Synthesis of globopentaose using a novel beta1,3-galactosyltransferase activity of the Haemophilus influenzae beta1,3-N-acetylgalactosaminyltransferase LgtD

We have previously described a bacterial system for the conversion of globotriaose (Gb3) into globotetraose (Gb4) by a metabolically engineered Escherichia coli strain expressing the Haemophilus influenzae lgtD gene encoding beta1,3-N-acetylgalactosaminyltransferase [Antoine, T., Bosso, C., Heyraud, A. Samain, E. (2005) Large scale in vivo synthesis of globotriose and globotetraose by high cell density culture of metabolically engineered Escherichia coli. Biochimie 87, 197-203]. Here, we found that LgtD has an additional beta1,3-galactosyltransferase activity which allows our bacterial system to be extended to the synthesis of the carbohydrate portion of globopentaosylceramide (Galbeta-3GalNAcbeta-3Galalpha-4Galbeta-4Glc) which reacts with the monoclonal antibody defining the stage-specific embryonic antigen-3. In vitro assays confirmed that LgtD had both beta1,3-GalT and beta1,3-GalNAcT activities and showed that differences in the affinity for Gb3 and Gb4 explain the specific and exclusive formation of globopentaose.     

Glucuronylation in Escherichia coli for the bacterial synthesis of the carbohydrate moiety of nonsulfated HNK-1

We have previously reported the large-scale synthesis of neolactotetraose (Galbeta-4GlcNAcbeta-3Galbeta-4Glc) from lactose in engineered Escherichia coli cells (Priem B, Gilbert M, Wakarchuk WW, Heyraud A and Samain E. 2002. A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria. Glycobiology. 12:235-240). In the present study we analyzed the adaptation of this system to glucuronylated oligosaccharides. The catalytic domain of mouse glucuronyl transferase GlcAT-P was cloned and expressed in an engineered strain which performed the in vivo synthesis of neolactotetraose. Under these conditions, efficient glucuronylation of neolactotetraose was achieved, but some residual neolactotetraose was still present. Although E. coli K-12 has an indigenous UDP-glucose dehydrogenase, the yield of glucuronylated oligosaccharides was greatly improved by the additional expression of the orthologous gene kfiD from E. coli K5. Glucuronylation of neolactohexaose and lactose was also observed. The final glucuronylated oligosaccharides are precursors of the brain carbohydrate motif HNK-1, involved in neural cell adhesion.     

A new fermentation process allows large-scale production of human milk oligosaccharides by metabolically engineered bacteria

When fed to a beta-galactosidase-negative (lacZ(-)) Escherichia coli strain that was grown on an alternative carbon source (such as glycerol), lactose accumulated intracellularly on induction of the lactose permease. We showed that intracellular lactose was efficiently glycosylated when genes of glycosyltransferase that use lactose as acceptor were expressed. High-cell-density cultivation of lacZ(-) strains that overexpressed the beta 1,3 N acetyl glucosaminyltransferase lgtA gene of Neisseria meningitidis resulted in the synthesis of 6 g x L(-1) of the expected trisaccharide (GlcNAc beta 1-3Gal beta 1-4Glc). When the beta 1,4 galactosyltransferase lgtB gene of N. meningitidis was coexpressed with lgtA, the trisaccharide was further converted to lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) and lacto-N-neoheaxose with a yield higher than 5 g x L(-1). In a similar way, the nanA(-) E. coli strain that was devoid of NeuAc aldolase activity accumulated NeuAc on induction of the NanT permease and the lacZ(-) nanA(-) strain that overexpressed the N. meningitidis genes of the alpha2,3 sialyltransferase and of the CMP-NeuAc synthase efficiently produced sialyllactose (NeuAc alpha 2-3Gal beta 1-4Glc) from exogenous NeuAc and lactose.     

