Kinetic studies on Korean serum cis-AB enzymes reveal diminished A and B transferase activities.

BACKGROUND: cis-AB enzymes are rare glycosyltransferases that synthesize both blood group A and B antigens. We have identified a large cohort of Korean cis-AB blood donors and studied the N-acetylgalactosaminyltransferase (glycosyltransferase A, GTA) and galactosyltransferase (glycosyltransferase B, GTB) activity of their cis-AB serum enzymes. MATERIALS AND METHODS: The cis-AB01 allele was identified by PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) in 60 donors collected at the Gwangju-Chonnam Red Cross Blood Center. Enzyme assays of this cis-AB enzyme were performed on available serum samples from 16 donors with the cis-AB01/O genotype and three with the cis-AB01/A genotype. RESULTS: In cis-AB donors with an O allele, both the GTA and GTB activity of the cis-AB enzyme were markedly reduced compared to normal A and B controls (29% and 27%, respectively). This is consistent with the behaviour predicted from kinetic studies of a recombinant model of the corresponding AAAB enzyme. CONCLUSION: Although variable, cis-AB enzymes feature reduced GTA and GTB activities. SUMMARY: Cis-AB enzymes feature variable but reduced GTA and GTB activities with relatively weaker GTB activity, consistent with the weak agglutination present on forward typing with anti-B.

Crystallization and preliminary X-ray diffraction analysis of two homologous antigen-binding fragments in complex with different carbohydrate antigens.

The antigen-binding fragments (Fab) of two murine monoclonal antibodies (mAb) S25-2 and S45-18, specific for carbohydrate epitopes in the lipopolysacchaide of the bacterial family Chlamydiaceae, have been crystallized in the presence and absence of synthetic oligosaccharides corresponding to their respective haptens. Crystals of both Fabs show different morphology depending on the presence of antigens. The sequence of mAb S45-18 was determined and shows a remarkable homology to that reported for mAb S25-2. These crystals offer an unparalleled opportunity to compare the structure and modes of binding of two homologous antibodies to similar but distinct carbohydrate epitopes.

Natural and recombinant A and B gene encoded glycosyltransferases.

The human blood group A and B synthesizing enzymes are glycosyltransferases that catalyse the transfer of a monosaccharide residue from UDP-GalNAc and UDP-Gal donors, respectively, to alphaFuc1,2-Gal terminated blood group H acceptors. Extensive investigations of their substrate specificity and physical properties have been carried out since their initial discovery. These studies demonstrated a rigid specificity for the acceptor structure, crossover in donor specificity and immunological similarity along with chromatographic differences. Cloning of the enzymes has shown that they are highly homologous, differing in only four of their 354 amino acids. Changing the residues Arg176-->Gly, Gly235-->Ser, Leu266-->Met and Gly 268-->Ala converts the enzyme specificity from blood group A to blood group B glycosyltransferase. Structure function investigations have been carried out by systematic interchange and modification of these four critical amino acids. These studies have shown that donor specificity is attributed to the last two amino acids. Mutants have also been produced with greatly enhanced turnover rates as well as hybrid A/B enzymes that catalyse both reactions efficiently.

Characterization of cysteine residues and disulfide bonds in proteins by liquid chromatography/electrospray ionization tandem mass spectrometry.

Cysteine residues and disulfide bonds are important for protein structure and function. We have developed a simple and sensitive method for determining the presence of free cysteine (Cys) residues and disulfide bonded Cys residues in proteins (<100 pmol) by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) in combination with protein database searching using the program Sequest. Free Cys residues in a protein were labeled with PEO-maleimide biotin immediately followed by denaturation with 8 M urea. Subsequently, the protein was digested with trypsin or chymotrypsin and the resulting products were analyzed by capillary LC/ESI-MS/MS for peptides containing modified Cys and/or disulfide bonded Cys residues. Although the MS method for identifying disulfide bonds has been routinely employed, methods to prevent thiol-disulfide exchange have not been well documented. Our protocol was found to minimize the occurrence of the thiol-disulfide exchange reaction. The method was validated using well-characterized proteins such as aldolase, ovalbumin, and beta-lactoglobulin A. We also applied this method to characterize Cys residues and disulfide bonds of beta 1,4-galactosyltransferase (five Cys), and human blood group A and B glycosyltransferases (four Cys). Our results demonstrate that beta 1,4-galactosyltransferase contains one free Cys residue and two disulfide bonds, which is in contrast to work previously reported using chemical methods for the characterization of free Cys residues, but is consistent with recently published results from x-ray crystallography. In contrast to the results obtained for beta 1,4-galactosyltransferase, none of the Cys residues in A and B glycosyltransferases were found to be involved in disulfide bonds.

