Secretion of a lactosaminoglycan-containing glycoprotein by peri-implantation sheep conceptuses

Sheep conceptuses from day 16 of pregnancy were cultured in the presence of [3H]glucosamine and [14C]leucine and a high-molecular-weight glycoprotein (HMWG) secreted into the culture medium was purified by a combination of anion-exchange and gel filtration chromatography. The HMWG was found to have a molecular weight between 800,000 and 900,000 and to be highly resistant to digestion with pronase. Characteristics of the carbohydrate portion of the purified glycoprotein were examined by selective chemical and enzymatic digestions and lectin binding studies. Mild alkaline reduction was ineffective in disassociating carbohydrate chains from the protein core. Furthermore, the protein was resistant to both O-glycanase and peptide:N-glycanase F. Harsh alkaline reduction caused the release of carbohydrates, however. After pronase digestion of these products, three molecular weight classes of carbohydrates were resolved by Sephadex G-25 chromatography. Two lines of evidence indicate that the HMWG contains lactosaminoglycan components. The intact molecule and two of the molecular weight classes of carbohydrates resolved by harsh alkaline reduction bind Datura stramonium lectin. Binding of HMWG to lectin could be partially inhibited by N-acetyllactosamine and completely inhibited by a mixture of N,N'-diacetylchitobiose and N,N',N"-triacetylchitotriose. Secondly, digestion with endo-beta-galactosidase causes the release of 16% of the [3H]glucosamine from the intact molecule. Therefore, the HMWG of the sheep conceptus is the first reported example of secretion of lactosaminoglycan-containing glycoprotein by peri-implantation embryos.     

The abundance of additional N-acetyllactosamine units in N-linked tetraantennary oligosaccharides of human Tamm-Horsfall glycoprotein is a donor-specific feature

Previously, treatment of Tamm-Horsfall glycoprotein (THp) from different donors with endo-beta-galactosidase has been shown to liberate a tetra- and a Sd(a)-active pentasaccharide, concluding the presence of N-linked carbohydrate chains containing additional N - acetyllactosamine units. These type of oligosaccharides were not found in a detailed structure elucidation of the carbohydrate moiety of THp of one male donor, suggesting a donor-specific feature for these type of structures. Therefore, THp was isolated from four healthy male donors and each subjected to endo-beta-galactosidase treatment in order to release these tetra- and Sd(a)-active pentasaccharide. Differences were observed in the total amount of released tetra- and Sda-active pentasaccharide of the used donors (42, 470, 478, 718 microg/100 mg THp), indicating that the presence of repeating N-acetyllactosamine units incorporated into the N-glycan moiety of THp is donor specific. Furthermore, a higher expression of the Sd(a) determinant on antennae which display N-acetyllactosamine elongation was observed, suggesting a better accessibility for the beta-N-acetylgalactosaminyltransferase. In order to characterize the N-glycans containing repeating N- acetyllactosamine units, carbohydrate chains were enzymatically released from THp and isolated. The tetraantennary fraction, which accounts for more than 33% of the total carbohydrate moiety of THp, was used to isolate oligosaccharides containing additional N - acetyllactosamine units. Five N-linked tetraantennary oligosaccharides containing a repeating N-acetyllactosamine unit were identified, varying from structures bearing four Sd(a) determinants to structures containing no Sd(a) determinant (see below). One compound was used in order to specify the branch location of the additional N- acetyllactosamine unit, and it appeared that only the Gal-6' and Gal-8' residues were occupied by a repeating N -acetyllactosamine unit.      

The distribution of repeating [Gal beta 1,4GlcNAc beta 1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1

The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species.     

Determination of the branch location of extra N-acetyllactosamine units in sialo N-linked tetraantennary oligosaccharides

An approach is presented for the determination of the branch location of 1 or 2 extra N-acetyllactosamine units in sialo N-linked carbohydrate chains from glycoproteins. Tetraantennary oligosaccharides containing extra N-acetyllactosamine units were digested with endo-beta-galactosidase, followed by treatment with N-acetyl-beta-glucosaminidase, yielding products which could be analysed by 1H-NMR spectroscopy, thereby giving conclusive data about the location of the extra units in the intact structures.     

