The mannose 6-phosphate receptor of Chinese hamster ovary cells. Compartmentalization of acid hydrolases in mutants with altered receptors

The localization of acid hydrolases was examined in Chinese hamster ovary cells with defective mannose 6-phosphate receptors; these mutants had been shown to exhibit reduced uptake and altered binding of exogenously added acid hydrolase (Robbins, A. R., Myerowitz, R., Youle, R. J., Murray, G. J., and Neville, D. M., Jr. (1981) J. Biol. Chem. 256, 10618-10622). Cells were grown in the presence of [3H]mannose, alpha-L-iduronidase and beta-hexosaminidase were immunoprecipitated sequentially, electrophoresed on polyacrylamide gels containing sodium dodecyl sulfate, and detected by fluorography. About 55% of the alpha-L-iduronidase and beta-hexosaminidase synthesized by the mutants in 12 h was found in the growth medium; parental cells secreted only approximately 15%. The mutants also secreted 2 to 6 times more alpha-mannosidase, beta-glucuronidase, and alpha-L-fucosidase than the parent as determined by measurements of enzyme activity. Intracellular levels of these enzymes were reduced in the mutants. The mutants secreted acid hydrolases in the precursor forms, within the cells these enzymes resided in lysosomes and were processed normally; thus, the mutants appeared aberrant only with respect to distribution of hydrolases between intracellular and extracellular compartments. [35S]methionine-labeled beta-hexosaminidase and alpha-L-iduronidase secreted by the mutants were taken up normally by both human fibroblasts and wild type CHO cells, and this uptake was inhibited by mannose 6-phosphate. Thus, the elevated secretion of acid hydrolases was not due to alteration of the mannose 6-phosphate recognition marker on the enzymes, but appears to result from alterations in the mannose 6-phosphate receptor.     

Identification of mannose uptake and catabolism genes in Corynebacterium glutamicum and genetic engineering for simultaneous utilization of mannose and glucose

Here, focus is on Corynebacterium glutamicum mannose metabolic genes with the aim to improve this industrially important microorganism’s ability to ferment mannose present in mixed sugar substrates. cgR_0857 encodes C. glutamicum’s protein with 36% amino acid sequence identity to mannose 6-phosphate isomerase encoded by manA of Escherichia coli. Its deletion mutant did not grow on mannose and exhibited noticeably reduced growth on glucose as sole carbon sources. In effect, C. glutamicum manA is not only essential for growth on mannose but also important in glucose metabolism. A double deletion mutant of genes encoding glucose and fructose permeases (ptsG and ptsF, respectively) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) was not able to grow on mannose unlike the respective single deletion mutants with mannose utilization ability. A mutant deficient in ptsH, a general PTS gene, did not utilize mannose. These indicate that the glucose-PTS and fructose-PTS are responsible for mannose uptake in C. glutamicum. When cultured with a glucose and mannose mixture, mannose utilization of manA-overexpressing strain CRM1 was significantly higher than that of its wild-type counterpart, but with a strong preference for glucose. ptsF-overexpressing strain CRM2 co-utilized mannose and glucose, but at a total sugar consumption rate much lower than that of the wild-type strain and CRM1. Strain CRM3 overexpressing both manA and ptsF efficiently co-utilized mannose and glucose. Under oxygen-deprived conditions, high volumetric productivity of organic acids concomitant with the simultaneous consumption of the mixed sugars was achieved by the densely packed growth-arrested CRM3 cells.     

Notch1 activation in mice causes arteriovenous malformations phenocopied by ephrinB2 and EphB4 mutants

Notch signaling is essential for embryonic vascular development in mammals and other vertebrates. Here we show that mouse embryos with conditional activation of the Notch1 gene in endothelial cells (Notch1 gain of function embryos) exhibit defects in vascular remodeling increased diameter of the dorsal aortae, and form arteriovenous malformations. Conversely, embryos with either constitutive or endothelial cell‐specific Notch1 gene deletion also have vascular defects, but exhibit decreased diameter of the dorsal aortae and form arteriovenous malformations distinctly different from the Notch1 gain of function mutants. Surprisingly, embryos homozygous for mutations of the ephrinB/EphB pathway genes Efnb2 and Ephb4 exhibit vascular defects and arteriovenous malformations that phenocopy the Notch1 gain of function mutants. These results suggest that formation of arteriovenous malformations in Notch1 gain of function mutants and ephrinB/EphB pathway loss of function mutant embryos occurs by different mechanisms. genesis 48:146–150, 2010. © 2010 Wiley‐Liss, Inc.     

