Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Asparagine-linked oligosaccharides containing poly-N-acetyllactosamine chains are preferentially bound by immobilized calf heart agglutinin

We have investigated the carbohydrate-binding specificity of a mammalian lectin, calf heart agglutinin, by determining the interaction of the immobilized lectin with a variety of complex-type Asn-linked oligosaccharides. Our results demonstrate that calf-heart agglutinin binds with high affinity to oligosaccharides containing the repeating disaccharide (3Gal beta 1-4GlcNAc beta 1)n or poly-N-acetyllactosamine sequence and that the presence of terminal beta-linked galactosyl residues is neither sufficient nor necessary for high affinity interactions.     

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.     

Structural requirements for the binding of oligosaccharides to immobilized lectin ofErythrina variegata (Linn) var.Orientalis

The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins withErythrina variegata agglutinin (EVA) were investigated by means of affinity chromatography on an EVA-Sepharose column. Some of the branched poly-N-acetyllactosamine-type oligosaccharides obtained from human erythrocyte band 3 glycoprotein were found to show high affinity to EVA-Sepharose, whereas complex-type oligosaccharides were shown to have low affinity. Hybrid type, oligomannose-type and unbranched poly-N-acetyllactosamine-type oligosaccharides bound very little or not at all to EVA-Sepharose. To further study the carbohydrate-binding specificity of this lectin, we investigated the interaction of immobilized EVA and oligosaccharide fragments obtained through partial hydrolysis from branched poly-N-acetyllactosamine-type oligosaccharides. Branched poly-N-acetyllactosamine-type oligosaccharides were subjected to limited hydrolysis with 0.1% trifluoroacetic acid at 100°C for 40 min and then separated on an amino-bonded silica column. One of pentasaccharides thus prepared strongly bound to the EVA-Sepharose column. Structural analysis of this pentasaccharide showed that the Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal sugar sequence, which is an l-antigen determinant, was essential for the high affinity binding of the oligosaccharides to the EVA-Sepharose column.Abbreviations EVA Erythrina variegata agglutinin - WGA wheat germ agglutinin - STA potato lectin - LEA tomato lectin - DSA Datura stramonium agglutinin - PBS 0.01 M sodium phosphate buffer, pH 7.3, containing 0.15 M NaCl - Galol galactitol     

Systematic fractionation of oligosaccharides of human immunoglobulin G by serial affinity chromatography on immobilized lectin columns

Human immunoglobulin G is known to contain 16 different biantennary complex-type asparagine-linked sugar chains, each of which occurs in a nonsialylated, monosialylated, or disialylated form. These oligosaccharides can be separated into 14 fractions by sequential affinity chromatography with Aleuria aurantia lectin (AAL)-Sepharose, RCA120-WG003, and E4-phytohemagglutinin-agarose columns. Twelve of them were found to contain a single oligosaccharide, while the fraction which passed through all three columns was shown to contain two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcOT. The fraction, which bound to the AAL-Sepharose column and passed through the remaining two lectin columns, also contained two oligosaccharides, GlcNAc beta 1----2Man alpha 1----6(+/- GlcNAc beta 1----4) (GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (Fuc alpha 1----6)GlcNAcOT. These results indicated that serial affinity chromatography with the three lectin columns can be used effectively to detect changes in the sugar chains of IgG resulting from diseases such as rheumatoid arthritis.     

Human galectin-1 recognition of poly-N-acetyllactosamine and chimeric polysaccharides

Human galectin-1 is a dimeric carbohydrate binding protein (Gal-1) (subunit 14.6 kDa) widely expressed by many cells but whose carbohydrate binding specificity is not well understood. Because of conflicting evidence regarding the ability of human Gal-1 to recognize N-acetyllactosamine (LN, Galbeta4GlcNAc) and poly-N-acetyllactosamine sequences (PL, [-3Galbeta4GlcNAcbeta1-]n), we synthesized a number of neoglycoproteins containing galactose, N-acetylgalactosamine, fucose, LN, PL, and chimeric polysaccharides conjugated to bovine serum albumin (BSA). All neoglycoproteins were characterized by MALDI-TOF. Binding was determined in ELISA-type assays with immobilized neoglycoproteins and apparent binding affinities were estimated. For comparison, we also tested the binding of these neoglycoconjugates to Ricinus communis agglutinin I, (RCA-I, a galactose-binding lectin) and Lycopersicon esculentum agglutinin (LEA, or tomato lectin), a PL-binding lectin. Gal-1 bound to immobilized Galbeta4GlcNAcbeta3Galbeta4Glc-BSA with an apparent K(d) of approximately 23 micro M but bound better to BSA conjugates with long PL and chimeric polysaccharide sequences (K(d)'s ranging from 11.9 +/- 2.9 microM to 20.9 +/- 5.1 micro M). By contrast, Gal-1 did not bind glycans lacking a terminal, nonreducing unmodified LN disaccharide and also bound very poorly to lactosyl-BSA (Galbeta4Glc-BSA). By contrast, RCA bound well to all glycans containing terminal, nonreducing Galbeta1-R, including lactosyl-BSA, and bound independently of the modification of the terminal, nonreducing LN or the presence of PL. LEA bound with increasing affinity to unmodified PL in proportion to chain length. Thus Gal-1 binds terminal beta4Gal residues, and its binding affinity is enhanced significantly by the presence of this determinant on long-chain PL or chimeric polysaccharides.     