Genetic engineering of Escherichia coli for the production of NI,NII-diacetylchitobiose (chitinbiose) and its utilization as a primer for the synthesis of complex carbohydrates

Chitinbiose was produced at more than 4 g L-1 by a high cell density culture of an Escherichia coli strain that co-expressed the rhizobial chitinoligosaccharide synthase gene nodC and a truncated form of the chitinase gene chiA which has been designed to be functionally produced in the E. coli cytoplasm. Chitinpentaose, which has previously been shown to be produced by the nodC protein in growing E. coli, was formed as an intermediate that was subsequently hydrolyzed into chitinbiose by the chitinase encoded by chiA. Chitinbiose was mainly recovered in the extracellular medium and to prevent its catabolism, the genes for the chitinbiose PTS permease had to be disrupted. When the additional gene lgtB for beta1,4-galactosyltransferase was expressed, intracellular chitinbiose was converted into the trisaccharide Galbeta-4GlcNAcbeta-4GlcNAc which could serve as acceptor for glycosyltransferase that recognize the terminal N-acetyllactosamine structure.     

The lactose analog GalNAcbeta1-->4Glc is present in bovine colostrum. Enzymatic basis for its occurrence.

We have isolated from bovine colostrum the lactose analog GalNAcbeta1-->4Glc. The enzymatic basis for its occurrence was studied by assaying the activities of GlcNAcbeta-R beta4-N-acetylgalactosaminyltransferase (beta4-GalNAcT) and GlcNAcbeta-R beta4-galactosyltransferase (beta4-GalT) in primary milk and several lactating bovine mammary gland fractions. As the beta4-GalNAcT, which appears to be tightly membrane bound, is induced by the milk protein alpha-lactalbumin (alpha-LA) to act on Glc, it is concluded that beta4-GalNAcT is responsible for the synthesis of GalNAcbeta1-->4Glc in the gland. The comparatively low level (15-20 mg/l) at which this disaccharide is produced may be due to the relatively poor interaction of beta4-GalNAcT with alpha-LA as well as to the fact that alpha-LA does not inhibit the action of the enzyme on N-acetylglucosaminides.     

Cooperative binding of the sugar substrates and allosteric regulatory protein (enzyme IIIGlc of the phosphotransferase system) to the lactose and melibiose permeases in Escherichia coli and Salmonella typhimurium

An Escherichia coli strain which overproduces the lactose permease was used to investigate the mechanism of allosteric regulation of this permease and those specific for melibiose, glycerol, and maltose by the phosphoenolpyruvate-sugar phosphotransferase system (PTS). Thio-beta-digalactoside, a high affinity substrate of the lactose permease, released the glycerol and maltose permeases from inhibition by methyl-alpha-d-glucoside. Resumption of glycerol uptake occurred immediately upon addition of the galactoside. The effect was not observed in a strain which lacked or contained normal levels of the lactose permease, but growth of wild-type E. coli in the presence of isopropyl-beta-thiogalactoside plus cyclic AMP resulted in enhanced synthesis of the lactose permease so that galactosides relieved inhibition of glycerol uptake. Thiodigalactoside also relieved the inhibition of glycerol uptake caused by the presence of other PTS substrates such as fructose, mannitol, glucose, 2-deoxyglucose, and 5-thioglucose. Inhibition of adenylate cyclase activity by methyl-alpha-glucoside was also relieved by thiodigalactoside in E. coli T52RT provided that the lactose permease protein was induced to high levels. Cooperative binding of sugar and enzyme III(Glc) to the melibiose permease in Salmonella typhimurium was demonstrated, but no cooperativity was noted with the glycerol and maltose permeases. These results are consistent with a mechanism of PTS-mediated regulation of the lactose and melibiose permeases involving a fixed number of allosteric regulatory proteins (enzyme III(Glc)) which may be titrated by the increased number of substrate-activated permease proteins. This work suggests that the cooperativity in the binding of sugar substrate and enzyme III(Glc) to the permease, demonstrated previously in in vitro experiments, has mechanistic significance in vivo. It substantiates the conclusion that PTS-mediated regulation of non-PTS permease activities involves direct allosteric interaction between the permeases and enzyme III(Glc), the postulated regulatory protein of the PTS.     

