Non-reducing Terminal Positions Research Articles

CD23 is a glycan-binding receptor in some mammalian species

CD23, the low-affinity IgE receptor found on B lymphocytes and other cells, contains a C-terminal lectin-like domain that resembles C-type carbohydrate-recognition domains (CRDs) found in many glycan-binding receptors. In most mammalian species, the CD23 residues required to form a sugar-binding site are present, although binding of CD23 to IgE does not involve sugars. Solid-phase binding competition assays, glycoprotein blotting experiments, and glycan array analysis employing the lectin-like domains of cow and mouse CD23 demonstrate that they bind to mannose, GlcNAc, glucose, and fucose and to glycoproteins that bear these sugars in nonreducing terminal positions. Crystal structures of the cow CRD in the presence of α-methyl mannoside and GlcNAcβ1–2Man reveal that a range of oligosaccharide ligands can be accommodated in an open binding site in which most interactions are with a single terminal sugar residue. Although mouse CD23 shows a pattern of monosaccharide and glycoprotein binding similar to cow CD23, the binding is weaker. In contrast, no sugar binding was observed in similar experiments with human CD23. The absence of sugar-binding activity correlates with accumulation of mutations in the gene for CD23 in the primate lineage leading to humans, resulting in loss of key sugar-binding residues. These results are consistent with a role for CD23 in many species as a receptor for potentially pathogenic microorganisms as well as IgE. However, the ability of CD23 to bind several different ligands varies between species, suggesting that it has distinct functions in different organisms.

The Intrinsic Relationship Between Structure and Function of the Sialyltransferase ST8Sia Family Members.

As a subset of glycosyltransferases, the family of sialyltransferases catalyze transfer of sialic acid (Sia) residues to terminal non-reducing positions on oligosaccharide chains of glycoproteins and glycolipids, utilizing CMP-Neu5Ac as the activated sugar nucleotide donor. In the four known sialyltransferase families (ST3Gal, ST6Gal, ST6GalNAc and ST8Sia), the ST8Sia family catalyzes synthesis of α2, 8-linked sialic/polysialic acid (polySia) chains according to their acceptor specificity. We have determined the 3D structural models of the ST8Sia family members, designated ST8Sia I (1), II(2), IV(4), V(5), and VI(6) using the Phyre2 server. Accuracy of these predicted models are based on the ST8Sia III crystal structure as the calculated template. The common structural features of these models are: (1) Their parallel templates and disulfide bonds are buried within the enzymes and are predominately surrounded by helices; (2) The anti-parallel β-sheets are located at the N-terminal region of the enzymes; (3) The mono-sialytransferases (mono-STs), ST8Sia I and ST8Sia VI, contain only a single pair of disulfide bonds, and there are no anti-parallel β-sheets in ST8Sia VI; (4) The Nterminal region of all of the mono-STs are located some distant away from their core structure; (5) These conformational features show that the 3D structures of the mono-STs are less compact than the two polySTs, ST8Sia II and ST8Sia IV, and the oligo-ST, ST8Sia III. These structural features relate to the catalytic specificity of the monoSTs; (6) In contrast, the more compact structural features of ST8Sia II, ST8Sia IV and ST8Sia III relate to their ability to catalyze the processive synthesis of oligo- (ST8Sia III) and polySia chains (ST8Sia II & ST8Sia IV); (7) Although ST8Sia II, III and IV have similar conformations in their corresponding polysialyltransferase domain (PSTD) and polybasic region (PBR) motifs, the structure of ST8Sia III is less compact than ST8Sia II and ST8Sia IV, and the amino acid components of the several three-residue-loops in the two motifs of ST8Sia III are different from that in ST8Sia II and ST8Sia IV. This is likely the structural basis for why ST8Sia III is an oligoST and not able to polysialylate and; (8) In contrast, essentially all amino acids within the threeresidue- loops in the PSTD of ST8Sia II and ST8Sia IV are highly conserved, and many amino acids in the loops and the helices of these two motifs are critical for NCAM polysialylation, as determined by mutational analysis and confirmed by our recent NMR results. In summary, these new findings provide further insights into the molecular mechanisms underlying polyST-NCAM recognition, polySTpolySia/ oligoSia interactions, and polysialylation of NCAM.