免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素

 报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果．哺乳动物红细胞能够特异地吸附凝集素．用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体．再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ结果显示,该凝集素的分子量约为７３ ｋ Ｄ．这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的．为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法．     

Comparison of the expression of cell surface poly-N-acetyllactosamine-type oligosaccharides in PC12 cells with those in its variant PC12D.

To explore the biological role of carbohydrate chains in the process of nerve cell differentiation, we carried out a characterization of the carbohydrate structure of glycoproteins by comparing conventional PC12 cells with variant cells (PC12D). In vitro metabolic labeling of cells with either [(3)H] glucosamine or [(3)H] threonine, together with tomato lectin staining, revealed that nerve growth factor (NGF) stimulation caused a decrease in the poly-N-acetyllactosamine synthesis of high-molecular-weight glycopeptides from PC12 cells. By comparison, the amount of glycopeptides with poly-N-acetyllactosamine from PC12D cells was already significantly low and it was not changed by NGF stimulation. By assaying the glycosyltransferases that participate in poly-N-acetyllactosamine synthesis, the decrease in the amount of the poly-N-acetyllactosamine in PC12D cells as well as NGF-stimulated PC12 cells could be accounted for by a reduction in the activity of poly-N-acetyllactosamine extension enzyme (GnT-i), because the amount of poly-N-acetyllactosamine in both cells precisely correlated with changes in GnT-i activity, whereas the activities of N-acetylglucosaminyltransferase V (GnT-V) and beta 1-4 galactosyltransferase remained unchanged. These results demonstrate that the decrease in poly-N-acetyllactosamine synthesis in PC12 cells occurred prior to neurite formation, whereas PC12D cells were insensitive to this effect. Next, we showed that GnT-i but not GnT-V catalyzed a rate-limiting reaction in the expression of poly-N-acetyllactosamine chains, especially in pheochromocytoma.     

Characterization of two protein disulfide isomerases from the endocytic pathway of bloodstream forms of Trypanosoma brucei

Proteins from the endocytic pathway in bloodstream forms of Trypanosome brucei are modified by the addition of linear poly-N-acetyllactosamine side chains, which permits their isolation by tomato lectin affinity chromatography. Antibodies against this tomato lectin binding fraction were employed to screen a cDNA expression library from bloodstream forms of T. brucei. Two cDNAs were prominent among those selected. These cDNAs coded for two putative protein disulfide isomerases (PDIs) that respectively contained one and two double-cysteine redox-active sites and corresponded to a single domain PDI and a class 1 PDI. Assays of the purified recombinant proteins demonstrated that both proteins possess isomerase activity, but only the single domain PDI had a reducing activity. These PDIs possess a number of unusual features that distinguish them from previously characterized PDIs. The expression of both is developmentally regulated, they both co-localize with markers of the endocytic pathway, and both are modified by N-glycosylation. The larger PDI possesses N-glycans containing poly-N-acetyllactosamine, a modification that is indicative of processing in the Golgi and suggests the presence of a novel trafficking pathway for PDIs in trypanosomes. Although generally PDIs are considered essential, neither activity appeared to be essential for the growth of trypanosomes, at least in vitro.     

The poly-N-acetyllactosamines attached to lysosomal membrane glycoproteins are increased by the prolonged association with the Golgi complex.