Designer biocatalysts for direct incorporation of exogenous galactose into globotriose

Galactose is ubiquitous. The synthesis of galactose-containing oligosaccharides using Leloir galactosyltransferase requires uridine diphosphate (UDP)-galactose as the precursor. Of all UDP-galactose synthesis pathways developed for in vitro synthesis, the salvage pathway represents the simplest route. In this study, for the first time, we designed and constructed an Escherichia coli strain to use salvage pathway for UDP-galactose synthesis, demonstrating effective and direct incorporation of exogenous galactose into globotriose (Gb3). Successful establishment of salvage pathway enabled a complete delineation of carbon and energy source. Consequently, the designed biocatalyst was able to achieve high yield synthesis from galactose (0.95 moles of Gb3/moles galactose consumed) and a high product titer (2 g/L) in shaker flask within 24 hr. Elimination of limitation in acceptor sugar via homologous overexpression of LacY, the transporter for lactose, further improved the synthesis, raising Gb3 titer to 6 g/L in 24 hr and 7.5 g/L in 48 hr. The design principles successfully demonstrated in this study could be broadly applied for synthesis of other galactose-containing oligosaccharides. This study also illustrates a valid strategy to overcome limitation in the transport of acceptor sugar. As lactose is one of the most important basal structures, the significant improvement in synthesis through its enhanced transport could be emulated in numerous other lactose-based oligosaccharides.     

The effects of osmotic stress on survival and alkaline phosphatase activity of Aeromonas hydrophila

Abstract Lactococcus lactis MG5267 is a plasmid-free strain in which the lactose operon is integrated in the bacterial chromosome. The chromosomal lac G gene which encodes phospho-β-galactosidase was inactivated by a double cross-over integration event. Unexpectedly, the resultant mutant was shown to retain a Lac-positive phenotype. The lysin gene from Listeria monocytogenes bacteriophage LM-4 was subsequently integrated into the chromosome of this strain such that expression of the heterologous gene was mediated by the lactose operon promoter. Expression of the lysin gene was shown to be regulated by growth on lactose. This represents an important strategy for the controlled and stabilised expression of biotechnologically useful genes in L lactis .     

Isolation and characterization of a novel Forssman active acidic glycosphingolipid with branched isoglobo-, ganglio-, and neolacto-series hybrid sugar chains

Equine kidney and spleen contain a Forssman active glycosphingolipid, and the structure of this glycolipid has been reported to be that of a globopentaosylceramide (GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1, 4Galbeta-1,4Glcbeta-1,1'Ceramide). We found that equine kidney contains several other anti-Forssman antibody-reactive glycosphingolipids. One of these acidic Forssman active glycosphingolipids was isolated and characterized by means of NMR, mass spectrometry, permethylation studies, and TLC-immunostaining. This glycolipid contains three moles of galactose, one mole of glucose, three moles of N-acetylgalactosamine, one mole of N-acetylglucosamine, and one mole of N-acetylneuraminic acid, and is stained on TLC with anti-Forssman antibodies and anti-GM2 ganglioside antibodies. HOHAHA and ROESY experiments and permethylation studies showed this glycolipid oligosaccharide to be branched at the innermost galactose; one chain has an isoglobo structure with a terminal Forssman disaccharide and the other chain is branched through the linkage of N-acetylglucosaminebeta-1,6 to the inner galactose. The nonreducing end of the GM2 trisaccharide is linked to this glucosamine. The structure of the oligosaccharide of the glycolipid presented here is a novel type, having branched isoglobo-, ganglio-, and neolacto-series oligosaccharides. Mass spectrometric analyses indicated the ceramide moiety of the glycolipid to be composed predominantly of hydroxy fatty acids (C20:0, C22:0, C23:0, C24:0, and C25:0) and hydroxysphinganine. GalNAcalpha-1,3GalNAcbeta-1,3Galalpha-1,3[GalNAcbet a-1, 4(NeuAcalpha-2,3)Galbeta-1,4GlcNAcbeta-1,6]Galbeta+ ++-1,4Glcbeta-1, 1'Ceramide     