Isolation and characterization of a new endo-beta-galactosidase from Diplococcus pneumoniae

An endo-beta-galactosidase, which hydrolyzes the internal beta-galactosidic linkages of R----GlcNAc (or GalNAc) beta 1----3Gal beta 1----4GlcNAc (or Glc), was isolated from the culture supernatant of Diplococcus pneumoniae. The enzyme, named endo-beta-galactosidase DII, hydrolyzed linear N-acetyllactosamine repeating structures in glycolipids and glycopeptides to release oligosaccharides. The specificity of endo-beta-galactosidase DII is the same as that of Escherichia freundii endo-beta-galactosidase as far as described above, but the following differences between these two enzymes were found: Branched lactosaminyl glycolipids and H-antigenic glycolipids were resistant to endo-beta-galactosidase DII, even when linear structure was present at the inner part. Throughout the enzymic hydrolysis, endo-beta-galactosidase DII released mostly small oligosaccharides (tetra-, tri-, and disaccharides) from substrates, suggesting that the enzyme split off the oligosaccharides stepwise from the nonreducing terminal. Lactosaminoglycans were partially hydrolyzed by endo-beta-galactosidase DII to produce small oligosaccharides as the major product and residual glycopeptides. The residual glycopeptides were readily hydrolyzed by E. freundii endo-beta-galactosidase to produce various sizes of oligosaccharides. Keratan sulfate was not degraded by endo-beta-galactosidase DII. These properties of endo-beta-galactosidase DII characterize it as a new endo-beta-galactosidase with a unique specificity.     

Structure of the acidic N-linked carbohydrate chains of the 55-kDa glycoprotein family (PZP3) from porcine zona pellucida.

N-linked carbohydrate chains of the major 55-kDa family, PZP3, of porcine zona pellucida glycoproteins are composed of neutral (28%) and acidic (72%) complex-type chains. The structures of the main components of the neutral chain have been established [Noguchi, S., Hatanaka, Y., Tobita, T. & Nakano, M. (1992) Eur. J. Biochem. 204, 1089-1100]. Here we report the structures of the acidic chains. Only two kinds of acidic fragments were released from PZP3 by endo-beta-galactosidase digestion following beta-elimination of O-linked chains. 500-MHz one-dimensional and two-dimensional 1H-NMR spectroscopy revealed their structures to be Sia alpha(2-3)Gal beta(1-4) [HSO3-6]GlcNAc beta(1-3)Gal and HSO3-6GlcNAc beta(1-3)Gal, showing that the sulfate-containing acidic chains are constructed with non-branched N-acetyllactosamine repeats which have sialic acid(s) at the non-reducing end(s) and sulfate at the C-6 position of GlcNAc residues. The acidic N-linked chains obtained from PZP3 by hydrazinolysis were separated into diantennary chains (34%) and tri- and tetra-antennary chains (66%) by concanavalin-A--agarose gel chromatography. The diantennary chains and their sialidase digests were fractionated by DEAE-HPLC. From the analyses of the endo-beta-galactosidase digests of each fraction, structures of the diantennary acidic chains were determined. They are classified into four groups. The first group is the sialylated chains without the sulfated N-acetyllactosamine repeating unit. The other three groups have the chains of various lengths differing in the number of monosulfated N-acetyllactosamine unit. These chains are extended from the Man alpha(1-3) branch of the trimannosyl core in the second group, from the Man alpha(1-6) branch in the third group, and from both branches in the fourth group. The structural features of the tri- and tetra-antennary acidic chains are also presented.     

Poly-LacNAc as an Age-Specific Ligand for Rotavirus P[11] in Neonates and Infants

Rotavirus (RV) P[11] is an unique genotype that infects neonates. The mechanism of such age-specific host restriction remains unknown. In this study, we explored host mucosal glycans as a potential age-specific factor for attachment of P[11] RVs. Using in vitro binding assays, we demonstrated that VP8* of a P[11] RV (N155) could bind saliva of infants (60.3%, N = 151) but not of adults (0%, N = 48), with a significantly negative correlation between binding of VP8* and ages of infants (P<0.01). Recognition to the infant saliva did not correlate with the ABO, secretor and Lewis histo-blood group antigens (HBGAs) but with the binding of the lectin Lycopersicon esculentum (LEA) that is known to recognize the oligomers of N-acetyllactosamine (LacNAc), a precursor of human HBGAs. Direct evidence of LacNAc involvement in P[11] binding was obtained from specific binding of VP8* with homopolymers of LacNAc in variable lengths through a glycan array analysis of 611 glycans. These results were confirmed by strong binding of VP8* to the Lec2 cell line that expresses LacNAc oligomers but not to the Lec8 cell line lacking the LacNAc. In addition, N155 VP8* and authentic P[11] RVs (human 116E and bovine B223) hemagglutinated human red blood cells that are known to express poly-LacNAc. The potential role of poly-LacNAc in host attachment and infection of RVs has been obtained by abrogation of 116E replication by the PAA-conjugated poly-LacNAc, human milk, and LEA positive infant saliva. Overall, our results suggested that the poly-LacNAc could serve as an age-specific receptor for P[11] RVs and well explained the epidemiology that P[11] RVs mainly infect neonates and young children.     