The poly-N-acetyllactosamines on neutrophils and monocytes have been shown to serve as ligands for various selectins present on endothelial cells and platelets. We have previously shown that only a limited number of glycoproteins contain poly-N-acetyllactosamine and found that lysosomal membrane glycoproteins (lamps) are the major glycoproteins carrying poly-N-acetyllactosamine. In order to understand the reason why only certain glycoproteins can be modified by poly-N-acetyllactosamine, we have utilized 21 degrees C incubation conditions, which were previously shown to cause the accumulation of glycoproteins at the trans-Golgi. HL-60 cells were labeled with [3H]galactose at 21 or 37 degrees C for 6 or 24 h, and lamp-1 and lamp-2 were immunoprecipitated. Upon examination by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, each lamp from HL-60 cells incubated at 21 degrees C exhibited a much broader, slower migrating band than that isolated from the cells incubated at 37 degrees C. The number of N-glycans containing poly-N-acetyllactosamine, estimated by their binding to tomato lectin column, increased approximately 30-50% after incubation at 21 degrees C than incubation at 37 degrees C. The analysis of oligosaccharides released by endo-beta-galactosidase digestion demonstrates that the amount of side chains containing three or more N-acetyllactosamine repeats increased about 100% after incubation at 21 degrees C, and methylation analysis confirmed these results. The same analysis and the results obtained by ion-exchange chromatography also provided evidence that the N-glycans of lamps are sialylated at 21 degrees C as much as at 37 degrees C. Pulse-chase experiments using [35S]methionine labeling indicated that the time necessary for processing of lamps is much longer at 21 degrees C than at 37 degrees C. These results therefore indicate that incubation at 21 degrees C causes the lamps to reside longer within the Golgi complex, and such longer residence allows lamps to acquire more polylactosaminoglycan. These results also suggest that the time necessary for moving through the Golgi complex is a critical factor for poly-N-acetyllactosamine formation.     

Isolation and characterization of an N-acetylgalactosamine specific lectin from Salvia sclarea seeds

Crude extracts from Salvia sclarea seeds were known to contain a lectin which specifically agglutinates Tn erythrocytes (Bird, G. W. G., and Wingham, G. (1974) Vox Sang. 26, 163-166). We have purified the lectin to homogeneity by ion-exchange chromatography and affinity chromatography. The agglutinin was found to be a glycoprotein of Mr = 50,000, composed of two identical subunits of Mr = 35,000 linked together by disulfide bonds. The purified lectin agglutinates specifically Tn erythrocytes and, at higher concentrations, also Cad erythrocytes. Native A, B, or O red blood cells are not agglutinated by the lectin and, even after treatment with sialidase or papain, these cells are not recognized. Tn red cells present 1.45 X 10(6) accessible sites to the lectin which binds to these erythrocytes with an association constant of 1.8 X 10(6) M-1. On Cad red cells, 1.73 X 10(6) sites are accessible to the lectin which binds with an association constant of 1.0 X 10(6) M-1. The carbohydrate specificity of the S. sclarea lectin has been determined in detail, using well defined monosaccharide, oligosaccharide, and glycopeptide structures. The lectin was found to be specific for terminal N-acetylgalactosamine (GalNAc) residues. It binds preferentially alpha GalNAc determinants either linked to Ser or Thr (as in Tn structures) or linked in 1-3 to a beta GalNAc or to an unsubstituted beta Gal. Although more weakly, the lectin binds beta GalNAc residues linked in 1-4 to a beta Gal (as in Cad structures). It does not recognize beta GalNAc determinants linked in 1-3 to a Gal (as in globoside) or the alpha GalNAc residues of blood group A structures.     

Affinity of osteogenin, an extracellular bone matrix associated protein initiating bone differentiation, for concanavalin A

Subcutaneous implantation of demineralized bone matrix results in bone differentiation. The bone inductive protein, osteogenin, was isolated recently by heparin affinity chromatography. The affinity of osteogenin for various lectins was examined to attain further purification and characterization. Osteogenin extracted from bovine bone matrix binds to concanavalin A (Con A) but not to wheat germ agglutinin or soybean lectin. The present data indicate that the bone inductive protein, osteogenin, is a glycoprotein. The use of a Con A Sepharose affinity column followed by preparative gel electrophoresis resulted in a greater than 250,000 fold purification of osteogenin.     

