Neuronal differentiation-dependent expression of the disialic acid epitope on CD166 and its involvement in neurite formation in Neuro2A cells

We previously demonstrated that alpha2,8-linked disialic acid (diSia) residues occur in several glycoproteins of mammalian brains (Sato, C., Fukuoka, H., Ohta, K., Matsuda, T., Koshino, R., Kobayashi, K., Troy, F. A., II, and Kitajima, K. (2000) J. Biol. Chem. 275, 15422-15431). The role of the diSia epitope on these glycoproteins is not known, whereas the importance of the diSia epitope on glycolipids is well documented in neurite formation. In this study, we demonstrated that the diSia epitope (Neu5Acalpha2 --> 8Neu5Acalpha2 --> 3Gal) on glycoproteins, but not on glycolipids, is involved in neurite formation in a mouse neuroblastoma cell line, Neuro2A, based on the following lines of evidence. First, the amount of diSia epitope on glycoproteins increased during retinoic acid-induced neurite formation. Second, retinoic acid treatment primarily increased the diSia epitope on a 100-kDa glycoprotein. We identified this protein as CD166 (SC1), an immunoglobulin superfamily cell adhesion molecule involved in neurite extension. Third, a monoclonal antibody against the diSia epitope specifically inhibited neurite formation. We also demonstrated that alpha2,8-sialyltransferase III mRNA expression increased 1.7-fold after the induction of neurite formation, suggesting that alpha2,8-sialyltransferase III is responsible for formation of the diSia epitope on CD166.     

Identification and adipocyte differentiation-dependent expression of the unique disialic acid residue in an adipose tissue-specific glycoprotein, adipo Q

Recently, we have shown that alpha 2,8-linked disialic acid (diSia) residue occurs in glycoproteins more frequently than ever recognized (Sato, C., Fukuoka, H., Ohta, K., Matsuda, T., Koshino, R., Kobayashi K., Troy, F. A., II, and Kitajima, K. (2000) J. Biol. Chem. 275, 15422--15431). In the course of identification of the diSia-containing glycoproteins in mammals, the 30-kDa glycoprotein was found in bovine serum. The 30-kDa glycoprotein was shown to be the bovine adipo Q, an adipocyte-specific protein, based on the partial amino acid sequences and the immuno-cross-reactivity with the recombinant mouse adipo Q. The bovine adipo Q was shown to have no N-linked but O-linked glycan(s) containing the diSia epitope, Neu5Ac alpha 2-->8Neu5Ac alpha 2-->3Gal. Furthermore, the diSia epitope was also found in the mouse adipo Q in serum as well as in the 3T3-L1 cells that are fully differentiated into adipocytes. Notably, among the known alpha 2,8-sialyltransferases, only the alpha 2,8-sialyltransferase III mRNA was detected in the 3T3-L1 cells at any stages of differentiation, and the recombinant alpha 2,8-sialyltransferase III could sialylate the purified bovine adipo Q. Thus, this study clearly provides the new findings that adipo Q is the diSia-containing glycoprotein and a physiological substrate of alpha 2,8-sialyltransferase III, whose substrates have not been identified so far.     

Structural studies of fertilization-associated carbohydrate-rich glycoproteins (hyosophorin) isolated from the fertilized and unfertilized eggs of flounder, Paralichthys olivaceus. Presence of a novel penta-antennary N-linked glycan chain in the tandem repeating glycopeptide unit of hyosophorin

New glycoproteins of 100-120 kDa were isolated from the unfertilized eggs of flounder, Paralichthys olivaceus. Compositionally indistinguishable glycopeptides of 6 kDa were also purified from the activated or fertilized eggs. These high and low molecular mass glycoproteins are characterized by high (about 85%) carbohydrate content. Although some heterogeneities exist in the amino acid sequences, the 6-kDa glycopeptides (decapeptides with single large N-linked glycan chains), isolated from the fertilized eggs are the repeating units of the high molecular mass glycoproteins. As judged from several distinctive features the 100-120-kDa glycoproteins are apparently major components of cortical alveoli of flounder eggs and are regarded as members of glycoproteins we have defined under the name of "hyosophorin" (Kitajima, K., Inoue, S., and Inoue, Y. (1989) Dev. Biol. 132, 544-553). Composition analysis, Smith degradation, hydrazinolysis-nitrous acid deamination, permethylation analysis, and 400-MHz 1H NMR spectroscopy provided evidence for the structure of a novel penta-antennary glycan chain attached to the repeating unit (decapeptide) of the protein core. The structure thus determined is: (Formula: see text). The presence of a unique class of carbohydrate-rich glycoproteins (H-hyosophorin) in the unfertilized eggs, their conversion to the repeating unit (L-hyosophorin) at fertilization, and the finding of a free glycan chain that was formed by scission between the GlcNAc and Asn residues of L-hyosophorin, in the fertilized eggs including embryos of 4-11-h postinsemination, support the view that these molecules may be important in fertilization and subsequent development.     