Isolation and Structural Characterization of an Oligosaccharide Produced by Bacillus subtilis in a Maltose-Containing Medium

Among 116 bacterial strains isolated from Korean fermented foods, one strain (SS-76) was selected for producing new oligosaccharides in a basal medium containing maltose as the sole source of carbon. Upon morphological characterization using scanning electron microscopy, the cells of strain SS-76 appeared rod-shaped; subsequent 16S rRNA gene sequence analysis revealed that strain SS-76 was phylogenetically close to Bacillus subtilis. The main oligosaccharide fraction B extracted from the culture supernatant of B. subtilis SS-76 was purified by high performance liquid chromatography. Subsequent structural analysis revealed that this oligosaccharide consisted only of glucose, and methylation analysis indicated similar proportions of glucopyranosides in the 6-linkage, 4-linkage, and non-reducing terminal positions. Matrix-assisted laser-induced/ionization time-of-flight/mass spectrometry and electrospray ionization-based liquid chromatography-mass spectrometry/mass spectrometry analyses suggested that this oligosaccharide consisted of a trisaccharide unit with 1,6- and 1,4-glycosidic linkages. The anomeric signals in the 1H-nuclear magnetic resonance spectrum corresponded to α-anomeric configurations, and the trisaccharide was finally identified as panose (α-D-glucopyranosyl-1,6-α-D-glucopyranosyl-1,4-D-glucose). These results suggest that B. subtilis SS-76 converts maltose into panose; strain SS-76 may thus find industrial application in the production of panose.

Development and characterization of a specific IgG monoclonal antibody toward the Lewis x antigen using splenocytes of Schistosoma mansoni-infected mice.

The parasitic blood fluke Schistosoma mansoni synthesizes immunogenic glycans containing the human Lewis x antigen (Le(x); Galactose-β1-4(Fucα1-3)N-acetylglucosamine-β-R, also called CD15), but the biological role(s) of this antigen in the parasites and in humans is poorly understood. To develop IgG-based monoclonal antibodies (mAbs) specific for Le(x), we harvested splenocytes from S. mansoni-infected Swiss Webster mice at Week 10 postinfection, when peak IgG responses to glycan antigens occur, and generated a panel of hybridomas secreting anti-glycan IgG that recognize periodate-sensitive epitopes in soluble egg antigens of the parasites, and also recognizes a neoglycoprotein containing a pentasaccharide with the Le(x) sequence. One murine mAb, an IgG3 designated F8A1.1, bound to glycoproteins and glycolipids from schistosome adults and human promyelocytic leukemic HL-60 cells that express Le(x) antigens, as assessed by a wide variety of approaches including immunofluorescence staining, confocal microscopy, flow cytometry and western blotting, as well as overlay assays of glycolipids after thin-layer chromatography. In contrast, F8A1.1 bound weakly to cercariae, 3-h schistosomula and human Jurkat cells. We also directly compared the glycan specificity of F8A1.1 with commercially available anti-CD15 IgG1 (clone W6D3) using a defined glycan microarray. The results demonstrated that F8A1.1 recognized glycans expressing Le(x) epitopes in a terminal nonreducing position, whereas anti-CD15 bound to glycans with multiple repeats of Le(x) epitopes, but not to glycans with a single, terminal Le(x) epitope. Our results show that F8A1.1 recognizes terminal Le(x) epitopes and can be used for identification, immunolocalization, immunoprecipitation and purification of Le(x)-containing glycoconjugates from schistosomes and mammalian cells.

Correlation between Sialyltransferases and cancer progression and malignant transformation

Sialyltransferases are a family of glycosyltransferases which catalyze the transfer of sialic acid from cytidine monophosphate N-acetylneuraminic acid(CMP-Neu5Ac)to non-reducing terminal positions on sugar chains of glycoconjugates.Studies show the expression of sialyltransferases in the normal cells and tumor cells has significant difference,They alter structure or quantity of sialylation of cell membrane terminal glycoconjugates.which can influence on tumor progression and transformation.Accordingly,research on the aberrant expression of sialyltransferases has great significance for tumor prevention and treatment. Key words: Sialyltransferases; Sialic acids; Neoplasm metastasis; Neoplasm invasiveness

Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

Human alpha one proteinase inhibitor (alpha1-PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28 degrees C. Eight milligrams per liter of active alpha1-PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant alpha1-PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF-MS, NSI-MS(n), and GC-MS. The MALDI and NSI- full MS spectra of permethylated N-glycans revealed that the N-glycans of both variants contain a series of high-mannose type glycans with 5-20 hexose units. Monosaccharide analysis showed that these were composed of N-acetylglucos-amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non-reducing position. The Galactofuranose-containing high-mannnose type N-glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger.