Temporal effect of prolactin on the activities of lactose synthetase, alpha-lactalbumin, and galactosyl transferase in mouse mammary gland explants

The onset of the prolactin (PRL) stimulation of lactose synthesis is between 4 and 8 hr after adding PRL to cultured mouse mammary tissues. The synthesis of lactose is catalyzed by the enzyme lactose synthetase, which is composed of two parts, alpha-lactalbumin and galactosyl transferase. In time-sequence studies, it was found that the activity of galactosyl transferase is enhanced by PRL in concert with the onset of the PRL stimulation of lactose synthesis. In contrast, the earliest detectable effect of PRL on alpha-lactalbumin activity occurred 24 hr after adding PRL to the cultures. It is, therefore, apparent that the rate-limiting component for the PRL stimulation of lactose synthesis in cultured mouse mammary tissues is galactosyl transferase activity.     

Purification of Shiga-like toxin 1 by pigeon egg white glycoproteins immobilized on Sepharose gels

The galabiose structure Galalpha1-4Gal is rarely found in natural glycoproteins, but is abundantly present in pigeon egg white proteins as Galalpha(1-4)Galbeta(1-4)GlcNAc termini. Pigeon ovalbumin, ovomucoid, or the whole egg white were immobilized on periodate-oxidized Sepharose CL-6B gels by reductive amination. These gels were found to bind Shiga-like toxin type 1 (SLT-1) specifically and efficiently. SLT-1 was eluted from the gel beads with 0.5 M melibiose, which was more efficient and milder than elution with 4.5 M MgCl(2). SLT-1 was purified to homogeneity from the crude extract of Escherichia coli SLT100 expressing SLT-1 by a single affinity chromatographic step in 83-88% yield. The capacity of the gel was estimated to be ca. 1mg toxin/ml gel. Interestingly, SLT-2 was not bound by these affinity gels containing Galalpha1-4Galbeta1-4GlcNAc termini. Since SLT-2 has been shown to bind to Galalpha1-4Galbeta1-4Glc-terminating compounds, our results suggest that Glc in globotriose moiety is important for binding SLT-2, and replacing the Glc with GlcNAc in this triose renders it ineffective for binding SLT-2.     

Chemical characterization of the oligosaccharides in beluga (Delphinapterus leucas) and Minke whale (Balaenoptera acutorostrata) milk

Carbohydrates were extracted from the milk of a beluga, Delphinopterus leucas (family Odontoceti), and two Minke whales, Balaenoptera acutorostrata (Family Mysticeti), sampled late in their respective lactation periods. Free oligosaccharides were separated by gel filtration and then neutral oligosaccharides were purified by preparative thin layer chromatography and gel filtration, while acidic oligosaccharides were purified by ion-exchange chromatography, gel filtration and high performance liquid chromatography (HPLC). Their structures were determined by 1H-NMR. In one of the Minke whale milk samples, lactose was a dominant saccharide, with Fuc(alpha1-2)Gal(beta1-4)Glc(2'-fucosyllactose), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc(lacto-N-neotetraose), GalNAc(alpha1-3)[Fuc(alpha1-2)]Gal(beta1-4)Glc(A-tetrasaccharide), Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (para lacto-N-neohexaose), Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl lacto-N-neotetraose), Neu5Ac(alpha2-6)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LST c) and Neu5Ac(alpha2-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (sialyl para lacto-N-neohexaose) also being found in the milk. The second Minke whale sample contained similar amounts of lactose, 2'-fucosyllactose and A-tetrasaccharide, but no free sialyl oligosaccharides. Sialyl lacto-N-neotetraose and sialyl para lacto-N-neohexaose are novel oligosaccharides which have not been previously reported from any mammalian milk or colostrum. These and other oligosaccharides of Minke whale milk may have biological significance as anti-infection factors, protecting the suckling young against bacteria and viruses. The lactose of Minke whale milk could be a source of energy for them. The beluga whale milk contained trace amounts of Neu5Ac(alpha2-3)Gal(beta1-4)Glc(3'-N-acetylneuraminyllactose), but the question of whether it contained free lactose could not be clarified. Therefore, lactose may not be a source of energy for suckling beluga whales.     

Glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase. A novel bifunctional enzyme in malaria parasites.

Plasmodium falciparum glucose 6-phosphate dehydrogenase (Pf Glc6PD), compared to other Glc6PDs has an additional 300 amino acids at the N-terminus. They are not related to Glc6PD but are similar to a family of proteins (devb) of unknown function, some of which are encoded next to Glc6PD in certain bacteria. The human devb homologue has recently been shown to have 6-phosphogluconolactonase (6PGL) activity. This suggests Pf Glc6PD may be a bifunctional enzyme, the evolution of which has involved the fusion of adjacent genes. Further functional analysis of Pf Glc6PD has been hampered because parts of the gene could not be cloned. We have isolated and sequenced the corresponding Plasmodium berghei gene and shown it encodes an enzyme (Pb Glc6PD) with the same structure as the P. falciparum enzyme. Pb Glc6PD is 950 amino acids long with significant sequence similarity in both the devb and Glc6PD domains with the P. falciparum enzyme. The P. berghei enzyme does not have an asparagine-rich segment between the N and C halves and it contains an insertion at the same point in the Glc6PD region as the P. falciparum enzyme but the insertion in the P. berghei is longer (110 versus 62 amino acids) and unrelated in sequence to the P. falciparum insertion. Though expression of this enzyme in bacteria produced largely insoluble protein, conditions were found where the full-length enzyme was produced in a soluble form which was purified via a histidine tag. We show that this enzyme has both Glc6PD and 6PGL activities. Thus the first two steps of the pentose phosphate pathway are catalysed by a single novel bifunctional enzyme in these parasites.     

Towards understanding the functional role of the glycosyltransferases involved in the biosynthesis of Moraxella catarrhalis lipooligosaccharide

The glycosyltransferase enzymes (Lgts) responsible for the biosynthesis of the lipooligosaccharide-derived oligosaccharide structures from Moraxella catarrhalis have been investigated. This upper respiratory tract pathogen is responsible for a spectrum of illnesses, including otitis media (middle ear infection) in children, and contributes to exacerbations of chronic obstructive pulmonary disease in elderly patients. To investigate the function of the glycosyltransferase enzymes involved in the biosynthesis of lipooligosaccharide of M. catarrhalis and to gain some insight into the mechanism of serotype specificity for this microorganism, mutant strains of M. catarrhalis were produced. Examination by NMR and MS of the oligosaccharide structures produced by double-mutant strains (2951lgt1/4Delta and 2951lgt5/4Delta) and a single-mutant strain (2951lgt2Delta) of the bacterium has allowed us to propose a model for the serotype-specific expression of lipooligosaccharide in M. catarrhalis. According to this model, the presence/absence of Lgt4 and the Lgt2 allele determines the lipooligosaccharide structure produced by a strain. Furthermore, it is concluded that Lgt4 functions as an N-acetylglucosylamine transferase responsible for the addition of an alpha-D-GlcNAc (1-->2) glycosidic linkage to the (1-->4) branch, and also that there is competition between the glycosyltransferases Lgt1 and Lgt4. That is, in the presence of an active Lgt4, GlcNAc is preferentially added to the (1-->4) chain of the growing oligosaccharide, instead of Glc. In serotype B strains, which lack Lgt4, Lgt1 adds a Glc at this position. This implies that active Lgt4 has a much higher affinity/specificity for the beta-(1-->4)-linked Glc on the (1-->4) branch than does Lgt1.     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal- ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.     