Deaminated neuraminic acid-rich glycoprotein of rainbow trout egg vitelline envelope. Occurrence of a novel alpha-2,8-linked oligo(deaminated neuraminic acid) structure in O-linked glycan chains

Deaminated neuraminic acid-rich glycoprotein (KDN-gp), first found and isolated from the vitelline envelope of rainbow trout eggs (Inoue, S., Kanamori, A., Kitajima, K., and Inoue, Y. (1988) Biochem. Biophys. Res. Commun. 153, 172-176), has been found to contain a number of O-linked glycan. Oligosaccharides were released by alkaline borohydride treatment of KDN-gp. Following fractionation by DEAE-Sephadex A-25 and thin-layer chromatography, a series of acidic oligosaccharides were obtained and analyzed for their chemical structures. The structure is based on composition analysis, methylation analysis, alkali-catalyzed "peeling" reactions, periodate oxidation, 400-MHz one- and two-dimensional 1H NMR spectroscopy, and molecular secondary ion mass spectrometry. The O-linked oligosaccharides isolated from KDN-gp have been shown to contain a common core trisaccharide Gal beta 1-3GalNAc alpha 1-3GalNAc in which the terminal Gal residue is blocked by a single residue of deaminated neuraminic acid (KDN) and the proximal GalNAc residue is substituted by alpha-2,8-linked oligo(KDN) chains. Structures of KDN-oligosaccharide chains in the glycoprotein are novel and expressed by the following general formula, where n = 0-5, for which data are available. [formula: see text]     

N-glycan structures from the major glycoproteins of pigeon egg white: predominance of terminal Galalpha(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.     

Peptide:N-glycosidase activity found in the early embryos of Oryzias latipes (Medaka fish). The first demonstration of the occurrence of peptide:N-glycosidase in animal cells and its implication for the presence of a de-N-glycosylation system in living organisms.

The recent discovery of free oligosaccharides typical for the complex type of glycan chains terminating with a free di-N-acetylchitobiosyl structure in certain fish eggs and early embryos (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Seko, A., Kitajima, K., Iwasaki, M., Inoue, S., and Inoue, Y. (1989) J. Biol. Chem. 264, 15922-15929; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) led us to find an enzyme responsible for detachment of N-linked glycan chains from glycoproteins by hydrolyzing the beta-aspartyl-glucosylamine linkage in Oryzias latipes embryos. The enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase or peptide:N-glycosidase (PNGase), was partially (2090-fold) purified, and the reaction site at which this enzyme acts was specified by analysis and identification of the reaction products. This is the first demonstration showing PNGase in animal sources, although the presence of PNGases was reported in a variety of plant extracts and bacteria. Thus, the commonality of this type of enzyme is now demonstrated, and the possible physiological role of PNGase in de-N-glycosylation as a basic biologic process is proposed.     

Fish egg glycophosphoproteins have species-specific N-linked glycan units previously found in a storage pool of free glycan chains.

Recent findings (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) of a relatively large quantity of complex-type free sialo-oligosaccharides in the unfertilized eggs of freshwater fish, Plecoglossus altivelis and Tribolodon hakonensis, prompted us to search for their progenitor glycoproteins. First we demonstrated a third occurrence of free sialoglycans in the unfertilized eggs of Medaka fish (Oryzias latipes). Next, in all three species studied, a uniformly high level of glycophosphoproteins (GPP) was identified and found to possess N-linked glycan units. The carbohydrate structures of the GPP were determined to be identical with those of the free glycans isolated from the unfertilized eggs of the respective fish species. Thus, the most likely candidate for the progenitor of free sialoglycans appeared to be the oocyte GPPs. This implies that the liberation of the free glycans by a putative peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase may represent a necessary biochemical event during vitellogenesis or oogenesis. The present results may provide insight into a new concept of a "protein N-glycosylation/de-N-glycosylation system" recently proposed by us (Seko, A., Kitajima, K., Inoue, Y., and Inoue, S. (1991) J. Biol. Chem. 266, 22110-22114).     

