Histidine 271 has a functional role in pig alpha-1,3galactosyltransferase enzyme activity

Alpha(1,3)Galactosyltransferase (GT) is a Golgi-localized enzyme that catalyzes the transfer of a terminal galactose to N-acetyllactosamine to create Galalpha(1,3)Gal. This glycosyltransferase has been studied extensively because the Galalpha(1,3)Gal epitope is involved in hyperacute rejection of pig-to-human xenotransplants. The original crystal structure of bovine GT defines the amino acids forming the catalytic pocket; however, those directly involved in the interaction with the donor nucleotide sugars were not characterized. Comparison of amino acid sequences of GT from several species with the human A and B transferases suggest that His271 of pig GT may be critical for recognition of the donor substrate, UDP-Gal. Using pig GT as the representative member of the GT family, we show that replacement of His271 with Ala, Leu, or Gly caused complete loss of function, in contrast to replacement with Arg, another basic charged residue, which did not alter the ability of GT to produce Galalpha(1,3)Gal. Molecular modeling showed that His271 may interact directly with the Gal moiety of UDP-Gal, an interaction possibly retained by replacing His with Arg. However, replacing His271 with amino acids found in alpha(1,3)GalNAc transferases did not change the donor nucleotide specificity. Thus His271 is critical for enzymatic function of pig GT.     

Immobilization of UDP-Galactose 4-Epimerase from Escherichia coli on the Yeast Cell Surface

UDP-galactose 4-epimerase (EC 5.1.3.2, Gal E) from Escherichia coli catalyzes the reversible reaction between UDP-galactose and UDP-glucose. In this study, the Gal E gene from E. coli, coding UDP-galactose 4-epimerase, was cloned into pYD1 plasmid and then transformed into Saccharomyces cerevisiae EBY100 for expression of Gal E on the cell surface. Enzyme activity analyses with EBY100 cells showed that the enzyme displayed on the yeast cell surface was very active in the conversion between UDP-Glc and UDP-Gal. It took about 3 min to reach equilibrium from UDP-galactose to UDP-glucose.     

Complement insufficiency limits efficacy in a xenograft model of hyperacute rejection for cancer therapy

Hyperacute rejection (HAR) is a rapid immunological response to an organ xenotransplant caused by recognition of endothelial galactose(α1,3)galactose (αGal) epitopes and complement-mediated cell lysis by host anti-αGal antibody (‘natural antibody’). The αGal epitope is synthesised by a galactosyl transferase ((α1,3)GT) which humans lack. Because human cells transduced with (α1,3)GT are sensitised to natural antibody/complement-mediated lysis in human serum, delivery of (α1,3)GT to tumour vasculature in patients is a potential therapeutic strategy, by mimicking the pathophysiology of organ rejection. We therefore sought to develop an animal model of HAR for cancer therapy. Nude/(α1,3)GT knock-out mice allowed the growth of human tumour xenografts and the use of ecotropic retrovirus producer cells to generate expression of αGal on tumour vasculature. Lysis of αGal-positive murine endothelial cells with rabbit complement in conjunction with murine anti-αGal antibody in vitro was used to define the conditions necessary for HAR. However, tumour growth retardation and destruction of αGal-positive tumour endothelium were minimal after their administration, despite sera retaining post hoc cytolytic activity with fresh complement. The major limitation of this experimental system, of relevance to other therapeutic approaches, results from the use of a xenograft, in which additional xenoreactivities lead to complement insufficiency. Development of a tractable preclinical model in which to evaluate HAR for cancer therapy requires a syngeneic experimental system.     