Effect of endo-beta-galactosidase on intact human erythrocytes.

Endo-beta-galactosidase, a glycosidase that hydrolyzes Gal beta 1-4 GlcNAc linkages in glycoconjugates, has been used to probe the plasma membrane of human erythrocytes. Coomassie blue staining of stroma components separated by sodium dodecyl sulfate-acrylamide gel electrophoresis indicates that treatment of red cells with endo-beta-galactosidase converts Protein 3, the anion transporter of the erythrocyte, to a more compact staining band. No other components detected by Coomassie staining are affected. Following labeling of red cells with galactose oxidase + NaB3H4, 45 to 50% of the [3H]galactose residues can be released by endo-beta-galactosidase. In contrast, only 5% of the label incorporated by treatment with periodate + NaB3H4, can be removed. [3H]Galactose residues are released from three components: Protein 3, Band 4.5, and the megaloglycolipids. The susceptibility of these components to endo-beta-galactosidase, together with the high content of Gal and GlcNAc present in Protein 3 and the megaloglycolipids, suggests that the erythrocyte membrane contains several components with N-acetyllactosamine repeating units, a structure commonly found in connective tissue glycoconjugates.     

Evidence for the presence of complex high-molecular mass N-linked oligosaccharides in intranuclear glycoproteins from HeLa cells.

Nonhistone proteins were extracted in 0.4 M NaCl from membrane-depleted nuclei of HeLa cells grown in the presence or the absence of [5,6-3H]fucose. Control experiments strongly suggest that most extracted proteins were indeed nuclear components. Several proteins, present in the 0.4 M NaCl nuclear extract, with M(r) ranging from 35,000 to 115,000 were identified on Western blots as fucosylated glycoproteins owing to their binding to the fucose-specific lectin, Ulex europeus agglutinin I. Results of experiments involving mild alkaline treatment and peptide N-glycosidase F digestion showed that the carbohydrate moieties of these fucosylated nuclear glycoproteins were N-linked to the polypeptide backbone. Analysis of the N-glycans revealed the presence of two populations of sialylated oligosaccharides on the basis of their relative molecular masses. The sensitivity of the high-M(r) oligosaccharides to endo-beta-galactosidase and their incorporation of [3H]glucosamine suggest that they could contain repeating N-acetyllactosamine units. [3H]Fucose incorporated into nuclei was confined to the nucleoli, as judged by autoradiography of sections cut through cells grown in the presence of [3H]fucose. Electron microscopy autoradiography showed that the fibrillar centers were never labeled, while silver grains were observed on the dense and the granular components of nucleoli. Taking into account of these data most nuclear fucosylated glycoproteins extracted in 0.4 M NaCl might be nucleolar ribonucleoproteins.     

alpha-D-galactose-bearing glycoproteins on the surface of stimulated murine peritoneal macrophages. Biochemical and immunochemical characterization of purified glycoproteins.

Two glycoproteins were isolated from lysates of thioglycollate-stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92-109 kDa and 115-125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92-kDa and 115-kDa glycoproteins had pI 5.2-5.4 and pI less than or equal to 4, respectively. Digestion of both glycoproteins with alpha-galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal alpha-D-galactosyl groups. After reduction with 2-mercaptoethanol, each glycoprotein fraction was sensitive to N-glycanase; the 115-kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92-kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N-glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N-glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex-type oligosaccharides and a small amount of high-mannose and/or hybrid-type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti-[i-(Den)] serum and their digestion with endo-beta-galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N-acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex-type oligosaccharides. The glycoprotein(s) fraction precipitated with anti-[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O-glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O-linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked alpha 2,6 to subterminal D-galactose or N-acetylgalactosamine residues, and alpha 2,3 to N-acetyllactosamine residues, respectively.     