Positive selection for resistance to 2-deoxyglucose gives rise, in Streptococcus salivarius, to seven classes of pleiotropic mutants, including ptsH and ptsl missense mutants

We have used the toxic non-metabolizabie glucose/ mannose analogue 2-deoxygiucose to isolate a comprehensive collection of mutants of the phosphoenoipyruvate:sugar phosphotransferase system from Streptococcus salivarius. To increase the range of possible mutations, we isolated spontaneous mutants on different media containing 2-deoxyglucose and various metabolizable sugars, either lactose, meli-biose, galactose or fructose. We found that the frequency at which 2-deoxygiucose-resistant mutants Were isolated varied according to the growth substrate. The highest frequency was obtained with the combination galactose and 2-deoxygiucose and was 15-fold higher than the rate observed with the mixture melibiose and 2-deoxygiucose, the combination that gave the lowest frequency. By combining results from: (i) Western biol analysis of III^Man, a specific component of the phosphoenolpyruvate:mannose phosphotransferase system in S. salivarius; (ii) rocket immunoelectrophoresis of HPr and EI, the two general energy-coupling proteins of the phosphotransferase system; and (iii) from gene sequencing, mutants could be assigned to seven classes. Class 1 was composed of strains devoid of III^Man_L, a low-molecular-weight form of III^Man_L (35200), class 2 was composed of strains exhibiting a reduced level of III^Man_L, class 3 was composed of strains devoid of both forms of III^Man (III^Man_L as well as III^Man_H, the high-molecular-weight form of III^Man (38900)), class 4 was composed of mutants bearing a mutation in ptsH, the gene encoding HPr, class 5 was composed of mutants bearing a mutation in ptsl, the gene encoding EI, class 6 was composed of 2-deoxygiucose-resistant strains without any apparent defect in PTS components, and class 7 was composed of strains possessing both forms of III^Man but abnormal levels of HPr and/or EI without any mutation in the ptsH and/or the ptsI genes. Preliminary characterization of representative strains of each class is reported.     

An Impaired Uptake of Glucosamine in Tunicamycin Resistant Chinese Hamster Ovary Cells

Tunicamycin resistant mutants (TM^R) were isolated and characterized from Chinese hamster ovary cells. One feature of this TM^R mutants was a marked decrease in incorporation of radioactive glucosamine, both into membrane glycoproteins and G protein of vesicular stomatitis virus.

The cellular uptake and incorporation into acid insoluble materials of various radioactive substances, including glucosamine, galactosamine, mannose, 2-deoxyglucose and leucine, was examined for the purpose of determination whether the reduced incorporation of radioactive glucosamine into glycoproteins was due to a defect in the glycosylation step or decreased uptake of glucosamine by cells.

While incorporation of glucosamine and 2-deoxyglucose into acid insoluble fractions was reduced strikingly in the mutants, the incorporation of mannose and leucine were the same as in the parent cells.

The uptake of glucosamine in TM^R cells was lower than that in the wild type cells, and the Km value for glucosamine uptake differed between the mutants and wild type cells. There was no obvious difference in the uptake of 2-deoxyglucose and mannose.     

Role of the phosphoenolpyruvate-dependent fructose phosphotransferase system in the utilization of mannose by Escherichia coli.

Mutants of Escherichia coli devoid of the membrane-spanning proteins PtsG and PtsMP, which are components of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and which normally effect the transport into the cells of glucose and mannose, do not grow upon or take up either sugar. Pseudorevertants are described that take up, and grow upon, mannose at rates strongly dependent on the mannose concentration in the medium (apparent Km > 5 mM); such mutants do not grow upon glucose but are derepressed for the components of the fructose operon. Evidence is presented that mannose is now taken up via the fructose-PTS to form mannose 6-phosphate, which is further utilized for growth via fructose 6-phosphate and fructose 1,6-bisphosphate.     