An Enzyme-Linked Lectin Assay for α1,3-Galactosyltransferase

UDP-Gal:Galβ1-4GlcNAc α1,3-galactosyltransferase (α3GalT) is responsible for the synthesis of carbohydrate xenoantigen Galα1-3Galβ1-4GlcNAc. In this work a convenient and sensitive assay system for quantification of α3GalT activity by enzyme-linked lectin assay (ELLA) with colorimetric detection is described. Microtiter plate wells whose surface had been coated with the polyacrylamide conjugate of the disaccharide Galβ1-4GlcNAc (acceptor) are incubated with α3GalT in the presence of “cold” UDP-Gal as glycosyl donor. Formation of product by enzymatic extension of the glycan chain is detected by the biotinylated plant lectin Viscum album agglutinin. The standard curve for correct quantification of α3GalT activity is completed after running standard assays with no (background) or known quantities of enzyme activity. Product formation detected in this manner is proportional to enzyme activity and the concentrations of the acceptor and the glycosyl-donor UDP-Gal. In accordance with the known specificity of α3GalT, no enzymatic conversion of Le^x into GalαLe^x was observed using this assay. Human αGal antibodies were isolated using a disaccharide-exposing affinity adsorbent and their specificity was studied. Relative to the application of these natural immunoglobulins as product-detecting tool, the ELLA proved to be more sensitive.     

Frameshift and nonsense mutations in a human genomic sequence homologous to a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase cDNA

We have previously isolated a murine UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase (alpha(1,3)-GT) cDNA (Larsen, R. D., Rajan, V. P., Ruff, M. M., Kukowska-Latallo, J., Cummings, R. D., and Lowe, J. B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8227-8231). This enzyme constructs the terminal alpha(1,3)-galactosyl linkage within the epitope Gal alpha 1----3Gal. This epitope is expressed by New World monkeys and many nonprimate mammals but generally not by Old World primates, anthropoid apes, or man. To investigate the molecular basis for the apparent species-specific absence of this enzyme and its oligosaccharide product, we have sequenced a human genomic DNA fragment homologous to the murine alpha(1,3)-GT cDNA. This fragment contains a 703-nucleotide region that shares 82% identity with a region of the murine cDNA encoding part of the enzyme's catalytic domain. The human sequence, however, has suffered deletion of single nucleotides at two separate positions, relative to the murine sequence. These frameshift mutations disrupt the translational reading frame that would otherwise maintain a 76% amino acid sequence identity between the human sequence and the murine alpha(1,3)-GT. Moreover, nonsense mutations exist within this disrupted reading frame that would truncate the human polypeptide, relative to the murine enzyme. We therefore propose that this human sequence represents a pseudogene and cannot determine expression of Gal alpha 1----3Gal epitopes on human cells.     

Ovarian cancer alpha 1,3-L-fucosyltransferase. Differentiation of distinct catalytic species with the unique substrate, 3'-sulfo-N-acetyllactosamine in conjunction with other synthetic acceptors.

Several N-acetyllactosamine (LacNAc) derivatives were tested as acceptors for alpha 1,3-L-fucosyltransferase present in human ovarian cancer sera and ovarian tumor. The enzyme of the soluble fraction of tumor was purified to apparent homogeneity by chromatography on bovine IgG glycopeptide-Sepharose followed by Sephacryl S-200 (M(r) < 67,000). As compared with 2'-methyl LacNAc, 3'-sulfo LacNAc was about 5-fold more sensitive in measuring alpha 1,3-fucosyltransferase in sera (Km, 3'-sulfo LacNAc, 0.12 mM; 2'-methyl LacNAc, 6.67 mM). When ovarian cancer serum was the enzyme source, either the sulfate group or a sialyl moiety at C-3' of LacNAc enhanced the acceptor ability (341 and 242%, respectively), whereas the sulfate group at C-2' or C-6' reduced the activity (22-36%); sulfate at C-6 or fucose at C-2' increased the activity (172 and 253%). The beta-benzylation of the reducing end, in general, increased the activity 2-3-fold. The enzyme of the soluble fraction of tumor exhibited more activity toward 3'-sulfo LacNAc (447%), 2'-fucosyl-LacNAc (436%), and 6-sulfo LacNAc (272%). Very low activity was observed with 3'-sialyl LacNAc (12.4%), 2'-sulfo LacNAc (33%), and 6'-sulfo LacNAc (5%); Fuc alpha 1,2Gal beta 1,3GlcNAc beta-O-p-nitrophenyl (166%), 2-methyl Gal beta 1,3GlcNAc beta-O-benzyl (204%), and 3-sulfo Gal beta 1,3GlcNAc (415%) also acted as acceptors, indicating the coexistence of alpha 1,3- and alpha 1,4-fucosyltransferase. The tumor particulate enzyme behaved entirely different, exhibiting low activity with 3'-sulfo LacNAc (39%) and 2'-fucosyl-LacNAc (148%); 3'-sialyl, 6'-sulfo, 6-sulfo, or 2'-sulfo LacNAc were 3, 43, 53, and 10% active, respectively. Thus, the ovarian cancer serum alpha 1,3-fucosyltransferase acts equally well on H-type 2,3'-sialyl LacNAc and 3'-sulfo LacNAc, but not on H-type 1. The enzyme of soluble tumor fraction acts on H-type 2,3'-sulfo LacNAc as well as H-type 1 but poorly on 3'-sialyl LacNAc. The tumor particulate enzyme acts on H-type 2 but poorly on 3'-sulfo or 3'-sialyl LacNAc and is inactive with H-type 1. When normal serum was examined with synthetic acceptors, > 80% activity was found as alpha 1,2-fucosyltransferase and the rest as alpha 1,3-fucosyltransferase. A screening of 21 ovarian cancer and 3 normal sera (3'-sulfo LacNAc as acceptor) showed 17-572% increase (average increase, 188%) of alpha 1,3-fucosyltransferase activity in cancer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression of alpha-gal epitopes on HeLa cells transduced with adenovirus containing alpha1,3galactosyltransferase cDNA