Presence of sulfate in N-glycosidically linked carbohydrate units of calf thyroid plasma membrane glycoproteins

Calf thyroid slices were found to incorporate [35S] sulfate into two major plasma membrane glycoproteins, which have been previously designated as GP-1 and GP-3 (Okada, Y., and Spiro, R. G. (1980) J. Biol. Chem. 255, 8865-8872). The 35S-glycoproteins were identified on the basis of their characteristic solubility and electrophoretic migration as well as their affinity for Bandeiraea simplicifolia I lectin. After pronase digestion of these glycoproteins, the 35S-label remained associated with the glycopeptides primarily on asparagine-linked carbohydrate units which were released by hydrazinolysis. Examination of the reduced radio-labeled products obtained by nitrous acid cleavage of the hydrazine-liberated oligosaccharides indicated that sulfate esters of N-acetylglucosamine occurred at three locations on the carbohydrate units; two 35S-monosaccharides (2,5-anhydromannitol 4- and 6-sulfate) and one 35S-disaccharide (beta-Gal(1----4)-2,5-anhydromannitol(6-SO4] were formed. The disaccharide is believed to be derived from an internal sulfated N-acetyllactosamine sequence while the monosaccharides most likely originate from 4- and 6-sulfated N-acetylglucosamine residues situated, respectively, at the non-reducing and reducing termini of the oligosaccharide units. Quantitation by NaB[3H]4 reduction of the sulfated saccharides obtained by nitrous acid treatment of hydrazine-released oligosaccharides from unlabeled GP-3 indicated that about 20% of the asparagine-linked carbohydrate units contain sulfate substituents.     

Reactivity of mucin-specific lectin from Sambucus sieboldiana with simple sugars, normal mucins and tumor-associated mucins. Comparison with other lectins.

Mucin-specific lectin from Sambucus sieboldiana (SSA-M) reacts in Western blotting and ELISA with mucins from porcine stomach, bovine and ovine submaxillary glands, the human milk fat globule membrane, in vitro human ovarian, breast and colonic tumor cell lines, and mucins produced in vivo in the ascites of patients with endometrial and ovarian tumors, but not with fetal bovine fetuin or human transferrin. Sialidase treatment of these mucins led to an increase in the binding of SSA-M, suggesting that sialic acid is not part of the binding site for this lectin. Furthermore, sialic acid did not inhibit lectin binding. Treatment of asialomucin with O-glycanase decreased the binding of SSA-M, confirming the reactivity of the lectin with an O-linked carbohydrate. Treatment of mucins with trifluoromethanesulfonic acid, which removes all but core carbohydrate, led to an increase in the binding of SSA-M, suggesting that the lectin reacts with O-linked core glycans. Indeed, the increased reactivity after sialidase treatment of ovine submaxillary mucin suggests the lectin reacts with peptide-linked N-acetylgalactosamine (GalNAc), since more than 98% of the glycan chains attached to this mucin are sialylated GalNAc. The binding of SSA-M to sialidase-treated porcine mucin was inhibited strongly by GalNAc and disaccharides containing galactose (lactose, melibiose, and N-acetyllactosamine) but not by free galactose (Gal), suggesting that the glycan for optimum binding is Gal beta(1-3)GalNAc. This pattern of inhibition was different to other core glycan-reactive lectins tested, indicating that SSA-M is distinct, and should be of use in the isolation and characterisation of mucins and O-linked glycans.     

Erythrocyte nucleoside and sugar transport. Endo-beta-galactosidase and endoglycosidase-F digestion of partially purified human and pig transporter proteins.

Nucleoside- and glucose-transport proteins isolated from human erythrocyte membranes were photoaffinity-labelled with [3H]nitrobenzylthioinosine and [3H]cytochalasin B, respectively, and subjected to endo-beta-galactosidase or endoglycosidase-F digestion. Without enzyme treatment the two radiolabelled transporters migrated on SDS/polyacrylamide gels with the same apparent Mr (average) of 55,000. Apparent Mr (average) values after endo-beta-galactosidase digestion were 47,000 and 48,000 for the nucleoside and glucose transporters respectively, and 44,000 and 45,000 respectively after endoglycosidase-F digestion. In contrast, endo-beta-galactosidase had no effect on the electrophoretic mobility of the nucleoside transporter isolated from pig erythrocytes. This transport system exhibited a higher Mr than the human protein, endoglycosidase-F treatment decreasing its apparent Mr (average) from 64,000 to 57,000. It is concluded that the human and pig erythrocyte nucleoside transporters are glycoproteins containing N-linked oligosaccharide. The data provide evidence of substantial carbohydrate and polypeptide differences between the human and pig erythrocyte nucleoside transporters, but evidence of molecular similarities between the human erythrocyte nucleoside and glucose transporters.     