Trypanosoma brucei glycoproteins contain novel giant poly-N-acetyllactosamine carbohydrate chains

The flagellar pocket of the bloodstream form of the African sleeping sickness parasite Trypanosoma brucei contains material that binds the beta-d-galactose-specific lectin ricin (Brickman, M. J., and Balber, A. E. (1990) J. Protozool. 37, 219-224). Glycoproteins were solubilized from bloodstream form T. brucei cells in 8 M urea and 3% SDS and purified by ricin affinity chromatography. Essentially all binding of ricin to these glycoproteins was abrogated by treatment with peptide N-glycosidase, showing that the ricin ligands are attached to glycoproteins via N-glycosidic linkages to asparagine residues. Glycans released by peptide N-glycosidase were resolved by Bio-Gel P-4 gel filtration into two fractions: a low molecular mass mannose-rich fraction and a high molecular mass galactose and N-acetylglucosamine-rich fraction. The latter fraction was further separated by high pH anion exchange chromatography and analyzed by gas chromatography mass spectrometry, one- and two-dimensional NMR, electrospray mass spectrometry, and methylation linkage analysis. The high molecular mass ricin-binding N-glycans are based on a conventional Manalpha1-3(Manalpha1-6)Manbeta1-4-GlcNAcbeta1-4GlcNAc core structure and contain poly-N-acetyllactosamine chains. A significant proportion of these structures are extremely large and of unusual structure. They contain an average of 54 N-acetyllactosamine (Galbeta1-4GlcNAc) repeats per glycan, linked mostly by -4GlcNAcbeta1-6Galbeta1-interrepeat linkages, with an average of one -4GlcNAcbeta1-3(-4GlcNAcbeta1-6)Galbeta1- branch point in every six repeats. These structures, which also bind tomato lectin, are twice the size reported for the largest mammalian poly-N-acetyllactosamine N-linked glycans and also differ in their preponderance of -4GlcNAcbeta1-6Galbeta1- over -4GlcNacbeta1-3Galbeta1- interrepeat linkages. Molecular modeling suggests that -4GlcNAcbeta1-6Galbeta1- interrepeat linkages produce relatively compact structures that may give these giant N-linked glycans unique physicochemical properties. Fluorescence microscopy using fluorescein isothiocyanatericin indicates that ricin ligands are located mainly in the flagellar pocket and in the endosomal/lysosomal system of the trypanosome.     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

Lectin-based affinity tag for one-step protein purification

The production of pure protein is indispensable for many applications in life sciences, however protein purification protocols are difficult to establish, and the experimental procedures are usually tedious and time-consuming. Therefore, a number of tags were developed to which proteins of interest can be fused and subsequently purified by affinity chromatography. We report here on a novel lectin-based affinity tag using the D-mannose-specific lectin LecB from Pseudomonas aeruginosa. A fusion protein was constructed consisting of yellow fluorescent protein and LecB separated by an enterokinase cleavage site. This protein was overexpressed in Escherichia coli Tuner (DE3), and the cell extract was loaded onto a column containing a mannose agarose matrix. Electrophoretically pure fusion protein at a yield of 24 mg/L culture was eluted with a D-mannose containing buffer The determination of equilibrium adsorption isotherms revealed an association constant of the lectin to the mannose agarose matrix of Ka = 3.26 x 10(5)/M. Enterokinase treatment of the purified fusion protein resulted in the complete removal of the LecB-tag. In conclusion, our results indicate that the lectin LecB of P. aeruginosa can be used as a tag for the high-yield one-step purification of recombinant proteins.     

Purification and cDNA cloning of UDP-GlcNAc:GlcNAcbeta1-3Galbeta1-4Glc(NAc)-R [GlcNAc to Gal]beta1,6N-acetylglucosaminyltransferase from rat small intestine: a major carrier of dIGnT activity in rat small intestine

A rat intestinal beta1,6N-acetylglucosaminyltransferase (beta1-6GnT) responsible for the formation of the beta1,6-branched poly-N-acetyllactosamine structure has been purified to apparent homogeneity by successive column chromatographic procedures using an assay wherein pyridylaminated lacto- N-triose II (GlcNAcbeta1-3Galbeta1-4Glc-PA) was used as an acceptor substrate and the reaction product was GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4Glc-PA. The purified enzyme catalyzed the conversion of the polylactosamine acceptor GlcNAcbeta1-3'LacNAc into GlcNAcbeta1-3'(GlcNAcbeta1-6') LacNAc (dIGnT activity), but it could not transfer GlcNAc to LacNAcbeta1-3'LacNAc (cIGnT activity). This enzyme could also convert mucin core 1 and core 3 analogs, Galbeta1-3GalNAcalpha1-O-paranitrophenyl (pNP) and GlcNAcbeta1-3GalNAcalpha1-O-pNP, into Galbeta1-3(GlcNAcbeta1-6) GalNAcalpha1-O-pNP (C2GnT activity) and GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-O-pNP (C4GnT activity), respectively. Based on the partial amino acid sequences of the purified protein, the cDNA encoding this enzyme was cloned. The COS-1 cells transiently transfected with this cDNA had high dI/C2/C4GnT activities in a ratio of 0.34:1.00:0.90, compared with non- or mock-transfected cells. The primary structure shows a significant homology with human and viral mucin-type core 2 beta1-6GnTs (C2GnT-Ms), indicating that this enzyme is the rat ortholog of human and viral C2GnT-Ms. This is the first identification and purification of this enzyme as a major carrier of dIGnT activity in the small intestine. This rat ortholog should mostly be responsible for making distal I-branch structures on poly-N-acetyllactosamine sequences in this tissue, as well as making mucin core 2 and core 4 structures, given that it also has high C2/C4GnT activities.     

Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica.

A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.     

Carbohydrate binding specificity of a beetle (Allomyrina dichotoma) lectin

Carbohydrate binding specificity of a lectin, allo A, isolated from a beetle (Allomyrina dichotoma), was investigated by means of lectin affinity chromatography. Sialylated complex-type and hybrid-type oligosaccharides/glycopeptides, and sialyllactose were retained by the column, whereas desialylated ones were retarded but not retained by the column. The association constants of allo A for biantennary oligosaccharides from human serum transferrin, determined by frontal analysis, were 8.0 X 10(5) M-1, 4.5 X 10(5) M-1, and 2.5 X 10(5) M-1 for disialo-, monosialo-, and asialo-oligosaccharides, respectively. Removal of the beta-galactose residues markedly reduced the association constant to 3.5 X 10(3) M-1. Furthermore, allo A was found to have no affinity for mucin-type glycopeptides carrying the sialylated Gal beta 1----3 GalNAc sugar sequence (Ka: 3.5 X 10(3) M-1). The results of this study indicated that allo A strongly binds to the trisaccharide structure, NeuAc alpha 2-3(6)Gal-beta 1-4GlcNAc, and that its binding potency is affected by the inner core structures of oligosaccharides and glycopeptides, because the presence of a bisecting N-acetyl-glucosamine residue and an alpha-fucose residue linked to the innermost N-acetylglucosamine residue reduced the association constants for oligosaccharides and glycopeptides.     

LAMP-1 in CHO cells is a primary carrier of poly-N-acetyllactosamine chains and is bound preferentially by a mammalian S-type lectin

Recent studies indicate that some mammalian S-type lectins bind preferentially to oligosaccharides containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1]n or poly-N-acetyllactosamine (PL) sequence. We report here our investigation on the distribution of these sequences in glycoproteins in Chinese hamster ovary (CHO) cells and the interaction of glycoproteins containing PL chains with an immobilized S-type lectin (L14) from calf heart tissue. Our results demonstrate that PL chains are carried by a few high molecular weight glycoproteins which are bound by tomato-lectin Sepharose and one of these was precipitated by antibody to LAMP-1 (a lysosomal-associated membrane glycoprotein). More importantly, these high molecular weight glycoproteins, including LAMP-1, were bound with high affinity by L14. These results indicate that mammalian S-type lectins are highly specific in their interactions with glycoproteins and that LAMPs carry important recognition sequences for these lectins.     

The canine renal parathyroid hormone receptor is a glycoprotein: characterization and partial purification

Covalent labeling of the canine renal parathyroid hormone receptor with [125I]bPTH(1-34) reveals several major binding components that display characteristics consistent with a physiologically relevant adenylate cyclase linked receptor. Through the use of the specific glycosidases neuraminidase and endoglycosidase F and affinity chromatography on lectin-agarose gels, we show here that the receptor is a glycoprotein that contains several complex N-linked carbohydrate chains consisting of terminal sialic acid and penultimate galactose in a beta 1,4 linkage to N-acetyl-D-glucosamine. No high mannose chains or O-linked glycans appear to be present. The peptide molecular weight of the deglycosylated labeled receptor is 62,000 [or 58,000 if the mass of bPTH(1-34) is excluded]. The binding of [125I]bPTH(1-34) to the receptor is inhibited in a dose-dependent fashion by wheat-germ agglutinin, but not by either succinylated wheat-germ agglutinin or Ricinus communis lectin, suggesting that terminal sialic acid may be involved in agonist binding. A combination of lectin affinity chromatography and immunoaffinity chromatography affords a 200-fold purification of the covalently labeled receptor.