Alpha1,3galactosyltransferase (alpha1,3GT) synthesizes alpha-gal epitopes (Gal(alpha)1-3Galbeta1-4GlcNAc-R) on glycoconjugates in nonprimate mammals but not in humans. Transduction of alpha1,3GT gene into human HeLa cells by an adenovirus vector allowed for accurate kinetics studies on the appearance of alpha1,3GT and of its product, the alpha-gal epitope, in the transduced cells. Mouse alpha1,3GT cDNA was inserted into a replication-defective adenovirus vector. This viral vector, designated Ad(alpha)GT, could be propagated in human 293 cells that have the viral E1 complementing gene. Transduction of HeLa cells resulted in immediate penetration of approximately 20 Ad(alpha)GT copies into each cell and the appearance of alpha1,3GT mRNA after 4h. Catalytic activity of alpha1,3GT was first detected in the cells after 6 h. The initial appearance of alpha-gal epitopes (approximately 6 x 10(4)/cell) on cell surface glycoconjugates was detected 10 h posttransduction, whereas 24 h posttransduction each cell expressed 2 x 10(6) epitopes. The activity of alpha1,3GT in cells transduced with approximately two copies of Ad(alpha)GT was eightfold lower than that in cells transduced with approximately 20 Ad(alpha)GT copies; however, the number of alpha-gal epitopes/cell remained closely similar. This implies that increased alpha1,3GT activity above a certain saturation level does not result in a corresponding increase in the carbohydrate product, possibly because of competing glycosyltransferases.     

Production of a recombinant carotenoid cleavage dioxygenase from grape and enzyme assay in water-miscible organic solvents

A recombinant carotenoid cleavage dioxygenase from Vitis vinifera L. was produced by Escherichia coli as a fusion with the glutathione-S-transferase (GST) protein under different bacterial growth conditions. The enzyme production was monitored by a GST assay. Addition of Triton X-100 prior to bacterial cell disruption doubled the release of soluble protein. A simple spectrophotometric enzyme assay was developed to measure carotenoid cleavage activity using lutein as substrate. Enzyme activity showed a 26-fold increase with the addition of 10% (v/v) acetone in the reaction mixture.     