The S-type lectin from calf heart tissue binds selectively to the carbohydrate chains of laminin.

We report that the S-type lectin in calf heart tissue, termed calf heart agglutinin (CHA), binds to immobilized mouse laminin in ligand blotting and solid-phase radioligand binding assays. When compared with other glycoproteins, radioiodinated CHA binds preferentially to immobilized laminin. The binding is saturable with a Kd of 9.2 x 10(-7) M and is competitively inhibited by nonradiolabeled CHA as well as a similar lectin from porcine heart tissue. Both lactose and N-acetyllactosamine are good inhibitors of binding to laminin but binding is not inhibited by heparin. Exoglycosidase treatments demonstrated that the binding of radioiodinated CHA to laminin is not dependent on terminal sialyl-, fucosyl-, beta- or alpha-linked galactosyl residues, whereas treatment of laminin with endo-beta-galactosidase significantly decreases the lectin binding. Thus, CHA binds selectively to the poly-N-acetyllactosamine chains on complex-type Asn-linked oligosaccharides in laminin.     

Identification of a 14-kDa laminin binding protein (HLBP14) in human melanoma cells that is identical to the 14-kDa galactoside binding lectin.

The carbohydrate moieties present on laminin play a crucial role in the multiple biological activities of this basement membrane glycoprotein. We report the identification of a human laminin binding protein with an apparent molecular mass of 14 kDa on sodium dodecyl sulfate-polyacrylamide gels that was found, after purification and amino acid microsequencing, to be identical to the previously described 14-kDa galactoside binding soluble L-14 lectin. We have designated this human laminin binding protein as HLBP14. HLBP14 was purified from human melanoma cells in culture by laminin affinity chromatography and gel electroelution. We demonstrate that HLBP14 binds specifically to the poly-N-acetyllactosamine residues of murine laminin and does not bind to other glycoproteins that do not contain such structures, such as fibronectin. HLBP14 was eluted from a murine laminin column by lactose, N-acetyllactosamine, and galactose but not by other control saccharides, including glucose, fucose, mannose, and melibiose. It did not bind to laminin treated with endo-beta-galactosidase. Lactose also eluted HLBP14 off a human laminin affinity column, implying that human laminin also contains poly-N-acetyllactosamine residues. On immunoblots, polyclonal antibodies raised against HLBP14 recognized HLBP14 as well as 31- and 67-kDa molecules that are also laminin binding proteins, indicating that these proteins share common epitopes. L-14, a dimeric lactose binding lectin, is expressed in a wide variety of tissues. Although the expression of this molecule has been linked to a variety of biological events, the elucidation of its specific functions has been elusive. The observation that HLBP14, a human cancer cell laminin binding protein, is identical to L-14 strongly suggests that the functions attributed to this lectin could be mediated, at least in part, through its ability to interact with the poly-N-acetyllactosamine residues of laminin. HLBP14 could potentially play a role during tumor invasion and metastasis by modulating the interactions between cancer cells and laminin.     

Blood group-active carbohydrate chains on the receptor for epidermal growth factor of A431 cells