The evolution of galactose α2,3-sialyltransferase: Cionaintestinalis ST3GAL I/II and Takifugu rubripes ST3GAL II sialylate Galβ1,3GalNAc structures on glycoproteins but not glycolipids

Sialyltransferases are a family of enzymes catalyzing the transfer of sialic acid residues to terminal non-reducing positions of oligosaccharide chains of glycoproteins and glycolipids. Although expression of sialic acid is well documented in animals of the deuterostomian lineage, sialyltransferases have been predominantly described for relatively recent vertebrate lineages such as birds and mammals. This study outlines the characterization of the only sialyltransferase gene found in the tunicate Ciona intestinalis, the first such report of a non-vertebrate deuterostomian sialyltransferase, which has been discussed as a possible orthologue of the common ancestor of galactose alpha2,3-sialyltransferases. We also report for the first time the characterization of a ST3Gal II gene from the bony fish Takifugu rubripes. We demonstrate that both genes encode functional alpha2,3-sialyltransferases that are structurally and functionally related to the ST3Gal family of mammalian sialyltransferases. However, characterization of the recombinant, purified forms of both enzymes reveal novel acceptor substrate specificities, with sialylation of the disaccharide Galbeta1-3GalNAc and asialofetuin, but not GM1 or GD1b observed. This is in contrast to the mammalian ST3Gal II that predominantly sialylates gangliosides. Taken together the ceramide binding/recognition site previously proposed for the mouse ST3Gal II might represent a unique feature of mammalian ST3Gal II that is missing in the evolutionary more distant fish and tunicate species reported here. This suggests that during the evolution of the ST3Gal II, probably following the separation of the teleosts, a significant shift in substrate specificity enabling the sialylation of gangliosides took place.

Soyasaponin I decreases the expression of α2,3-linked sialic acid on the cell surface and suppresses the metastatic potential of B16F10 melanoma cells

The transfer of sialic acids to the non-reducing terminal positions on sugar chains of glycoconjugates is catalyzed by sialyltransferases (STs). Increased sialylation is correlated with oncogenic transformation and metastatic potential. ST inhibitors may be potentially valuable as anti-cancer and anti-metastatic agents. In this study, we evaluated the effects of soyasaponin I (Ssa I), a known inhibitor of STs, on tumor metastasis through studying a highly metastatic cancer cell line B16F10. Ssa I specifically inhibited the expression of α2,3-linked sialic acids without affecting other glycans on the B16F10 cell surface. We also found that Ssa I decreased the migratory ability of cells, enhanced cell adhesion to extracellular matrix proteins. Finally, a pulmonary metastasis assay demonstrated that alteration of glycosylation in this way significantly reduced the ability of tumor cells to distribute to the lungs of mice. Collectively, these findings suggested that α2,3-linked sialic acids may play an important role in metastasis potential of B16F10 cells.

N-Glycan Structures from the Major Glycoproteins of Pigeon Egg White: PREDOMINANCE OF TERMINAL Galα (1)Gal

N-Glycans from major glycoproteins of pigeon egg white (ovotransferrin, ovomucoid, and ovalbumins) were enzymatically released and were reductively aminated with 2-aminopyridine, separated, and structurally characterized by mass spectrometry and a three-dimensional mapping technique using three different columns of high performance liquid chromatography (HPLC) (Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, 139-146). Twenty-five major N-glycan structures, all of them hitherto unknown, were identified as pyridylamino derivatives. Of these, 13 were neutral, 10 were monosialyl, and 2 were disialyl oligosaccharides. All N-glycans contain from one to four Galalpha(1,4)Galbeta(1,4) sequences at the nonreducing terminal positions and are devoid of fucose residues. N-Acetylneuraminic acids were alpha(2,6)-linked only to beta-galactose. The HPLC profiles of the N-glycans from four different glycoproteins were qualitatively very similar to each other, but not identical in the peak distributions. Monosialyl glycans were most abundant in all four glycoproteins, followed by neutral glycans. Disialyl glycans were lowest in ovotransferrin, and highest in ovomucoid. Triantennary structures with bisecting GlcNAc were predominant in ovotransferrin, and tetra-antennary (with and without bisecting GlcNAc-containing) structures were predominant in other glycoproteins. Penta-antennary structures (with a sialic acid and without bisecting GlcNAc residue) were also found in small quantities in all four glycoproteins. In contrast to the chicken egg white counterparts, which contain mostly high mannose and hybrid types, all N-glycan structures in the major pigeon egg white glycoproteins are complex type.