Assessment of the two Helicobacter pylori alpha-1,3-fucosyltransferase ortholog genes for the large-scale synthesis of LewisX human milk oligosaccharides by metabolically engineered Escherichia coli

We previously described a bacterial fermentation process for the in vivo conversion of lactose into fucosylated derivatives of lacto-N-neotetraose Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)Glc (LNnT). The major product obtained was lacto-N-neofucopentaose-V Gal(beta1-4)GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc, carrying fucose on the glucosyl residue of LNnT. Only a small amount of oligosaccharides fucosylated on N-acetylglucosaminyl residues and thus carrying the LewisX group (Le(X)) was also produced. We report here a fermentation process for the large-scale production of Le(X) oligosaccharides. The two fucosyltransferase genes futA and futB of Helicobacter pylori (strain 26695) were compared in order to optimize fucosylation in vivo. futA was found to provide the best activity on the LNnT acceptor, whereas futB expressed a better Le(X) activity in vitro. Both genes were expressed to produce oligosaccharides in engineered Escherichia coli (E. coli) cells. The fucosylation pattern of the recombinant oligosaccharides was closely correlated with the specificity observed in vitro, FutB favoring the formation of Le(X) carrying oligosaccharides. Lacto-N-neodifucohexaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)[Fuc(alpha1-3)]Glc represented 70% of the total oligosaccharide amount of futA-on-driven fermentation and was produced at a concentration of 1.7 g/L. Fermentation driven by futB led to equal amounts of both lacto-N-neofucopentaose-V and lacto-N-neofucopentaose-II Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)Gal(beta1-4)Glc, produced at 280 and 260 mg/L, respectively. Unexpectedly, a noticeable proportion (0.5 g/L) of the human milk oligosaccharide 3-fucosyllactose Gal(beta1-4)[Fuc(alpha1-3)]Glc was produced in futA-on-driven fermentation, underlining the activity of fucosyltransferase FutA in E. coli and leading to a reassessment of its activity on lactose. All oligosaccharides produced by the products of both fut genes were natural compounds of human milk.     

Characterization and specific assay for a galactoside beta-3-galactosyltransferase of human kidney

Two galactosyltransferases identified as UDP-galactose:lactose (lactosylceramide) alpha-4- and beta-3-galactosyltransferases [Bailly P. et al. (1986) Biochem. Biophys. Res. Commun. 141, 84-91] have been characterized in human kidney microsomes. Using methyl beta-D-galactoside as acceptor substrate, we have determined the experimental conditions (pH 5.0, 4 mM Cd2+) in which only the beta-3-galactosyltransferase activity is detectable. The reaction product has been characterized by chemical methods and glycosidase studies. Under these experimental conditions, some of the enzyme properties have been further investigated. Apparent Km values are for UDP-galactose, 0.170 mM; for lactose, 242 mM; and for lactosylceramide, 2.5 mM. Acceptor specificity studies suggest that the beta-3-galactosyltransferase is specific for terminal Gal beta 1-4Glc(NAc) residues and responsible for elongation of oligosaccharide chains in glycolipids. Competition studies with lactose and N-acetylgalactosamine as acceptor substrates indicate that the transferase described here can be distinguished from the UDP-galactose:2-acetamide-2-deoxy-D-galactose beta-3-galactosyltransferase and therefore represents a novel enzyme capable of synthesizing unusual carbohydrate structures similar to those which accumulate in certain neurological diseases.     