Drosophila purine auxotrophy: New alleles ofadenosine2 exhibiting a complex visible phenotype

New mutant alleles of theadenosine2 locus (ade2; 2–17.7) have been isolated using the eye-color phenotype exhibited by the prototype auxotrophic alleleade2 ¹ as the screening criterion. The new mutants form a single complementation group, suggesting that they all exhibit purine auxotrophy and defective formylglycineamide ribotide amidotransferase enzyme, likeade2 ¹. Tests carried out on particular new alleles confirm these suggestions. The new mutants all exhibit more extreme physical defects than the prototype. They have wing abnormalities like mutants defective in pyrimidine biosynthesis and reduced bristles like those defective in protein synthesis; thus they exhibit the combined visible phenotype ofrudimentary wings,rosy eyes, andbobbed bristles. Cytogenetic analysis places the locus in the interband proximal to26B1-2.This work was supported by NSERC Operating Grant A3269 to D.N., an Alberta Heritage Foundation for Medical Research Postdoctoral Fellowship to S.Y.K.T., and National Institute on Aging Grant AG00029 to D.P.     

The role of DNA base excision repair in filamentation in Escherichia coli K-12 adhered to epithelial HEp-2 cells

Base excision repair (BER) is dedicated to the repair of oxidative DNA damage caused by reactive oxygen species generated by chemical and physical agents or by metabolism which can react with DNA and cause a variety of mutations. Epithelial cells are typically the first type of host cell to come into contact with potential microbial invaders. In this work, we have evaluated whether the adherence to human epithelial cells causes DNA damage and associated filamentation. Experiments concerning adherence to HEp-2 cells were carried out with mutants deficient in BER that were derived from Escherichia coli K-12. Since the removal of mannose during bacterial interaction with HEp-2 cells allows adhesion through mannose-sensitive adhesins, the experiments were also performed in the presence and the absence of mannose. Our results showed enhanced filamentation for the single xth (BW9091) and triple xth nfo nth (BW535) mutants in adherence assays with HEp-2 cells performed without d-mannose. The increased filamentation growth was inhibited by complementation of BER mutants with a wild type xth gene. Moreover, we measured SOS induction of bacteria adhered to HEp-2 cells in the presence and absence of d-mannose through of SOS-chromotest assay and we observed a higher β-galactosidase expression in the absence of mannose. In this context, data showed evidence that bacterial attachment to HEp-2 epithelial surfaces can generate DNA lesions and SOS induction.     

Expression of an inducible enzyme II fructose and activation of a cryptic enzyme II glucose in glucose-grown cells of spontaneous mutants of Streptococcus salivarius lacking the low-molecular-mass form of IIIman, a component of the phosphoenolpyruvate:mannose phosphotransferase system.

We have reported previously that the phosphoenolpyruvate:mannose phosphotransferase system (mannose PTS) of Streptococcus salivarius, consisting of an Enzyme II mannose (EIIman) and two forms of Enzyme III mannose (IIIman) with Mr values of 38,900 and 35,200, respectively, concomitantly transports and phosphorylates mannose, as well as glucose and fructose. In this paper, we report the presence, in S. salivarius, of alternative specific fructose and glucose PTSs encoded by inducible and cryptic genes, respectively. Protein phosphorylation experiments conducted with [32P]phosphoenolpyruvate have allowed us to identify by SDS-PAGE and autoradiography the EII fructose (EIIfru) (Mr 57,500) and the EII glucose (EIIglc) (Mr 58,700). No proteins corresponding to IIIfru or IIIglc could be detected. EIIfru phosphorylated fructose on the C-1 position rather than, as with the constitutive mannose PTS, on the C-6 position. Growth on fructose resulted in the induction of EIIfru as well as an increase of 1-phosphofructokinase activity. Nevertheless, the genes encoding these proteins were independently regulated. Studies carried out with spontaneous mutants lacking the low-molecular-mass form of IIIman (mutants A37, G29 and B31) showed that EIIfru was expressed in glucose-grown cells of strains G29 and B31, but not in strain A37, whereas the cryptic gene encoding EIIglc was activated in all three mutant strains. The results obtained with the mutants suggest that the three spontaneous mutants were not all mutated on the gene encoding IIIman although all of them lacked IIIman.     