Polysialic acid is associated with sodium channels and the neural cell adhesion molecule N-CAM in adult rat brain.

We have studied alpha 2,8-linked polysialic acid (polySia) and the neural cell adhesion molecule (N-CAM) in the adult rat brain by immunohistochemistry and Western blot analysis. Both molecules were widely distributed but not ubiquitous. Various brain regions showed colocalization of polySia and N-CAM. Strong immunoreactivity for polySia was seen in regions which were negative for N-CAM, such as the main and accessory olfactory bulbs. Immunohistochemical evidence for the heterogeneity of polySia expression in different brain regions was confirmed by immunoblotting. We present evidence that N-CAM is not the only polySia bearing protein in adult rat brain. Specifically, immunoprecipitation using the polySia-specific monoclonal antibody mAb 735 precipitated not only N-CAM isoforms carrying polySia, but also the sodium channel alpha subunit. Immunoblotting using sodium channel alpha subunit antibody (SP20) revealed a smear from 250 kDa upwards. PolySia removal using an endoneuraminidase specific for alpha 2,8-linked polysialic acid of 8 or more residues long, reduced this smear to a single band at 250 kDa. Thus both N-CAM and sodium channels carry homopolymers of alpha 2,8-linked polysialic acid in adult rat brain.     

Development of a 1H NMR structural-reporter-group concept for the primary structural characterisation of alpha-D-glucans

An NMR study of proton chemical shift patterns of known linear alpha-D-glucopyranose di- and trisaccharide structures was carried out. Chemical shift patterns for (alpha1-->2)-, (alpha1-->3)-, (alpha1-->4)- and (alpha1-->6)-linked D-glucose residues were analysed and compared to literature data. Using these data, a 1H NMR structural-reporter-group concept was formulated to function as a tool in the structural analysis of alpha-D-glucans.     

Identification of disialic acid-containing glycoproteins in mouse serum: a novel modification of immunoglobulin light chains, vitronectin, and plasminogen

Serum glycoproteins are involved in various biologic activities, such as the removal of exogenous antigens, fibrinolysis, and metal transport. Some of them are also useful markers of inflammation and disease. Although the amount of sialic acid increases following inflammation, little attention has been paid to the presence of linkage-specific epitopes in serum, especially the alpha2,8-linkage. In a previous study, we demonstrated that four components in mouse serum contain alpha2,8-linked disialic acid (diSia), based on immunoreactivity with monoclonal antibody 2-4B, which is specific to N-glycolylneuraminic acid (Neu5Gc)alpha2-->(8Neu5Gc alpha2-->)(n-1), n > or = 2 [Yasukawa et al., (2005) Glycobiology, 15, 827-837]. In this study, we purified three components, 30-, 70-, and 120-kDa gp, and identified them as an immunoglobulin (Ig) light chain, vitronectin, and plasminogen, respectively, using matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analyses. Modifications of these proteins with alpha2,8-linked diSia were chemically confirmed by fluorometric C7/C9 analyses and mild acid hydrolysates-fluorometric anion-exchange chromatography analyses. We also demonstrated that the IgG, IgM, and IgE light chains are commonly modified with alpha2,8-linked diSia. In addition, both mouse and rat vitronectin contained diSia, and the amount of disialylation in vitronectin dramatically decreased after hepatectomy. These results indicate that a novel diSia modification of serum glycoproteins is biologically important for immunologic events and fibrinolysis.     

Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.

Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin.     