The molecular basis for galalpha(1,3)gal expression in animals with a deletion of the alpha1,3galactosyltransferase gene

The production of homozygous pigs with a disruption in the GGTA1 gene, which encodes alpha1,3galactosyltransferase (alpha1,3GT), represented a critical step toward the clinical reality of xenotransplantation. Unexpectedly, the predicted complete elimination of the immunogenic Galalpha(1,3)Gal carbohydrate epitope was not observed as Galalpha(1,3)Gal staining was still present in tissues from GGTA1(-/-) animals. This shows that, contrary to previous dogma, alpha1,3GT is not the only enzyme able to synthesize Galalpha(1,3)Gal. As iGb3 synthase (iGb3S) is a candidate glycosyltransferase, we cloned iGb3S cDNA from GGTA1(-/-) mouse thymus and confirmed mRNA expression in both mouse and pig tissues. The mouse iGb3S gene exhibits alternative splicing of exons that results in a markedly different cytoplasmic tail compared with the rat gene. Transfection of iGb3S cDNA resulted in high levels of cell surface Galalpha(1,3)Gal synthesized via the isoglobo series pathway, thus demonstrating that mouse iGb3S is an additional enzyme capable of synthesizing the xenoreactive Galalpha(1,3)Gal epitope. Galalpha(1,3)Gal synthesized by iGb3S, in contrast to alpha1,3GT, was resistant to down-regulation by competition with alpha1,2fucosyltransferase. Moreover, Galalpha(1,3)Gal synthesized by iGb3S was immunogenic and elicited Abs in GGTA1 (-/-) mice. Galalpha(1,3)Gal synthesized by iGb3S may affect survival of pig transplants in humans, and deletion of this gene, or modification of its product, warrants consideration.     

Synthesis of alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) on human tumor cells by recombinant alpha1,3galactosyltransferase produced in Pichia pastoris.

This study describes the processing of human tumor cells or cell membranes to express alpha-gal epitopes (Galalpha1-3Gal-beta1-4GlcNAc-R) by the use of New World monkey (marmoset) recombinant alpha1,3galactosyltransferase (ralpha1,3GT), produced in the yeast Pichia pastoris. Such tumor cells and membranes may serve, in cancer patients, as autologous tumor vaccines that are targeted in vivo to antigen-presenting cells by the anti-Gal antibody. This ralpha1,3GT lacks transmembrane and cytoplasmic domains, ensuring its solubility without detergent. It is effectively produced in P. pastoris under constitutive expression of the P(GAP) promoter and is secreted into the culture medium in a soluble, truncated form fused to a (His)(6) tag. This tag enables the simple affinity purification of ralpha1,3GT on a nickel-Sepharose column and elution with imidazole. The purified enzyme appears in SDS-PAGE as two bands with the size of 40 and 41 kDa and displays the same acceptor specificity as the mammalian native enzyme. ralpha1,3GT is very effective in synthesizing alpha-gal epitopes on membrane-bound carbohydrate chains and displays a specific activity of 1.2 nM membrane bound alpha-gal epitopes/min/mg. Incubation of very large amounts of human acute myeloid leukemia cells (1 x 10(9 )cells) with neuraminidase, ralpha1,3GT, and UDP-Gal resulted in the synthesis of approximately 6 x 10(6 )alpha-gal epitopes per cell. Effective synthesis of alpha-gal epitopes could be achieved also with as much as 2 g cell membranes prepared from the tumor of a patient with ovarian carcinoma. These data imply that ralpha1,3GT produced in P. pastoris is suitable for the synthesis of alpha-gal epitopes on bulk amounts of tumor cells or cell membranes required for the preparation of autologous tumor vaccines.     

Molecular characterization of a novel UDP-galactose:fucoside alpha3-galactosyltransferase that modifies Skp1 in the cytoplasm of Dictyostelium