Mutants of the pentose‐fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor

Mutants of Pichia stipitis NRRL Y‐7124 able to tolerate and produce ethanol from hardwood spent sulfite liquor (HW SSL) were obtained by UV mutagenesis. P. stipitis cells were subjected to three successive rounds of UV mutagenesis, each followed by screening first on HW SSL gradient plates and then in diluted liquid HW SSL. Six third generation mutants with greater tolerance to HW SSL as compared to the wild type (WT) were isolated. The WT strain could not grow in HW SSL unless it was diluted to 65% (v/v). In contrast, the third generation mutants were able to grow in HW SSL diluted to 75% (v/v). Mutants PS301 and PS302 survived even in 80% (v/v) HW SSL, although there was no increase in cell number. All the third generation mutants exhibited higher growth rates but significantly lower growth yields on xylose or glucose compared to the WT. The mutants fermented 4% (w/v) glucose as efficiently as the WT and fermented 4% (w/v) xylose more efficiently with a higher ethanol yield than the WT. In a medium containing 4% (w/v) each of xylose and glucose, all the third generation mutants utilized glucose as efficiently and xylose more efficiently than the WT. This resulted in higher ethanol yield by the mutants. The mutants retained the ability to utilize galactose and mannose and ferment them to ethanol. Arabinose was consumed slowly by both the mutants and WT with no ethanol production. In 60% (v/v) HW SSL, the mutants utilized and fermented glucose, mannose, galactose and xylose while the WT could not ferment any of these sugars. Biotechnol. Bioeng. 2009; 104: 892–900. © 2009 Wiley Periodicals, Inc.     

Genetics of somatic mammalian cells. XIV. Genetic analysis in vitro of auxotrophic mutants

Genetic analysis has been carried out on auxotrophic mutants produced by treatment of Chinese hamster ovary and the Chinese hamster lung cells with mutagenic agents in vitro. Thirty-six different mutants were subjected to complementation analysis and biochemical tests. The different mutations studied result in growth requirements for proline; glycine; glycine or folinic acid; adenine or several of its precursors; inositol; adenine plus thymidine; and glycine plus adenine plus thymidine. The mutants which require glycine fall into four different complementation classes while those requiring adenine or hypoxanthine form two different complementation classes. The biochemical blocks of the latter two classes both occur somewhere in the steps involved in conversion of 5-phosphoribosyl-1-pyrophosphate to 5-aminoimidazole 4-carboxylic acid ribonucleotide. The auxotrophic mutants described exhibit all-or-none responses to their specific nutrilite supplements and are stable with respect to reversion. They involve alterations in ten different genes, and hence form a useful set of mutants for a variety of genetic studies. All the auxotrophies produced in a single exposure to a mutagen are due to single gene mutations, even when multiple nutritional requirements were produced. All the mutations studied are recessive.     

Genetic and biochemical studies of phosphomannose isomerase deficient mutants ofSaccharomyces cerevisiae

Summary Three mannose-negative mutants ofSaccharomyces cerevisiae have been isolated. These mutants showed growth inhibition when mannose was added to a growth medium containing glycerol or fructose. Crosses between wild type mutants showed segregation of 2⁺/2⁻. Crosses between the mutants themselves showed that they were closely linked. Two mutants (XM3 and D2) showed characteristics of allelic structural alteration of phosphomannoseisomerase. Mutant D4 had a deficiency of phosphomannoseisomerase activity, but with a normal thermostability. Revertants from D4 had a normal thermostability.     

Postreplication repair-defective mutants of Drosophila melanogaster fall into two classes

Summary Primary cell cultures derived from embryos of a control stock of Drosophila melanogaster respond to ultraviolet light within the first hour after exposure with a decline in thymidine incorporation and a decline in the ability to form newly synthesized (nascent) DNA in long segments. Cells derived from two nonallelic excision-defective mutants (mei-9 and mus201) exhibit the same quantitative decline in both phenomena as do control cells. In contrast, cells from five nonallelic postreplication repair-defective mutants (mei-41, mus101, mus205, mus302 and mus310) respond to ultraviolet light by synthesizing nascent DNA in abnormally short segments. Two of these five mutants (mus302 and mus310) also exhibit unusually low thymidine incorporation levels after irradiation, whereas the other three mutants display the normal depression of incorporation.These results indicate that excision repair does not influence the amount or the length of nascent DNA synthesized in Drosophila cells within the first hour after exposure to ultraviolet light. Of the five mutations that diminish postreplication repair, only two reduce the ability of irradiated cells to synthesize normal amounts of DNA.Abbreviation used UV ultraviolet light — principal wavelength 254 nm     

Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses

Mannose‐capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAMobserved in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine‐induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co‐stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL‐10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low‐dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.     