The antigens expressed on the carbohydrate chains of the receptor for epidermal growth factor of A431 cells were studied by immunoblotting with monoclonal antibodies. Blood group A and the Type 1 based blood group ALeb and Lea antigens were detected as well as antigens associated with unsubstituted, monofucosylated and difucosylated Type 2 blood group chains. The Lea and the difucosylated Type 2 antigen activities were abolished by treating the blotted receptor with endo-beta-galactosidase, indicating that they are expressed on backbone structures of poly-lacto/neolacto type. (The term 'poly-lacto/neolacto' is used here to describe oligosaccharide backbone structures consisting of repeating Type 1, Gal beta 1-3GlcNAc (lacto) or Type 2, Gal beta 1-4GlcNAc (neolacto) sequences.) The glycosidic linkage of oligosaccharides to protein was investigated using Pronase digests of the receptor biosynthetically labelled with [3H]glucosamine or [3H]fucose. The oligosaccharides were alkali-resistant, consistent with N- rather than O-glycosidically linked chains. A proportion of [3H]fucose-labelled glycopeptides was susceptible to endo-beta-galactosidase, confirming the immunoblotting experiment using antibodies against the Lea and the difucosylated Type 2 antigenic determinants. Oligosaccharides were released from the [3H]fucose- and [3H]-glucosamine-labelled glycopeptides by hydrazinolysis. Chromatography of the oligosaccharides on Bio-Gel P6 and Concanavalin A columns indicated a spectrum of oligosaccharides which include those of high mannose type labelled with [3H]glucosamine, and a mixture of oligosaccharides labelled with [3H]fucose and [3H]glucosamine of bi- and multiantennary complex types of which a subpopulation is susceptible to digestion with endo-beta-galactosidase.     

Characterization of oligosaccharide ligands expressed on SW1116 cells recognized by mannan-binding protein. A highly fucosylated polylactosamine type N-glycan

Mannan-binding protein (MBP) is a C-type serum lectin and activates complement through the lectin pathway when it binds to ligand sugars such as mannose, N-acetylglucosamine, and fucose on microbes. In addition, the vaccinia virus carrying the human MBP gene was shown to exhibit potent growth inhibitory activity toward human colorectal carcinoma, SW1116, cells in nude mice. We have proposed calling this activity MBP-dependent cell-mediated cytotoxicity (MDCC) (Ma, Y., Uemura, K., Oka, S., Kozutsumi, Y., Kawasaki, N., and Kawasaki, T. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 371-375). In this study, the MBP ligands on the surface of SW1116 cells were characterized. Initial experiments involving plant lectins and anti-Lewis antibodies as inhibitors of MBP binding to SW1116 cells indicated that fucose plays a crucial role in the interaction. Subsequently, Pronase glycopeptides were prepared from whole cell lysates, and oligosaccharides were liberated by hydrazinolysis. After being tagged by pyridylamination, MBP ligand oligosaccharides were isolated with an MBP affinity column, and then their sequences were determined by mass spectrometry and tandem mass spectrometry after permethylation, in combination with endo-beta-galactosidase digestion and chemical defucosylation. The MBP ligands were shown to be large, multiantennary N-glycans carrying a highly fucosylated polylactosamine type structure. At the nonreducing termini, Le(b)/Le(a) or tandem repeats of the Le(a) structure prevail, a substantial proportion of which are attached via internal Le(x) or N-acetyllactosamine units to the trimannosyl core. The structures characterized are unique and distinct from those of other previously reported tumor-specific carbohydrate antigens. It is concluded that MBP requires clusters of tandem repeats of the Le(b)/Le(a) epitope for recognition.     

Differentiation-associated decrease in the proportion of fucosylated polylactosaminoglycans of CaCo-2 human colonic adenocarcinoma cells.

CaCo-2 cells are human colonic adenocarcinoma cells which can differentiate spontaneously into enterocytes when maintained confluent for extended periods of time. Cells kept in culture for 4 days (rapidly growing), 7-9 days (early confluence) and 19-22 days (late confluence) were incubated for 24 h with L-[5,6-3H]fucose or D-[6-3H]glucosamine in order to examine the changes in glycoprotein carbohydrate structure that occur during this differentiation. Labelled glycopeptides obtained by exhaustive Pronase digestion of the cell-surface and cell-pellet fractions were fractionated on Bio-Gel P-6. A high-Mr glycopeptide fraction which was excluded from Bio-Gel P-6 was present in all cases. These glycopeptides were then fractionated by affinity chromatography on Datura stramonium agglutinin-agarose. The glycopeptides which were specifically bound to the lectin column were largely degraded by endo-beta-galactosidase, thereby indicating that they consisted of fucosylated polylactosaminoglycans. The proportion of labelled polylactosaminoglycans decreased with increasing time in culture, whereas sucrase activity, which is characteristic of differentiated enterocytes, increased. These results demonstrate that a relatively large decrease in the proportion of fucosylated polylactosaminoglycans occurs with differentiation of CaCo-2 cells.     