A monoclonal antibody against meningococcus group B polysaccharides distinguishes embryonic from adult N-CAM

The neural cell adhesion molecules (N-CAM) occur chiefly in two molecular forms that are selectively expressed at various stages of development. Highly sialylated forms prevalent in embryonic and neonatal brain are gradually replaced by less sialylated forms as development proceeds. Here we describe a monoclonal antibody raised against the capsular polysaccharides of meningococcus group B (Men B) which specifically distinguishes embryonic N-CAM from adult N-CAM. This antibody recognizes alpha 2-8-linked N-acetylneuraminic acid units (NeuAc alpha 2-8). Immunoblot together with immunoprecipitation experiments with cell lines or tissue extracts showed that N-CAM are the major glycoproteins bearing such polysialosyl units. Moreover we could not detect any sialoglycolipid reactive with this antibody in mouse brain or in the neural cell lines examined. By indirect immunofluorescence staining this anti-Men B antibody decorated cells such as AtT20 (D16/16), which expressed the embryonic forms of N-CAM, but not cells that expressed the adult forms. In primary cultures this antibody allowed us to follow the embryonic-to-adult conversion in individual cells. In addition, the existence of cross-reactive polysialosyl structures on Men B and N-CAM in embryonic brain cells for caution in efforts to develop immunotherapy against neonatal meningitis.     

Quantitative mapping of the N-linked sialyloligosaccharides of recombinant erythropoietin: combination of direct high-performance anion-exchange chromatography and 2-aminopyridine derivatization.

A rapid quantitative analysis of the sialylated N-linked oligosaccharides of recombinant erythropoietin (EPO) expressed in Chinese hamster ovary (CHO) cells has been developed. The procedure utilizes a glycoamidase (glycopeptidase F) to release all of the N-linked oligosaccharides from the native glycoprotein, followed by direct chromatographic analysis using high-performance anion-exchange chromatography (HPAEC) with pulsed amperometric detection. The eight sialyloligosaccharides isolated from HPAEC were characterized by derivatizing with 2-aminopyridine followed by two-dimensional HPLC mapping of the pyridylaminated asialooligosaccharides (Tomiya et al., 1988, Anal. Biochem. 171, 73-90). Seven kinds of complex-type asialooligosaccharides were identified ranging from a biantennary structure to N-acetyllactosamine-extended tetraantennary structure. Approximately 3% of the terminal galactose residues of the oligosaccharides released from EPO were not sialylated whereas 97% contained an alpha(2-->3)-linked sialic acid. Quantitative oligosaccharide mapping of four different lots of EPO from CHO cells was performed to quantify the molar balance and distribution of the N-linked oligosaccharides. The sialyloligosaccharides were distributed with approximately 5% disialylated (single type), 20% trisialylated (six types), and 75% tetrasialylated (four types) oligosaccharides with an average molar recovery of 85% starting from 750 pmol of EPO.     

Polysialic Acid Is Present in Mammalian Semen as a Post-translational Modification of the Neural Cell Adhesion Molecule NCAM and the Polysialyltransferase ST8SiaII

Fertilization in animals is a complex sequence of several biochemical events beginning with the insemination into the female reproductive tract and, finally, leading to embryogenesis. Studies by Kitajima and co-workers (Miyata, S., Sato, C., and Kitajima, K. (2007) Trends Glycosci. Glyc, 19, 85–98) demonstrated the presence of polysialic acid (polySia) on sea urchin sperm. Based on these results, we became interested in the potential involvement of sialic acid polymers in mammalian fertilization. Therefore, we isolated human sperm and performed analyses, including Western blotting and mild 1,2-diamino-4,5-methylenedioxybenzene-HPLC, that revealed the presence α2,8-linked polySia chains. Further analysis by a glyco-proteomics approach led to the identification of two polySia carriers. Interestingly, besides the neural cell adhesion molecule, the polysialyltransferase ST8SiaII has also been found to be a target for polysialylation. Further analysis of testis and epididymis tissue sections demonstrated that only epithelial cells of the caput were polySia-positive. During the epididymal transit, polySia carriers were partially integrated into the sperm membrane of the postacrosomal region. Because polySia is known to counteract histone as well as neutrophil extracellular trap-mediated cytotoxicity against host cells, which plays a role after insemination, we propose that polySia in semen represents a cytoprotective element to increase the number of vital sperm.     