Skp1 is a nucleocytoplasmic protein that is post-translationally modified by a pentasaccharide, Gal alpha1,Gal alpha1,3Fuc alpha1,2Gal-beta1,3GlcNAc alpha1O-, at a 4-hydroxylated derivative of Pro-143 in the amebazoan Dictyostelium discoideum. An enzymatic activity that catalyzes formation of the Gal alpha1,3Fuc linkage by transfer of Gal from UDP-alphaGal to Fuc alpha1,2Gal beta1,3GlcNAc alpha1O-benzyl, or the corresponding glycoform of Skp1, was described previously in cytosolic extracts of Dictyostelium. A protein GT78 associated with this activity has been purified to chromatographic homogeneity. In-gel tryptic digestion followed by nano-liquid chromatography-mass spectrometry on a quadrupole time-of-flight geometry instrument with data-dependent tandem mass spectrometry acquisition yielded a number of peptide fragmentation spectra, nine of which were manually de novo sequenced and found to map onto a predicted 3-exon gene of unknown function on chromosome 4. GT78 is predicted to comprise 648 amino acids with an N-terminal glycosyltransferase and a C-terminal beta-propeller domain. Overexpression of GT78 with a His6-tag resulted in a 120-fold increase in GalT-activity in cytosolic extracts, and purified His6-GT78 exhibited alpha3GalT-activity toward a synthetic acceptor substrate. Expression of the truncated N-terminal region confirmed the predicted catalytic activity of this domain. Disruption of the GT78 gene led to a loss of enzyme activity in extracts and accumulation of the non-galactosylated isoform of Skp1 in cells. GT78 therefore represents the Skp1 alpha3GalT, and its mechanism conforms to the sequential model of Skp1 glycosylation in the cytoplasm shown for earlier enzymes in the pathway. Informatics studies suggest that related catalytic domains are expressed in the Golgi or cytoplasm of plants, other protozoans, and animals.     

Functional Identification of a Hydroxyproline-O-galactosyltransferase Specific for Arabinogalactan Protein Biosynthesis in Arabidopsis

Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [¹⁴C]Gal from UDP-[¹⁴C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)₇ and anhydrous hydrogen fluoride-deglycosylated d(AO)₅₁. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [¹⁴C]Gal to (AO)₇, and the resulting product co-eluted with (AO)₇ by reverse-phase HPLC. Acid hydrolysis of the [¹⁴C]Gal-(AO)₇ product released ¹⁴C-radiolabel as Gal only. Base hydrolysis of the [¹⁴C]Gal-(AO)₇ product released a ¹⁴C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg²⁺ or Mn²⁺ for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function.     

Mucin biosynthesis revisited. The enzymatic transfer of Gal in beta 1,3 linkage to the GalNAc moiety of the core structure R1-GlcNAc beta 1,6GalNAc alpha-O-R2.

Synthetic glycosides containing the core, -Glc-NAc beta 1,6GalNAc alpha-, acted as acceptors for beta-galactosyltransferase of human ovarian tumor. A significant amount of Gal was transferred from UDP-Gal (100 nmol) to the alpha-benzylglycoside of LacNAc beta 1,6GalNAc (LGBn) (25.1 nmol of Gal) and the alpha-ortho-nitrophenylglycosides of LacNAc beta 1,6GalNAc (22.0 nmol of Gal), GlcNAc beta 1,6GalNAc (15.5 nmol of Gal), and Fuc alpha 1,3GlcNAc beta 1,6GalNAc (25.9 nmol of Gal); LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn (where Bn is benzyl) was almost inactive (only 1.2 nmol of Gal), indicating the Gal transfer to the alpha-GalNAc moiety. The product from LGBn was isolated in microgram quantities and identified by fast atom bombardment mass spectrometry as LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn. The alpha GalNAc:beta 1,3Gal transferase was present in high concentration in ovarian tumor tissue (ovarian cancer serum----1.4; ascitic fluid----0.9; tumor----17.4). Asialo Cowper's gland mucin (ACGM) at 5 mg/ml reaction mixture inhibited the transfer of Gal to LGBn (25.2 and 53.4% respectively for 2 and 18 h incubation at 37 degrees C); inhibition by LGBn was 13.4 and 24.5%, respectively. In contrast to the inhibition by ACGM (25.2-31.6%), there was substantial increase (13.4-35.7%) in the inhibition by LGBn, when the incubation for 2 h at 37 degrees C was continued for 40 h at 4 degrees C, indicating the high affinity of LGBn for the enzyme at lower temp. Km for LGBn in presence of ACGM was 7.6 mM and in absence, 2.7 mM; Km for ACGM (M(r) 200,000) in presence of LGBn was 16.1 microM and Ki for ACGM (as the inhibitor) was 41.7 microM. In comparison with two normal ovarian tissues, the enzyme was found to be low (55-67%) in three ovarian tumors and high (146-260%) in two ovarian and one uterus tumors, as measured with ACGM; the synthetic acceptors showed similar activities. The enzyme had nearly the same extent of activity in the pH range 6-8. Fuc alpha 1,3GlcNAc beta 1,6GalNAc alpha-O-ONP had the highest affinity for the enzyme. The present study demonstrates the feasibility of beta 1,3Gal attachment on alpha GalNAc, which has already been substituted by beta 1,6GlcNAc, then elongated by beta 1,4Gal and also terminated by alpha 1,3Fuc.     