Effects of Glucose Availability on Expression of the Key Genes Involved in Synthesis of Milk Fat,Lactose and Glucose Metabolism in Bovine Mammary Epithelial Cells

As the main precursor for lactose synthesis, large amounts of glucose are required by lactating dairy cows. Milk yield greatly depends on mammary lactose synthesis due to its osmoregulatory property for mammary uptake of water. Thus, glucose availability to the mammary gland could be a potential regulator of milk production. In the present study, the effect of glucose availability on expression of the key genes involved in synthesis of milk fat, lactose and glucose metabolism in vitro was investigated. Bovine mammary epithelial cells (BMEC) were treated for 12 h with various concentrations of glucose (2.5, 5, 10 or 20 mmol/L). The higher concentrations of glucose (10–20 mmol/L) did not affect the mRNA expression of acetyl-CoA carboxylase, diacyl glycerol acyl transferase, glycerol-3 phosphate acyl transferase and α-lactalbumin, whereas fatty acid synthase, sterol regulatory element binding protein-1 and beta-1, 4-galactosyl transferase mRNA expression increased at 10 mmol/L and then decreased at 20 mmol/L. The content of lactose synthase increased with increasing concentration of glucose, with addition of highest value at 20 mmol/L of glucose. Moreover, the increased glucose concentration stimulated the activities of pyruvate kinase and glucose-6-phosphate dehydrogenase, and elevated the energy status of the BMEC. Therefore, it was deduced that after increasing glucose availability, the extra absorbed glucose was partitioned to entering the synthesis of milk fat and lactose by the regulation of the mRNA expression of key genes, promoting glucose metabolism by glycolysis and pentose phosphate pathway as well as energy status. These results indicated that the sufficient availability of glucose in BMEC may promote glucose metabolism, and affect the synthesis of milk composition.     

Specificity of binding of three soluble rat lung lectins to substituted and unsubstituted mammalian beta-galactosides

Binding of a series of mammalian glycoconjugates to three soluble rat lung lectins was determined with a quantitative assay. The three lectins, RL-14.5, RL-18, and RL-29, had a similar apparent affinity for lactose and associated with the same critical determinants, which included positions 4 and 6 of Gal and part of Glc. Derivatization at position 3 of Glc in lactose markedly reduced reactivity with the three lectins. For RL-14.5 and RL-29 the determinant extended specifically to the 3-hydroxyl of Glc which must be equatorial. In contrast, the stereochemical requirements for RL-18 were less specific, and Gal beta 1-3GalNAc bound as well as lactose. For RL-29 activity was markedly enhanced by GalNAc alpha 1-3 substitution on Gal, a modification which had little effect with RL-18 and inhibited binding to RL-14.5. Combinations of these residues in larger oligosaccharides and glycopeptides did not substantially enhance binding above that which might be expected from the sum of the constituent beta-galactoside residues. Although these lectins showed overlapping specificities, their binding properties are sufficiently different to suggest selective interactions with naturally occurring mammalian glycoconjugates.     

Enzymes involved in carbohydrate metabolism and their role on exopolysaccharide production in Streptococcus thermophilus

The role of the enzymes uridine-5'-diphospho-(UDP) glucose pyrophosphorylase and UDP galactose 4-epimerase in exopolysaccharide production of Gal⁻ ropy and non-ropy strains of Streptococcus thermophilus in a batch culture was investigated. Growth of the ropy and non-ropy strains was accompanied by total release of the galactose moiety from lactose hydrolysis in modified Bellinker broth with lactose as the only carbon source. This was associated with a greater exopolysaccharide production by the ropy strain. The polymer produced by both strains in cultures with lactose or glucose as carbon sources contained glucose, galactose and rhamnose, indicating that glucose was used as a carbon source for bacterial growth and for exopolysaccharide formation. UDP-glucose pyrophosphorylase activity was associated with polysaccharide production during the first 12 h in a 20 h culture in the ropy strain, but not in the non-ropy strain. UDP-galactose 4-epimerase was not associated with exopolysaccharide synthesis in any strain. The evidence presented suggests that the glucose moiety from lactose hydrolysis is the source of sugar for heteropolysaccharide synthesis, due to a high UDP-glucose pyrophosphorylase activity.