Enzymatic synthesis of blood group A and B trisaccharide analogues.

Glycosyltransferases A and B utilize the donor substrates UDP-GalNAc and UDP-Gal, respectively, in the biosynthesis of the human blood group A and B trisaccharide antigens from the O(H)-acceptor substrates. These enzymes were cloned as synthetic genes and expressed in Escherichia coli, thereby generating large quantities of enzyme for donor specificity evaluations. The amino acid sequence of glycosyltransferase A only differs from glycosyltransferase B by four amino acids, and alteration of these four amino acid residues (Arg-176-->Gly, Gly-235-->Ser, Leu-266-->Met and Gly-268-->Ala) can change the donor substrate specificity from UDP-GalNAc to UDP-Gal. Crossovers in donor substrate specificity have been observed, i.e., the A transferase can utilize UDP-Gal and B transferase can utilize UDP-GalNAc donor substrates. We now report a unique donor specificity for each enzyme type. Only A transferase can utilize UDP-GlcNAc donor substrates synthesizing the blood group A trisaccharide analog alpha-D-Glcp-NAc-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2 )7CH3 (4). Recombinant blood group B was shown to use UDP-Glc donor substrates synthesizing blood group B trisaccharide analog alpha-D-Glcp-(1-->3)-[alpha-L-Fucp-(1-->2)]-beta-D-Galp-O-(CH2) 7CH3 (5). In addition, a true hybrid enzyme was constructed (Gly-235-->Ser, Leu-266-->Met) that could utilize both UDP-GlcNAc and UDP-Glc. Although the rate of transfer with UDP-GlcNAc by the A enzyme was 0.4% that of UDP-GalNAc and the rate of transfer with UDP-Glc by the B enzyme was 0.01% that of UDP-Gal, these cloned enzymes could be used for the enzymatic synthesis of blood group A and B trisaccharide analogs 4 and 5.

Genetically tagged putative differentiation intermediates derived from undifferentiated HT29 human colon carcinoma cells.

The HT29 colonic carcinoma cell line has proven to be a very practical tool for modelling aspects of colonic cell differentiation and toxification by chemotherapeutic agents. As an approach to subclone and clarify molecular events involved in sublineage maturation, non-differentiated HT29 cells were electroporated with a dominant marker gene (NeoR) to convey aminoglycoside resistance (G418R). Transfectants surviving passage in glucose-G418 medium were >200 times the abundance of transient G418R cells of controls. Genomic analysis showed that each clonal type was unique in NeoR integration pattern while mitochondrial DNA copy was relatively unchanged. All of the randomly generated NeoR clones resembled the parental phenotype, but some over-produced the mucin, secretory cell type or the cell death phenotype after culturing in 2 mM sodium butyrate medium. Re-exposure to glucose medium restored the parental-like phenotype.

Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli.

The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli. The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity.

Synthesis of the acceptor analog alphaFuc(1-->2)alphaGal-O(CH2)7 CH3: a probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase.

We report the chemical synthesis of alphaFuc(1-->2)alphaGal-O(CH2)7CH3 (1) an analog of the natural blood group (O)H disaccharide alphaFuc(1-->2)betaGal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog (alphaGal(-->3)alphaFuc(1-->2)]alphaGal-O(CH2)7CH3+ ++ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide alphaFuc(1-->2)betaGal-O(CH2)7CH3 (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.