Evaluation of the Galα1-3Gal Epitope as a Host Modification Factor Eliciting Natural Humoral Immunity to Enveloped Viruses

Human sera contain high levels of natural antibody (Ab) to Galα1-3Gal, a terminal glycosidic structure expressed on the surface of cells of mammals other than Old World primates. Incorporation of this determinant onto retroviral membranes by passage of viruses in cells encoding α-1-3-galactosyltransferase (GT) renders retroviruses sensitive to lysis by natural Ab and complement in normal human serum (NHS). Plasma membrane-budding viruses representing four additional virus groups were examined for their sensitivities to serum inactivation after passage through human cell lines that lack a functional GT or human cells expressing recombinant porcine GT. The inactivation of lymphocytic choriomeningitis virus (LCMV) by NHS directly correlated with host modification of the virus via expression of Galα1-3Gal and was blocked by incorporation of soluble Galα1-3Gal disaccharide into the inactivation assay. GT-deficient mice immunized to make high levels of Ab to Galα1-3Gal (anti-Gal Ab) were tested for resistance to LCMV passaged in GT-expressing cells. Resistance was not observed, but in vitro analyses of the mouse immune sera revealed that the antiviral activity of the sera was insufficient to eliminate LCMV infectivity on its natural targets of infection, macrophages, which express receptors for Ab and complement. Newcastle disease virus and vesicular stomatitis virus (VSV) were inactivated by NHS regardless of cell passage history, whereas Sindbis virus (SV) passaged in human cells resisted inactivation. Both VSV and SV passaged in Galα1-3Gal-expressing human cells incorporated this sugar moiety onto their major envelope glycoproteins. SV passaged in mouse cells expressing Galα1-3Gal was moderately sensitive to inactivation by NHS. These results indicate that enveloped viruses expressing Galα1-3Gal differ in their sensitivities to NHS and that a potent complement source, such as that in NHS, is required for efficient inactivation of sensitive viruses in vitro and in vivo.     

The wbbD gene of E. coli strain VW187 (O7:K1) encodes a UDP-Gal: GlcNAc{alpha}-pyrophosphate-R {beta}1,3-galactosyltransferase involved in the biosynthesis of O7-specific lipopolysaccharide

In this work, we demonstrate that the wbbD gene of the O7 lipopolysaccharide (LPS) biosynthesis cluster in Escherichia coli strain VW187 (O7:K1) encodes a galactosyltransferase involved in the synthesis of the O7-polysaccharide repeating unit. The galactosyltransferase catalyzed the transfer of Gal from UDP-Gal to the GlcNAc residue of a GlcNAc-pyrophosphate-lipid acceptor. A mutant strain with a defective wbbD gene was unable to form O7 LPS and lacked this specific galactosyltransferase activity. The normal phenotype was restored by complementing the mutant with the cloned wbbD gene. To characterize the WbbD galactosyltransferase, we used a novel acceptor substrate containing GlcNAcalpha-pyrophosphate covalently bound to a hydrophobic phenoxyundecyl moiety (GlcNAc alpha-O-PO(3)-PO(3)-(CH(2))(11)-O-phenyl). The WbbD galactosyltransferase had optimal activity at pH 7 in the presence of 2.5 mM MnCl(2). Detergents in the assay did not increase glycosyl transfer. Digestion of enzyme product by highly purified bovine testicular beta-galactosidase demonstrated a beta-linkage. Cleavage of product by pyrophosphatase and phosphatase, followed by HPLC and NMR analyses, revealed a disaccharide with the structure Gal beta1-3GlcNAc. Our results conclusively demonstrate that WbbD is a UDP-Gal: GlcNAcalpha-pyrophosphate-R beta1,3-galactosyltransferase and suggest that the novel synthetic glycolipid acceptor may be generally applicable to characterize other bacterial glycosyltransferases.     