Massive glycation of protein HC, a low molecular weight lipocalin, in non-diabetic individuals

Human protein HC is a member of the lipocalin superfamily with unique properties since it carries a covalently bound fluorescent chromophore mediating the linkage of the major part of protein HC to several plasma proteins, with IgA as the dominating complex partner. Native protein HC displays characteristic absorption and fluorescence spectra similar to those of glycated proteins with advanced glycosylation end products (AGEs). In vitro glycation of protein HC induces the formation of fibril aggregates with a corresponding increase of absorption in the visible region of the spectrum. Boronate-affinity chromatography and a novel galactosyltransferase assay indicate that protein HC is modified with residues of glucose exposed in a terminal non-reducing position which is typical of glycated proteins. The glycation level of several isolated batches of protein HC as measured by both assays was around 35%, which represents the highest level described for human plasma-derived proteins from healthy individuals.

Lack of modulation of the galectin-3 and asialoglycoprotein receptor transcription in hepatocarcinoma of transgenic mice

Galectin-3 and asialoglycoprotein receptor are lectins belonging to the classes of soluble lectins and of membrane C type lectins respectively. Conflicting results have been reported concerning their transcription level in the time course development of tumours. In the present study we investigated the abnormalities and the transcription levels of galectin-3 and asialoglycoprotein receptor genes in liver-targeted SV40 large T transgenic mice related to normal mice. In the strain expressing the highest level of large T, 100% of the male mice reproducibly developed an hepatocarcinoma. We provide evidence that the galectin-3 and asialoglycoprotein receptor genes are stable in such mice. The galectin-3 gene is weakly transcribed and its level is identical and constant in normal and transgenic mice, suggesting a lack of involvement in the development of large T-induced hepatocarcinoma. The asialoglycoprotein receptor gene is actively transcribed and its level remains high all along the development of the tumour; therefore, in such an hepatocarcinoma the asialoglycoprotein receptor could be used to take up drugs, genes or oligonucleotides associated with glycosylated carriers bearing galactose residues in a terminal non-reducing position.

The specificity of an α-(1 → 2)- l-galactosyltransferase from albumen glands of the snail Helix pomatia

The specificity of an l-galactosyltransferase ( l-Gal-T) from albumen glands of the snail Helix pomatia has been studied. This enzyme transfers l-Gal from GDP- l-Gal to various disaccharides with β-linked d-Gal in terminal non-reducing position, forming an α-(1 → 2) linkage. The subterminal residue and the type of interglycosidic linkage proved to be of minor importance. However, the branched trisaccharide β- d-Gal-(1 → 3)-[β- d-Gal-(1 → 6)]-β- d-Gal-(1 → O)Me is a very poor acceptor. The specificity of the l-Gal-T correlates well with the equimolar occurrence of l-Gal and the structural element → 2)-Gal-(1 → found in the storage polysaccharide of this snail. Since l-Fuc is also transferred from its GDP-activated form, the membrane preparations of the albumen glands can be used to synthesize fucosylated oligosaccharides. © 1997 Elsevier Science Ltd.

Characterization of five type II arabinogalactan-protein fractions from red wine of increasing uronic acid content

Five arabinogalactan-protein conjugates (AGP) were separated from red wine by two successive anion-exchange chromatography steps and further purified to apparent homogeneity by affinity and size-exclusion chromatography. Together they represent more than 40% of total wine polysaccharides, confirming the abundance of AGPs in red wine. The five purified fractions had a common arabinogalactan core with characteristics typical of wine type II AGPs, but differed mainly in their uronic acid content, as evidenced by differences in the strength of their binding to the anion-exchanger. Their uronic acid content and glycosidic linkage composition revealed that the three less acidic AGPs contained from 3 to 7% glucuronic acid, half in terminal non-reducing positions and half in terminal Rha p-(1 → 4)-Glc pA-(1 → sequences. The two more acidic AGP-containing fractions contained both glucuronic (6.1 and 13.3%, respectively) and galacturonic (1.9 and 2.3%, respectively) acid in association with 2- and 2,4-linked rhamnose, indicating the presence of AG-rhamnogalacturonan fragments.