Sequential interchange of four amino acids from blood group B to blood group A glycosyltransferase boosts catalytic activity and progressively modifies substrate recognition in human recombinant enzymes.

The human blood group A and B glycosyltransferase enzymes are highly homologous and the alteration of four critical amino acid residues (Arg-176 --> Gly, Gly-235 --> Ser, Leu-266 --> Met, and Gly-268 --> Ala) is sufficient to change the enzyme specificity from a blood group A to a blood group B glycosyltransferase. To carry out a systematic study, a synthetic gene strategy was employed to obtain their genes and to allow facile mutagenesis. Soluble forms of a recombinant glycosyltransferase A and a set of hybrid glycosyltransferase A and B mutants were expressed in Escherichia coli in high yields, which allowed them to be kinetically characterized extensively for the first time. A functional hybrid A/B mutant enzyme was able to catalyze both A and B reactions, with the kcat being 5-fold higher for the A donor. Surprisingly, even a single amino acid replacement in glycosyltransferase A with the corresponding residue from glycosyltransferase B (Arg-176 --> Gly) produced enzymes with glycosyltransferase A activity only, but with very large (11-fold) increases in the kcat and increased specificity. The increases observed in kcat are among the largest obtained for a single amino acid change and are advantageous for the preparative scale synthesis of blood group antigens.

Expression of a recombinant human glycosyltransferase from a synthetic gene and its utilization for synthesis of the human blood group B trisaccharide.

A 1034-bp synthetic gene encoding the human blood group B glycosyltransferase, which catalyzes the transfer of galactose from UDP-Gal to Fuc alpha(1-2)Gal beta-OR to give the blood group B determinant Gal alpha(1-3)[Fuc alpha(1-2)]Gal beta-OR (where R is a glycoprotein or glycolipid), has been expressed in Escherichia coli by replacing its membrane-anchoring domain with an ompA bacterial secretory signal. The active enzyme was purified from the periplasm using UDP-hexanolamine affinity chromatography and used in the synthesis of preparative amounts of the human blood group B trisaccharide antigen. The substrate specificity and kinetics of the recombinant enzyme were comparable to the enzyme from human sera. Thus we have achieved the construction of a completely synthetic glycosyltransferase gene and its successful expression.

Expression and characterization of secreted forms of rubella virus E2 glycoprotein in insect cells.

Two different forms of rubella virus E2 glycoproteins were expressed in insect cells: intact wild-type E2 and a soluble form of E2 (E2 delta Tm) glycoprotein, in which the C-terminal membrane-anchor domain was deleted. E2 delta Tm behaved as a secretory protein and was secreted abundantly (5 mg/liter) from insect cells. In contrast to wild-type E2 (36 kDa), E2 delta Tm was secreted into the media and was detected as two species (33 and 30 kDa). Lectin binding assays in conjunction with glycosidase analyses revealed that both intracellular wild-type E2 and E2 delta Tm contained only N-linked glycans, while the two secreted forms of E2 delta Tm were found to differ in their glycosylation, with the 30-kDa form having only N-linked glycans while the 33-kDa species had both N-linked and O-linked glycans. The secreted E2 delta Tm species were purified by precipitation between 20 and 40% saturation with (NH4)2SO4 and retained full antigenicity. The levels of antibodies elicited in mice immunized with purified E2 delta Tm showed that the immunogenicity of secreted E2 delta Tm compared favorably to that of natural virion E2.

Expression and characterization of a soluble rubella virus E1 envelope protein.

Individual specific antigenic rubella virus (RV) structural proteins are required for accurate serological diagnosis of acute and congenital rubella infections as well as rubella immune status. The RV envelope glycoprotein E1 is the major target antigen and plays an important role in viral-specific immune responses. The native virion is difficult to produce in large quantities and the protein subunits are also difficult to isolate without loss of antigenicity. The production of a soluble RV E1 (designated E1 delta Tm) using the baculovirus-insect cell expression system is described. In contrast to wild-type RV E1, the genetically engineered E1 delta Tm protein lacks a transmembrane anchor. It behaved as a secretory protein and was secreted abundantly from insect cells. Pulse-chase studies were used to examine the synthesis, glycosylation, and secretion of E1 delta Tm by the insect cells. The secreted E1 delta Tm protein was purified from serum-free medium by one-step immunochromatography. The purified E1 delta Tm protein retained full antigenicity and may be a convenient source of E1 protein for use in diagnostic assay and rubella vaccine development.