Identification of an exo-ß-1,3-d-galactanase from Fusarium oxysporum and the synergistic effect with related enzymes on degradation of type II arabinogalactan

An exo-ß-1,3-d-galactanase (Fo/1,3Gal) was purified from the culture filtrate of Fusarium oxysporum 12S. A cDNA encoding Fo/1,3Gal was isolated by in vitro cloning. Module sequence analysis revealed a “GH43_6” domain and a “CBM35_galactosidase-like” domain in Fo/1,3Gal. The recombinant enzyme (rFo/1,3Gal) expressed in Pichia pastoris degraded ß-1,3-galactan and ß-1,3-galactobiose (Gal2), and released only galactose (Gal). In contrast, the enzyme did not hydrolyze p-nitrophenyl ß-d-galactopyranoside, ß-1,4-Gal2, or ß-1,6-Gal2. The enzyme also showed low activity towards native type II arabinogalactans such as larchwood arabinogalactan (LWAG) and gum arabic. Using LWAG as substrate, rFo/1,3Gal released Gal, ß-1,6-Gal2, ß-1,6-galactotriose (Gal3), and ß-1,6-Gal3 substituted with a single arabinofuranose residue accompanied with unidentified oligosaccharides, indicating that the enzyme can by-pass the branching points of ß-1,3-galactan backbones. A time course analysis of products released by rFo/1,3Gal on LWAG revealed that ß-1,6-Gal2 is the main side chain in LWAG and that the activity of rFo/1,3Gal was decreased when degrees of polymerization of side chains increase. rFo/1,3Gal worked synergistically with three other recombinant F. oxysporum enzymes (ß-1,6-galactanase, ß-l-arabinopyranosidase, and α-l-arabinofuranosidase) that degrade side chains, on the degradation of LWAG. However, the synergism was much lower than anticipated, probably because LWAG have longer side chains than the three enzymes used are able to remove or ß-1,3-galactan main chain is interrupted with glycosidic linkages that are different from the ß-1,3-galactosyl linkage. Affinity gel electrophoresis revealed that rFo/1,3Gal specifically bound to ß-1,3-galactan.     

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

A soluble and active form of recombinant human ST6Gal I was expressed in Escherichia coli. The gene encoding the soluble form of ST6Gal I lacking the membrane and cytosolic regions was introduced into a bacterial expression vector, pMAL-p2X, fused in frame with a maltose-binding protein (MBP) tag. Low-temperature cultivation at 13C during IPTG-induction significantly improved both solubility and MBP-tagging of the recombinant enzyme expressed in bacteria. The supernatant prepared by disruption of the cells demonstrated sialic acid transfer activity to both an oligosaccharide and a glycoprotein, asialofetuin, indicating that the enzyme expressed in bacteria is soluble and active. The MBP-tagged enzyme was efficiently purified by a combination of cation-exchange column and amylase-conjugated agarose column chromatography. The purified recombinant enzyme exerted enzymatic activity even in the absence of detergents in the reaction mixture. Acceptor substrate specificity of the enzyme was marginally different from that of rat liver ST6Gal I. These observations suggest that membrane and cytosolic regions of ST6Gal I may affect the properties of the enzyme. The purified recombinant enzyme was applied to convert desialylated fetuin to resialylated fetuin. Lectin blotting demonstrated that resialylated fetuin possesses a single Neu5Ac 2-6 residue. The resialylated fetuin efficiently blocked hemagglutination induced by influenza virus strain A/Memphis/1/71 (H3N2), indicating that resialylated carbohydrate chains on the protein are so active as to competitively inhibit virus-receptor interaction. In conclusion, soluble recombinant ST6Gal I obtained using our bacterial expression system is a valuable tool to investigate the molecular mechanisms of biological and pathological interactions mediated via carbohydrates. Published in 2005.The authors contributed equally to this work.