Minoru Ujita

Poly-N-acetyllactosamine Synthesis in BranchedN-Glycans Is Controlled by Complemental Branch Specificity of i-Extension Enzyme and β1,4-Galactosyltransferase I

Poly-N-acetyllactosamine is a unique carbohydrate that can carry various functional oligosaccharides, such as sialyl Lewis X. It has been shown that the amount of poly-N-acetyllactosamine is increased inN-glycans, when they contain Galβ1→4GlcNAcβ1→6(Galβ1→4GlcNAcβ1→2)Manα1→6 branched structure. To determine how this increased synthesis of poly-N-acetyllactosamines takes place, the branched acceptor was incubated with a mixture of i-extension enzyme (iGnT) and β1,4galactosyltransferase I (β4Gal-TI). First,N-acetyllactosamine repeats were more readily added to the branched acceptor than the summation of poly-N-acetyllactosamines formed individually on each unbranched acceptor. Surprisingly, poly-N-acetyllactosamine was more efficiently formed on Galβ1→4GlcNAcβ1→2Manα→R side chain than in Galβ1→4GlcNAcβ1→6Manα→R, due to preferential action of iGnT on Galβ1→4GlcNAcβ1→2Manα→R side chain. On the other hand, galactosylation was much more efficient on β1,6-linked GlcNAc than β1,2-linked GlcNAc, preferentially forming Galβ1→4GlcNAcβ1→6(GlcNAcβ1→2)Manα1→6Manβ →R. Starting with this preformed acceptor,N-acetyllactosamine repeats were added almost equally to Galβ1→4GlcNAcβ1→6Manα→R and Galβ1→4GlcNAcβ1→2Manα→R side chains. Taken together, these results indicate that the complemental branch specificity of iGnT and β4Gal-TI leads to efficient and equal addition ofN-acetyllactosamine repeats on both side chains of GlcNAcβ1→6(GlcNAcβ1→2)Manα1→6Manβ→R structure, which is consistent with the structures found in nature. The results also suggest that the addition of Galβ1→4GlcNAcβ1→6 side chain on Galβ1→4GlcNAcβ1→2Man→R side chain converts the acceptor to one that is much more favorable for iGnT and β4Gal-TI.

Poly-N-acetyllactosamine Extension inN-Glycans and Core 2- and Core 4-branchedO-Glycans Is Differentially Controlled by i-Extension Enzyme and Different Members of the β1,4-Galactosyltransferase Gene Family

Poly-N-acetyllactosamines are attached to N-glycans, O-glycans, and glycolipids and serve as underlying glycans that provide functional oligosaccharides such as sialyl LewisX. Poly-N-acetyllactosaminyl repeats are synthesized by the alternate addition of β1,3-linked GlcNAc and β1,4-linked Gal by i-extension enzyme (iGnT) and a member of the β1,4-galactosyltransferase (β4Gal-T) gene family. In the present study, we first found that poly-N-acetyllactosamines inN-glycans are most efficiently synthesized by β4Gal-TI and iGnT. We also found that iGnT acts less efficiently on acceptors containing increasing numbers of N-acetyllactosamine repeats, in contrast to β4Gal-TI, which exhibits no significant change. In O-glycan biosynthesis,N-acetyllactosamine extension of core 4 branches was found to be synthesized most efficiently by iGnT and β4Gal-TI, in contrast to core 2 branch synthesis, which requires iGnT and β4Gal-TIV. Poly-N-acetyllactosamine extension of core 4 branches is, however, less efficient than that of N-glycans or core 2 branches. Such inefficiency is apparently due to competition between a donor substrate and acceptor in both galactosylation andN-acetylglucosaminylation, since a core 4-branched acceptor contains both Gal and GlcNAc terminals. These results, taken together, indicate that poly-N-acetyllactosamine synthesis inN-glycans and core 2- and core 4-branchedO-glycans is achieved by iGnT and distinct members of the β4Gal-T gene family. The results also exemplify intricate interactions between acceptors and specific glycosyltransferases, which play important roles in how poly-N-acetyllactosamines are synthesized in different acceptor molecules.