Characterization of glycoprotein glycan receptors for Escherichia coli F17 fimbrial lectin

The specificity of N-acetylglucosamine-binding F17 fimbrial lectin of coli (also called FY or Att25) for the oligosacharide structures was investigated by mannose-resistant hemagglutination inhibition tests and direct binding assays. The linkage position of N-acetylglucosamine influenced its affinity in the preferential order of βt1–3 > β1–6 > β1–4 β > β1–2. Minimal GlcNAc β1–3Gal β1-sequence strongly bound to F17 lectin, whether located in terminal nonreducing position or internally in carbohydrate moieties. F17 lectin specifically interacted with this unit in O-glycosidically linked oligosaccharides of the bovine glycophorins and intestinal mucins. On the contrary, GlcNAc β1- NAsn, GlcNAc β1-6Man β- and GlcNAc β1-4GlcNAc- of N-glycosylated proteins failed to bind to the lectin. Our findings emphasized the presence of multiple F17 mucosal receptor complex in the newborn calf intestines. Furthermore, the density of receptors for F17 fimbrial lectin seemed to depend on the age of the calf and the intestinal segment.

Sequential nuclear magnetic resonance assignment of beta-1,2-linked mannooligosaccharides isolated from the phosphomannan of the pathogenic yeast Candida albicans NIH B-792 strain.

The H-1 and H-2 signals of beta-1,2-linked mannooligosaccharides isolated from the phosphomannan of Candida albicans NIH B-792 strain by mild acid hydrolysis were assigned by a sequential NMR assignment method that combines two-dimensional 1H-1H correlated spectroscopy (COSY) and two-dimensional nuclear Overhauser enhancement and exchange spectroscopy (NOESY). The results indicated that the H-1 and H-2 of each beta-1,2-linked mannopyranose unit show largely different signals compared with those of the alpha-linked ones and that the correlation between linkages and signals could not be explained by a conventional additivity rule. Furthermore, a regular proportional downfield shift of the H-1 signal was observed in the order of the mannose unit from the reducing terminal except those of the reducing and nonreducing terminal positions. Although the 1H NMR spectra of these oligosaccharides were complicated due to the presence of a large portion of the beta-anomer from the reducing terminal mannose unit, reduction of the oligosaccharides with NaBH4 to the corresponding alcohols gave simple and more readily interpretable 1H NMR spectra. Unexpectedly, however, a shift of H-1 signals by this reduction occurred not only on the second mannose unit but also on the third and fourth mannose units from the modified reducing terminal group of each oligosaccharide alcohol. This result indicates that the reducing terminal mannose unit is able to affect up to the fourth mannose unit from the reducing terminal. The presence of a long-distance interresidue NOE also suggests that the beta-1,2-linked mannooligosaccharides have a compactly folded conformation in solution.

Non-reducing terminal linkage position determination in intact and permethylated synthetic oligosaccharides having a penultimate amino sugar: Fast atom bombardment ionization, collisional-induced dissociation and tandem mass spectrometry

Certain linkage positions in oligosaccharides can be discerned by collision-activated dissociation mass spectrometry, rationalized by molecular modelling. Previous work on synthetic oligosaccharides has suggested that daughter ion patterns can distinguish among intact compounds which terminate in alpha-L-fucose and have a penultimate amino sugar. The current study indicates that these observations can be extended to oligosaccharides terminating in beta-D-galactose. In addition, we have observed that protonated, ammoniated and lithiated molecular ions all produce linkage-specific daughter ion spectra in these two sets of oligosaccharides. Sodiated molecular ions could be fragmented usefully under high collision energy offset conditions; potassiated ions were stable and not dissociable under conditions available in a triple-quadrupole instrument. We also show linkage discernment among the permethylated set of these six synthetic oligosaccharides. Methylated derivatives of this set of compounds give more useful product ions, including a 3-linkage specific ion. A novel relationship was noted by a plot of collision energy against (daughter ion/parent ion) ratio, which gave a unique slope for each of the non-reducing terminal linkage positions 3, 4 and 6 in the set of six compounds. The slope of this plot is related to the ability of each linkage position in the oligosaccharide to absorb collisional energy. Rotational freedom of the individual glycosidic linkage is hypothesized to play a role in this phenomenon.

Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin.