Isolation, molecular and biological properties of a lectin from rice embryo: relationship with wheat germ agglutinin properties

Rice lectin (Oryza sativa, var. Balilla 28) was purified from defatted embryos by aqueous acid extraction at pH 1.3 followed by ammonium sulfate precipitation between 2 and 4 M, affinity chromatography on agarose-p-aminophenyl-beta-D-N-acetylglucosamine, and gel filtration on AcA 54. The homogeneity of the lectin was checked by polyacrylamide gel electrophoresis, gel filtration, and immunodiffusion. The amino acid analysis revealed a high half-cystine content (9%) and a low aromatic and hydrophobic amino acid content. The lectin contained neither neutral carbohydrates nor amino sugars. The isoelectric point was estimated to be 8.1. The molecular weight of rice lectin was estimated to be 38,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions showed two polypeptides with Mr 19,000 and 15,000. The circular dichroism spectrum of rice lectin in far ultraviolet was characterized by a positive maximum at 228 nm and a negative band at 203 nm suggesting the presence of a beta-pleated sheet and the absence of alpha-helix. Rice lectin had no human blood group specificity and agglutinated rabbit erythrocytes more efficiently than erythrocytes from other animal species. Furthermore, agglutination was enhanced by trypsin treatment of erythrocytes. The erythroagglutinating activity was very high since the minimal concentration needed to agglutinate erythrocytes was 0.05 micrograms/ml. Although [methyl-3H]thymidine incorporation was stimulated in human lymphocytes, rice lectin could not be considered as a mitogenic lectin since it stimulated neither blast transformation nor lymphocyte proliferation. The saccharide specificity of rice lectin was related to N-acetylglucosamine and its oligomers: N,N',N"-triacetylchitotriose was the most powerful inhibitor. Furthermore, the N-acetylneuraminic acid was not a specific rice determinant. Finally, the double immunodiffusion method revealed a cross-reactivity between rice lectin and wheat germ agglutinin, indicating that these lectins were closely antigenically related. The analogies and differences between biological and immunological properties of rice lectin and wheat germ agglutinin are discussed and the possibility of their evolution from a common ancestor is put forward.     

Poly-N-acetyllactosamine structures on murine cell surface T200 glycoprotein participate in natural killer cell binding to YAC-1 targets

Cell-surface murine T200 glycoprotein has been implicated in the binding of NK cells to certain susceptible tumor targets. The existence of poly-N-acetyllactosamine structures on T200 glycoprotein and the ability of lactosamine-type oligosaccharides to inhibit NK cell-mediated cytotoxicity suggest that these structures may also be important in NK-target binding. To further identify and characterize these structures, relevant saccharides and reconstituted membrane liposomes containing fractionated effector cell membrane proteins were tested for their ability to block conjugate formation. Under base line conditions, the majority of plastic-non-adherent, Percoll-fractionated, NK-enriched splenocytes that formed conjugates with NK-susceptible YAC-1 targets functioned as lytic effectors in a single-cell cytotoxicity assay. These effectors were blocked in their ability to bind to YAC-1 targets by the addition of N-acetyllactosamine [Gal(beta 1,4)-GlcNAc] and chitobiose [GlcNAc(beta 1,4)GlcNAc], but not by saccharides lacking lactosamine-type linkages. Liposomes prepared from octyl-beta-D-glucopyranoside-extracted YAC-1 and NK-enriched effector cell membranes interfered with conjugate formation, whereas liposomes prepared from NK-insensitive P815 cells were inconsequential. Surface radiolabeled effector cell membrane proteins were fractionated by tomato lectin-Sepharose 4B (poly-N-acetyllactosamine-specific) column chromatography. Tomato lectin-bound material was enriched in a glycoprotein identical with T200, which, when incorporated into liposomes, was a potent inhibitor of effector-target binding. This inhibitory capacity was abrogated by treatment of liposomes with Ly-5 mAb (T200 mAb) or the lactosamine-specific enzyme endo-beta-galactosidase. When T200 was purified by mAb affinity chromatography and incorporated into liposomes, it was a potent inhibitor of conjugate formation, an effect that was blocked by pretreatment of T200-containing liposomes with Ly-5 mAb or endo-beta-galactosidase. These data provide additional evidence that T200 can mediate binding of NK cells to YAC-1 targets, and that poly-N-acetyllactosamine-type structures on NK cell surface T200 glycoprotein are important in the binding process.