Asparagine-linked glycoprotein biosynthesis in rat brain: identification of glucosidase I, glucosidase II, and and endomannosidase (glucosyl mannosidase)

Previous studies from this laboratory provided evidence, largely based upon the presence of a novel alpha-D-mannosidase, suggesting that the biosynthesis of N-linked glycoproteins may be different in brain as compared to other tissues (Tulsiani, D. R. P., and Touster, O. (1985) J. Biol. Chem. 260, 13,081-13,087). In the present report we describe studies on the enzymes involved in early processing reactions. These studies indicate that the brain, like other tissues, contains glucosidases I and II. The two glucosidases were separated as distinct activities with some overlapping by chromatography on a DE-52 column. The differential inhibition studies and substrate specificity studies support our conclusion that, as in other tissues, rat brain glucosidase I cleaves alpha 1,2-linked terminal glucosyl residues, whereas glucosidase II prefers alpha 1,3-linked glucosyl residues. In addition to these two processing glucosidases, we have characterized an endo enzyme (glucosyl mannosidase) in rat brain. The endomannosidase cleaves a disaccharide (glucosyl alpha 1,3-mannose) from monoglucosylated oligosaccharides (GlcMan7-9GlcNAc). Little or no activity was observed when di- or triglucosylated oligosaccharide was used as a substrate. The pH optimum of the glucosyl mannosidase is 6.2-6.8. The enzyme appears to be an intrinsic microsomal membrane component, since washing of the microsomal membranes with salt solution did not release the enzyme in soluble form. A mixture of Triton X-100 and sodium deoxycholate is required for complete solubilization of the enzyme. The solubilized enzyme is eluted from a Bio-Gel A-1.5m column as a single peak with an apparent molecular weight of 380,000.     

Studies on the uronic acid-containing glycoproteins of Fusarium sp. M7-1: IV. Isolation and identification of four novel oligosaccharide units derived from the acidic polysaccharide chain.

Four novel oligosaccharide units were isolated from the acetolysis products of the acidic polysaccharide chain derived from the glycoproteins of Fusarium sp. M7-1. Their chemical structures were resolved mainly by 1H-NMR spectrometry in combination with methylation analysis and mass spectrometry. The results indicate that these oligosaccharide units originated from the side chains, GlcNAc alpha 1-->4GlcA alpha 1-->2(GlcNac alpha 1-->4)GlcA alpha 1-->2Gal, GlcNAc alpha 1-->4GlcA alpha 1-->2(GlcNAc alpha 1-->4)GlcA alpha 1-->2(GlcNac alpha 1-->4)GlcA alpha 1-->2Gal, ChN<--P--> 6Man beta 1-->4GlcA alpha 1-->2Gal, and Man beta 1-->2(ChN<--P-->6)Man beta 1-->4GlcA alpha 1-->2Gal linked together with the other units reported previously [Jikibara et al. (1992) J. Biochem. 111, 236-243] through beta 1-->6galactofuranoside linkages in the acidic polysaccharide chain.     

Structure and Immunochemistry of the Cell Wall Mannans from Saccharomyces chevalieri, Saccharomyces italicus, Saccharomyces diastaticus, and Saccharomyces carlsbergensis

The mannans of Saccharomyces chevalieri, S. italicus, S. diastaticus, and S. carlsbergensis, were acetolyzed, and the fragments were separated by gel filtration. All gave similar acetolysis fingerprints, which were distinguished from S. cerevisiae by the presence of a pentasaccharide component in addition to the mono-, di-, tri-, and tetrasaccharides. All oligosaccharide fragments were composed of mannose in alpha-linkage. From methylation analysis and other structural studies, the disaccharide was shown to be alphaMan(1 --> 2)Man; the trisaccharide was shown to be a mixture of alphaMan(1 --> 2)alphaMan (1 --> 2)Man and alphaMan(1 --> 3)alphaMan(1 --> 2)Man; the tetrasaccharide was alphaMan(1 --> 3)alphaMan(1 --> 2)alphaMan(1 --> 2)Man; and the pentasaccharide was alphaMan(1 --> 3)alphaMan(1 --> 3)alphaMan(1 --> 2)alphaMan(1 --> 2)Man. The ratios of the different fragments varied slightly from strain to strain. Mannanase digestion of two of the mannans yielded polysaccharide residues that were unbranched (1 --> 6)-linked polymers, thus establishing the structural relationship between these mannans and that from S. cerevisiae. Antisera raised against the various yeasts cross-reacted with the mannans from each, and also with S. cerevisae mannan. The mannotetraose and mannopentaose acetolysis fragments gave complete inhibition of the precipitin reactions, which indicated that, in these systems as in the S. cerevisiae system, the terminal alpha(1 --> 3)-linked mannose unit was the principal immunochemical determinant on the cell surface.     