Fetuin derivatives with enzymatically altered oligosaccharide units were tested for their ability to inhibit pertussis toxin-mediated agglutination of goose erythrocytes and the binding of 125I-labeled fetuin to pertussis toxin-coated polystyrene tubes. Fetuin oligosaccharides were sequentially degraded by treatment with: neuraminidase (asialofetuin) followed by beta-galactosidase (asialoagalactofetuin) and, lastly, with beta-N-acetylhexosaminidase (asialoagalacto-a[N-acetylglucosamino]fetuin). Asialofetuin retained only 19 and 53% of the inhibitory activity of native fetuin in the hemagglutination and 125I-fetuin binding assays, respectively. Asialoagalactofetuin showed no further reduction of inhibition in the hemagglutination system and, instead, resulted in partial recovery of inhibition in the 125I-fetuin-pertussis toxin binding assay. Asialoagalacto-a[N-acetylhexosamino]fetuin showed a further decrease in ability to inhibit pertussis toxin binding in both assays. The inhibitory activity of asialoagalactofetuin could be restored to that of native fetuin by adding back D-galactose with UDP-Gal:D-glucosyl-1,4-beta-galactosyltransferase, followed by the addition of terminal sialic acid residues with CMP-N-acetylneuraminic acid:beta-D-galactosyl-1,4-N-acetyl-beta-D-glucosamine-alpha-2,6-N- acetylneuraminyltransferase. The data suggested that a requirement for pertussis toxin binding to fetuin may be the presence of acetamido-containing sugar groups in the nonreducing terminal position of fetuin's oligosaccharides.

An arabinogalactan from the culture medium of Rubus fruticosus cells in suspension

A water-soluble arabinogalactan, isolated from the extracellular medium of suspension-cultured cells of Rubus fruticosus, contained arabinose, rhamnose, galactose, and also protein (6.5%) and uronic acid (2.5%). Methylation analysis of the arabinogalactan and the arabinose-free product obtained by mild acid hydrolysis showed that the polysaccharide was a typical arabino-3,6-galactan in which rhamnose and glucuronic acid occupied non-reducing terminal positions. Successive Smith-degradations combined with methylation analysis and 13C-n.m.r. spectroscopy revealed that the arabinogalactan contained a main chain of (→3)-linked β- d-galactopyranosyl residues with a high degree of branching at positions 6 by (1→6)-linked d-galactopyranosyl side-chains of various lengths, in which several contiguous residues were substituted at positions 3. The polymer is thus an arabinogalactan-protein belonging to the galactans of Type II.

The reactivity of galactose oxidase with snail galactans, galactosides and d-galactose-composed oligosaccharides

The enzymic oxidation of snail galactans, of their first and second Smith degradation products and of some structurally related polysaccharides was studied. Lymnaea stagnalis galactan after one cycle of Smith degradation reacted best and native Helix pomatia galactan was almost inactive. Investigations on the structural requirements for oligosaccharides to bind to galactose oxidase showed that the branched tetrasaccharide, Gal-β-1 → 6-[Gal-β-1 → 3]-Gal-β-1 → 1 l-Gro, in the terminal nonreducing position was the most complementary structure in the native galactan to associate with the enzyme. All nitrophenyl α-galactosides reacted better, and the ortho-form was 10-times more potent compared with this tetrasaccharide, indicative of the involvement of a hydrophobic region in binding. However, the β-linked isomers were only equally or less reactive than galactose. The enzymic oxidation determined colorimetrically by transferring the peroxide formed to o-dianisidine ceased at a maximum typical for each substrte and independent of the reaction time. When the absolute turn-over rates of ortho- and para-nitrophenyl α-galactoside and for the β-isomer were determined by HPLC, it could be demonstrated that the oxidation had not finished at the maximum of the colour reaction, but proceeded until the substrate was consumed. The initial speed of the colour reaction paralleled the absolute oxidation rate.

Biosynthesis of the Fucose-Containing Xyloglucan Nonasaccharide by Pea Microsomal Membranes

Pea microsomal membranes catalyze the transfer of [(14)C]fucose (Fuc) from GDP-[U-(14)C]fucose, with or without added unlabeled UDP-glucose (Glc), UDP-xylose (Xyl) or UDP-galactose (Gal), to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, [(14)C] fucose residues occur exclusively in a fragment corresponding in size to the xyloglucan nonasaccharide, Glc(4) Xyl(3) Gal Fuc. This fragment contains a single labeled fucose residue per oligomer, alpha-linked in a terminal nonreducing position. By comparison, in incubations where GDP-[(14)C] fucose is absent and replaced by UDP-[(3)H]xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products is an octasaccharide. In the presence of both GDP-[(14)C]fucose and UDP-[(3)H]xylose, a nonasaccharide containing the two labels is produced. Fucose and xylose residues are transferred within a few minutes to acceptor molecules of molecular weight up to 300,000. Such products do not elongate detectably over 60 minutes of incubation. The data support the conclusion that the nonasaccharide subunit of xyloglucan may be generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis is dependent on the inclusion of exogenous GDP-fucose.