Immunochemical studies of Rhizobium mutants

Summary It was shown in gel diffusion and in immunoelectrophoresis tests that somatic antigens of smooth cultures were more complex than those of rough mutants. Somatic antigens ofR. trifolii 24 contained glucuronic acid, glucosamine, galactose, glucose, mannose, xylose or fucose, ribose, and 2-keto-3-deoxyoctonate. Arabinose was identified in some preparations. The antigens of the smooth strain contained a large amount of rhamnose whereas those of the rough mutants were deficient in that sugar.     

Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin β-subunit by disruption of disulfide bonds

The human chorionic gonadotropin β-subunit (hCGβ) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGβ produced in Chinese hamster ovary (CHO) cells: wild-type hCGβ (βWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of βWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled βWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGβ molecule. Abbreviations: hCGβ, human chorionic gonadotropin β-subunit; βWT, wild type hCGβ; CHO, Chinese hamster ovary; Endo-H, endoglycosidase H; Endo-F, endoglycosidase F This revised version was published online in November 2006 with corrections to the Cover Date.     

Hexose phosphate metabolism and exopolysaccharide formation inPseudomonas cepacia

When grown on solid medium containing excess glucose, glucose dehydrogenase-deficient (Gcd^–) mutants ofPseudomonas cepacia 249 formed large amounts of an exopolysaccharide comprised of galactose, glucose, mannose, glucuronic acid, and rhamnose. The Gcd⁺ parent strain failed to accumulate comparable amounts of exopolymer from glucose because of its rapid conversion of glucose to gluconic and 2-ketogluconic acids and its lower content of enzymes related to glucose-1-phosphate synthesis. Both Gcd⁺ and Gcd^– strains ofP. cepacia accumulated exopolymer when substrates such as mannitol and glycerol were substituted for glucose. A survey of clinical isolates from patients with cystic fibrosis indicated that there was no correlation between ability ofP. cepacia to colonize the respiratory tracts of such individuals and increased capacity to form exopolymer related to glucose dehydrogenase deficiency.     

A PTS EII mutant library in Group A Streptococcus identifies a promiscuous man‐family PTS transporter influencing SLS‐mediated hemolysis

The Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram‐positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate‐dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)‐mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared with parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC‐encoding genes in the GAS MGAS5005 genome and subjected this library to metabolic and hemolysis assays to functionally characterize each EIIC. It was found that a few EIIs had a very limited influence on PTS sugar metabolism, whereas others were fairly promiscuous. The mannose‐specific EII locus, encoded by manLMN, was expressed as a mannose‐inducible operon that exhibited the most influence on PTS sugar metabolism, including mannose. Importantly, components of the mannose‐specific EII also acted to prevent the early onset of SLS‐mediated hemolysis. Interestingly, these roles were not identical in two different M1T1 GAS strains, highlighting the possible versatility of the PTS to adapt to strain‐specific needs.     

Biosynthesis of Glycoproteins in the Human Pathogenic Fungus Sporothrix Schenckii: Synthesis of Dolichol Phosphate Mannose and Mannoproteins by Membrane-Bound and Solubilized Mannosyl Transferases

A membrane fraction obtained from the filamentous form of Sporothrix schenckii was able to transfer mannose from GDP-Mannose into dolichol phosphate mannose and from this inTermediate into mannoproteins in coupled reactions catalyzed by dolichol phosphate mannose synthase and protein mannosyl transferase(s), respectively. Although the transfer reaction depended on exogenous dolichol monophosphate, membranes failed to use exogenous dolichol phosphate mannose for protein mannosylation to a substantial extent. Over 95% of the sugar was transferred to proteins via dolichol phosphate mannose and the reaction was stimulated several fold by Mg²⁺ and Mn²⁺. Incubation of membranes with detergents such as Brij 35 and Lubrol PX released soluble fractions that transferred the sugar from GDP-Mannose mostly into mannoproteins, which were separated by affinity chromatography on Concanavilin A–Sepharose 4B into lectin-reacting and non-reacting fractions. All proteins mannosylated in vitro eluted with the lectin-reacting proteins and analytical electrophoresis of this fraction revealed the presence of at least nine putative mannoproteins with molecular masses in the range of 26–112 kDa. The experimental approach described here can be used to identify and isolate specific glycoproteins mannosylated in vitro in studies of O-glycosylation.