reached its maximum activity during this period. After the 8th day of hypoxia, oxygenase activity increased while deaminase activity decreased. The NADPH-dependent electron-transport system necessary for the microsomal oxygenase activity was largely inactivated after the 10th day of hypoxia, while oxygenase activity was not affected. The particulate porphobilinogen oxygenase could be solubilized from the bone marrow microsomes with 1% deoxycholate or 0.5 M KCl. In addition, the oxygenase was also released by freezing and thawing the microsomes isolated from bone marrow of rats which had been submitted to an erythropoietic stress (hypoxia or phenylhydrazine). The enzyme solubilized with deoxycholate or KCl showed a high molecular weight form and a low molecular weight form (Mr 25 000). The former could be transformed into the latter either by treatment with 2 M KCl or by succinylation. When the oxygenase was solubilized by freezing and thawing a third molecular weight form (Mr 50 000) also appeared. The solubilized enzyme could be succinylated without loss of its catalytic activity, while the membrane-bound enzyme could not be succinylated.

Changes in the Activities of Pyrrolooxygenases during the Germination of Wheat Grains.

Porphobilinogen oxygenase, skatole pyrrolooxygenase, and tryptophan pyrrolooxygenase were found in the different parts of germinating wheat (Triticum aestivum) grain seedlings. In the embryos of grains germinated for 24 hours, the activities of PBG oxygenase and skatole pyrrolooxygenase were inhibited by a labile inhibitor. Tryptophan pyrrolooxygenase activity was not inhibited. Embryos of grains germinated for 48 hours showed higher activities for the three enzymes. The latter were also present in the radicles and coleoptiles of 96-hour germinated wheat grains. A DEAE-cellulose analysis of a crude enzymic preparation from embryos allowed the separation of two molecular forms of the three pyrrolooxygenases. The more cationic forms of porphobilinogen oxygenase and skatole pyrrolooxygenase were associated with the inhibitor. This form of porphobilinogen oxygenase had allosteric kinetics while the more anionic form had Michaelis kinetics. Both forms of skatole pyrrolooxygenase had Michaelis kinetics. The activity of tryptophan pyrrolooxygenase was highest in seedling roots and was found to be inhibited in seedling young leaves. This enzyme oxidized tryptophanyl dipeptides, as well as a nonapeptide, to N-formylkynurenine-containing peptides. The pyrrolooxygenase also oxidized the tryptophanyl residues of lysozyme, chymotrypsin, and trypsin.

Induction of porphobilinogen oxygenase and porphobilinogen deaminase in rat blood under conditions of erythropoietic stress.

Porphobilinogen is the substrate of two enzymes: porphobilinogen deaminase and porphobilinogen-oxygenase. The first one transforms it into the metabolic precursors of heme and the second diverts it from this metabolic pathway by oxidizing porphobilinogen to 5-oxopyrrolinones. Rat blood is devoid of porphobilinogen-oxygenase under normal conditions while it carries porphobilinogen-deaminase activity. When the rats were submitted to hypoxia (pO2 = 0.42 atm) for 18 days, the activity of porphobilinogen-oxygenase appeared at the tenth day of hypoxia and reached the maximum at the 14-16th day. It decreased to a half after 2 days (half-life of the enzyme) and disappeared after 4 days of return to normal oxygen pressure. Porphobilinogen-deaminase activity increased after the first day of hypoxia, reached a maximum at the 14-16th day and did not decrease to normal values until the 15th day after return to normal oxygen pressure. The activities of both porphobilinogen-oxygenase and porphobilinogen-deaminase were induced by administration of erythropoietin. When rats were made anaemic with phenylhydrazine, porphobilinogen-oxygenase activity also appeared in the blood cells. Although the reticulocyte concentration was higher when compared to that obtained under hypoxia, the activities of the oxygenase obtained under both conditions were comparable. Porphobilinogen-deaminase activity was always closely related to the reticulocyte content. The appearance of porphobilinogenase-oxygenase under the described erythropoietic conditions was due to a de novo induction of the enzyme, as shown by its inhibition with actinomycin D and cycloheximide. Porphobilinogen-oxygenase as well as porphobilinogen-deaminase were present in the rat bone marrow under normal conditions. Their activities increased in phenylhydrazine treated rats. The properties and kinetics of porphobilinogen-oxygenase from the rat blood and bone marrow were determined and found it differ in several aspects.