Glucan-Binding Activity of Silkworm 30-kDa Apolipoprotein and Its Involvement in Defense against Fungal Infection

The silkworm Bombyx mori 30-kDa lipoproteins (6G1 and 19G1), major components of the hemolymph, were shown to bind to glucans. 6G1 apolipoprotein was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant 6G1 apolipoprotein specifically bound to β-glucan, but not to chitin, mannan, peptidoglycan, or oligosaccharide chains on glycoproteins. The β-glucan binding of the recombinant 6G1 was inhibited by laminaribiose and laminarin, a soluble glucan, but not by lipopolysaccharide or insect blood sugar, trehalose at physiological concentration. Furthermore, the recombinant 6G1 was shown to participate in the activation of prophenoloxidase cascade and to interfere with hyphal growth of the entomopathogenic fungus Paecilomyces tenuipes, injected into pupae of B. mori. These results suggest that 6G1 lipoprotein plays a role in the protection of B. mori against invading fungi.

Molecular cloning of the mouse osteoglycin-encoding gene.

Osteoglycin (OG) is a glycoprotein that was first isolated from bovine bone. The deduced amino acid (aa) sequence from the cDNA analysis showed that a precursor of OG has consensus leucine-rich repeats. In this study, we have isolated from a mouse limb-bud library cDNA clones encoding a 298-aa OG. This molecule shows 85 and 86% homology to human and bovine OG, respectively. Furthermore, the C-terminal two-thirds of the protein shows 48% homology to the corresponding portion of chick proteoglycan (PG)-Lb, a PG that has been shown to be preferentially expressed in the zone of flattened chondrocytes in the developing limb cartilage. Northern blot analysis of various mouse tissues revealed a 3.7-kb transcript in a limited number of these tissues.

Xylosidases associated with the cell surface of Penicillium herquei IFO 4674.

Penicillium herquei IFO 4674 is a filamentous fungus that produces a large amount of hydrolases for fibrous polysaccharides. We purified two beta-xylosidases, S1 and S2. The molecular masses of S1 and S2 determined by MALDI-TOF-MS were 103,700 and 37,460 Da. The optimum pHs of S1 and S2 were 4.0 and 6.5, respectively. By several kinds of alcohols, especially glycerol, S1 was activated while S2 was unaffected or inhibited. S1 had a transxylosylation activity, while S2 did not. The s2 gene encoding xylosidase S2 was cloned by PCR with primers designed on the basis of partial amino acid sequences of S2. The s2 consisted of 1005 by encoding 335 amino acids (37,433 Da) and had no secretion signal sequence. The deduced amino acid sequence shows a high identity to that of Bacteroides ovatus xylosidase/arabinosidase (56%), which is a member of the family 43 glycoside hydrolase.

Specific Binding of Silkworm Bombyx mori 30-kDa Lipoproteins to Carbohydrates Containing Glucose

Insect lectins are important as part of nonspecific self-defense, but their antifungal mechanisms remain to be elucidated. Fungi contain glucans on the cell surface and insect glucan-binding proteins are considered to be essential for antifungal mechanisms. We purified glucose-binding proteins from hemolymph of pupae of the silkworm Bombyx mori, and the amino acid sequence analysis showed that their two proteins are 30-kDa lipoproteins, major components of B. mori hemolymph. These lipoproteins specifically bound to glucose and glucans, suggesting that they are involved in insect self-defense systems.