Expression of soluble forms of rubella virus glycoproteins in mammalian cells.

Rubella virus (RV) virions contain two envelope glycoproteins, E1 and E2. Removal of hydrophobic regions in their carboxyl termini by genetic engineering caused them to be secreted rather than maintained in cell membranes of transfected COS cells. Truncated E2 was secreted in the absence of E1, whereas E1 lacking its transmembrane domain required coexpression of E2 for export from the cell. Secreted E2 was found to contain both O-linked and N-linked complex glycans, whereas secreted E1 retained virus neutralization and hemagglutination epitopes, suggesting the possibility of using soluble RV antigens as subunit vaccines and for serodiagnostic purposes. Stable Chinese hamster ovary cell lines secreting RV E1 were constructed for large scale preparation of recombinant E1.

Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

The role of N-linked glycosylation in processing and intracellular transport of rubella virus glycoprotein E2 has been studied by expressing glycosylation mutants of E2 in COS cells. A panel of E2 glycosylation mutants were generated by oligonucleotide-directed mutagenesis. Each of the three potential N-linked glycosylation sites was eliminated separately as well as in combination with the other two sites. Expression of the E2 mutant proteins in COS cells indicated that in rubella virus M33 strain, all three sites are used for the addition of N-linked oligosaccharides. Removal of any of the glycosylation sites resulted in slower glycan processing, lower stability, and aberrant disulfide bonding of the mutant proteins, with the severity of defect depending on the number of deleted carbohydrate sites. The mutant proteins were transported to the endoplasmic reticulum and Golgi complex but were not detected on the cell surface. However, the secretion of the anchor-free form of E2 into the medium was not completely blocked by the removal of any one of its glycosylation sites. This effect was dependent on the position of the deleted glycosylation site.

Overexpression of Cu-Zn superoxide dismutase in Drosophila does not affect life-span.

Aging and disease processes may be due to deleterious and irreversible changes produced by free radical reactions. The enzyme copper-zinc superoxide dismutase (Cu-Zn SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) performs a protective function by scavenging superoxide radicals. The Cu-Zn SOD gene (Sod) cloned from Drosophila melanogaster was introduced via P element-mediated transformation into the germ line. Homozygous lines carrying additional copies of the Sod gene were recovered and characterized. Increases in Sod transcripts and enzyme activity were observed in the transformed lines, indicating that all of the sequence information required for gene expression is contained on the inserted gene fragment. The effects of additional SOD on oxygen free radical metabolism and longevity were investigated. Additional SOD did not markedly affect oxygen metabolism or longevity.

Cloning, sequence analysis and chromosomal localization of the Cu-Zn superoxide dismutase gene of Drosophila melanogaster.

The Cu-Zn superoxide dismutase (SOD) cDNA and genomic DNA from Drosophila melanogaster have been isolated. The sequence of the coding region for the Sod gene as well as 413 bp of the 5'-untranslated region, 247 bp of the 3'-flanking DNA, and the single 725-bp intron have been determined [Seto et al., Nucleic Acids Res. 15 (1987) 10601]. The sequence reveals an additional C-terminal triplet coding for valine not found in the mature SOD protein. The nucleotide sequence of the coding region has 56% and 57% homology compared with the corresponding human and rat Sod genes, respectively. The codon usage for the Sod gene is similar to that found for other Drosophila genes. The gene hybridizes to position 68A4-9 on Drosophila polytene chromosomes. In addition, in wild-type Drosophila the Sod cDNA hybridizes to a 0.7-0.8-kb transcript which is greatly diminished in a Sod 'null' mutant that produces only 3.5% of the SOD protein.