The immobilized leukoagglutinin from the seeds of Maackia amurensis binds with high affinity to complex-type Asn-linked oligosaccharides containing terminal sialic acid-linked alpha-2,3 to penultimate galactose residues

We recently reported that the purified leukoagglutinin (designated MAL) from the seeds of the leguminous plant Maackia amurensis is a potent leukoagglutinin for the mouse lymphoma cell line BW5147 (Wang, W.-C., and Cummings, R. D. (1987) Anal. Biochem. 161,80). We and others have shown that this lectin is a weak hemagglutinin of human erythrocytes (Kawaguchi, T., Matsumoto, I., and Osawa, T. (1974) J. Biol. Chem. 249, 2786). We now report that leukoagglutination by MAL is inhibited by low concentrations of 2,3-sialyllactose (NeuAc alpha 2,3Gal beta 1,4Glc), but it is not inhibited by either 2,6-sialyllactose (NeuAc alpha 2,6Gal beta-1,4Glc), lactose, or free NeuAc. To further study the carbohydrate-binding specificity of this lectin, we investigated the interactions of immobilized MAL with glycopeptides prepared from the mouse lymphoma cell line BW5147 and from purified glycoproteins. We found that immobilized MAL interacts with high affinity with complex-type tri- and tetraantennary Asn-linked oligosaccharides containing outer sialic acid residues linked alpha 2,3 to penultimate galactose residues. Glycopeptides containing sialic acid linked only alpha 2,6 to penultimate galactose did not interact detectably with the immobilized lectin. Our analyses indicate that the interactions of complex-type Asn-linked chains with the lectin are dependent on sialic acid linkages and are not dependent on either the branching pattern of the mannose residues or the presence of poly-N-acetyllactosamine sequences.     

Importance of glycosidases in mammalian glycoprotein biosynthesis

Processing glycosidases play an important role in N-glycan biosynthesis in mammalian cells by trimming Glc(3)Man(9)GlcNAc(2) and thus providing the substrates for the formation of complex and hybrid structures by Golgi glycosyltransferases. Processing glycosidases also play a role in the folding of newly formed glycoproteins and in endoplasmic reticulum quality control. The properties and molecular nature of mammalian processing glycosidases are described in this review. Membrane-bound alpha-glucosidase I and soluble alpha-glucosidase II of the endoplasmic reticulum remove the alpha1,2-glucose and alpha1,3-glucose residues, respectively, beginning immediately following transfer of Glc(3)Man(9)GlcNAc(2) to nascent polypeptides. The alpha-glucosidases participate in glycoprotein folding mediated by calnexin and calreticulin by forming the monoglucosylated high mannose oligosaccharides required for the interaction with the chaperones. In some mammalian cells, Golgi endo alpha-mannosidase provides an alternative pathway for removal of glucose residues. Removal of alpha1,2-linked mannose residues begins in the endoplasmic reticulum where trimming of mannose residues in the endoplasmic reticulum has been implicated in the targeting of malfolded glycoproteins for degradation. Removal of mannose residues continues in the Golgi with the action of alpha1, 2-mannosidases IA and IB that can form Man(5)GlcNAc(2) and of alpha-mannosidase II that removes the alpha1,3- and alpha1,6-linked mannose from GlcNAcMan(5)GlcNAc(2) to form GlcNAcMan(3)GlcNAc(2). These membrane-bound Golgi enzymes have been cloned and shown to have very distinct patterns of tissue-specific expression. There are also broad specificity alpha-mannosidases that can trim Man(4-9)GlcNAc(2) to Man(3)GlcNAc(2), and provide an alternative pathway toward complex oligosaccharide formation. Cloning of the remaining alpha-mannosidases will be required to evaluate their specific functions in glycoprotein maturation.     

N-Glycan structures of squid rhodopsin.

To determine the glycoforms of squid rhodopsin, N-glycans were released by glycoamidase A digestion, reductively aminated with 2-aminopyridine, and then subjected to 2D HPLC analysis [Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y. & Tomiya, N. (1995) Anal. Biochem.226, 139-146]. The major glycans of squid rhodopsin were shown to possess the alpha1-3 and alpha1-6 difucosylated innermost GlcNAc residue found in glycoproteins produced by insects and helminths. By combined use of 2D HPLC, electrospray ionization-mass spectrometry and permethylation and gas chromatography-electron ionization mass spectrometry analyses, it was revealed that most (85%) of the N-glycans exhibit the novel structure Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4(Galbeta1-4Fucalpha1-6)(Fucalpha1-3)GlcNAc.