Immunologically cross-reactive 57 kDa and 53 kDa glycoprotein antigens of bovine milk fat globule membrane : isoforms with different N-linked sugar chains and differential glycosylation at early stages of lactation

Two glycoprotein antigens with molecular masses of 57 kDa (MGP57) and 53 kDa (MGP53) were co-purified from bovine milk fat globule membrane (MFGM) by immunoaffinity chromatography using a monoclonal antibody raised against the MFGM. Their N-terminal sequences of 22 amino acids determined were identical, and the sequence was homologous (about 60% identical) to the deduced amino acid sequence of mouse milk fat globule epidermal growth factor (EGF) factor 8 (MFG-E8) (Ref. [12], Stubbs, J.D. et al., Proc. Natl. Acad. Sci. USA, 87, 8417-8421, 1990). This suggests that MGP57/53 are bovine MFGM components 15/16 (PAS-6 and PAS-7), which have recently been reported to be bovine homologs of MFG-E8. N-Glycanase treatment of these glycoproteins reduced their molecular masses, and consequently the enzymatically deglycosylated MGP57 and MGP53 converged on a single band of 50 kDa as measured by SDS-PAGE, indicating that the polypeptide portions of these two distinct glycoprotein antigens are very similar or identical and that their N-linked sugar chains contributed to minor difference in their molecular masses. Western blot analyses using lectins also revealed that they were differentially glycosylated; MGP57 was stained with concanavalin A (Con A) more strongly than MGP53, whereas MGP 53 was stained well with soybean agglutinin (SBA). Reactivity with SBA remarkably increased during early stages of lactation. Two-dimensional gel electrophoresis showed that MGP57 and MGP53 were electrically heterogeneous; from day 9 after parturition, both glycoproteins fell in almost the same range of isoelectric points between 6.4 and 7.6, also, such glycoproteins from day 1 after parturition were more acidic, probably due to terminal sialylation of their sugar chains.

Carbohydrate binding specificity of the recombinant chitin-binding domain of human macrophage chitinase.

The chitin-binding domain of human macrophage chitinase was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and assayed for its binding activity. The purified recombinant chitin-binding domain bound to chitin, but not to glucan, xylan, or mannan. The binding of the recombinant chitin-binding domain to chitin was inhibited by N-acetylglucosamine, di-N-acetylchitobiose, and hyaluronan, but not by N-acetylgalactosamine or chondroitin. Furthermore, a solid-phase binding assay showed that the recombinant domain interacts specifically with hyaluronan and hybrid-type N-linked oligosaccharide chains on glycoproteins, and that the oligosaccharide-binding characteristics are similar to those of wheat germ agglutinin, a lectin that binds to chitin. The results suggest that human chitinase chitin-binding domain may be involved in tissue remodeling through binding to polysaccharides or extracellular matrix glycoproteins, and this recombinant protein can be used to elucidate biological functions of the enzyme.

A change in soybean agglutinin binding patterns of bovine milk fat globule membrane glycoproteins during early lactation

Milk fat globule membrane (MFGM) glycoproteins were prepared from bovine milk at different stages of early lactation. Western blot analyses using several lectins revealed that reactivity of MFGM glycoproteins, especially 47K and 80K bands, to soybean agglutinin (SBA) remarkably increased during the lactation, while no change was observed for Ricinus communis agglutinin‐I (RCA‐I) binding. Sialidase treatment of MFGM glycoproteins revealed that the number of SBA‐positive bands and the amount of SBA‐positive oligosaccharides in these bands are increased during the lactation. Since SBA binds N‐acetylgalactosamine terminated oligosaccharides, the results indicated that N‐acetylgalactosaminylation of bovine MFGM glycoproteins is stimulated during the lactation.

Carbohydrate Binding Specificity of Recombinant Human Macrophage β-Glucan Receptor Dectin-1

Human macrophage dectin-1, a type II transmembrane β-glucan receptor, was expressed as a fusion protein with an N-terminal hexahistidine tag and glutathione S-transferase in an Escherichia coli cell-free translation system, and assayed for binding specificity. Recombinant dectin-1 specifically bound to some β-glucans, but not to other carbohydrates. The β-glucan binding of recombinant dectin-1 was inhibited by laminarin, a soluble β-glucan, and by laminarioligosaccharides, but not by other carbohydrates. These results suggest that recombinant human dectin-1 can be used as a useful probe in identifying ligands in humans and tonic foods due to its strict binding specificity.