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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.

The constitutively active receptor (CAR) is a crucial regulator of genes encoding for enzymes active in drug/steroid oxidation, conjugation, and transport. In our attempt to isolate the endogenous inhibitory ligand(s) for the mouse CAR, we found surprisingly that the inhibitory activity was associated with di- and tri-isopropylated phenyl phosphates that were present in livers of untreated mice. Trans-activation experiments in mammalian cells with synthetic compounds verified that mouse CAR was inhibited by various isopropylated phenyl phosphates (40-80%). Such triaryl phosphates are widely used as fire retardants, lubricants, and plasticizers, and some of them are known to disturb reproduction by currently unknown mechanisms. Equipped with the finding that these compounds could interact with mouse CAR, we proceeded to determine their functional effects on other nuclear receptors. Human CAR and pregnane X receptor (PXR) were variably activated (2-5-fold) by triaryl phosphates while mouse PXR, peroxisome proliferator-activated receptor-alpha, and vitamin D receptor were refractory. Among steroid hormone receptors, the human androgen receptor was inhibited by triphenyl phosphate and di-ortho-isopropylated phenyl phosphate (40-50%) and activated by di- and tri-para-substituted phenyl phosphates (2-fold). Our results add to the list of CAR and PXR activators and suggest steroid-dependent biological pathways that may contribute to the reproductive effects of triaryl phosphates.

The GalNAcbeta1-4GlcNAc determinant (LdN) occurs in some human and bovine glycoconjugates and also in lower vertebrates and invertebrates. It has been found in unsubstituted as well as terminally substituted forms at the distal end of conjugated glycans, but it has not been reported previously at truly internal positions of polylactosamine chains. Here, we describe enzyme-assisted conversion of LdNbeta1-OR oligosaccharides into GlcNAcbeta1-3GalNAcbeta1-4GlcNAcbeta1-OR. The extension reactions, catalyzed by human serum, were modeled after analogous beta3-GlcNAc transfer processes that generate GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-OR. The newly synthesized GlcNAcbeta1-3GalNAc linkages were unambiguously identified by nuclear magnetic resonance data, including the appropriate long-range correlations in heteronuclear multiple bond correlation spectra. The novel GlcNAcbeta1-3'LdN determinant proved to be a functional acceptor for several mammalian glycosyltransferases, suggesting that human polylactosamines may contain internal LdN units in many distinct forms. The GlcNAcbeta1-3'LdN determinant was unusually resistant toward jackbean beta-N-acetylhexosaminidase; the slow degradation should lead to a convenient method for the search of putative internal LdN determinants in natural polylactosamine chains.

Analysis of nucleotide sugar metabolism is essential in studying glycosylation in cells. Here we describe practical methods for both extraction of nucleotide sugars from cell lysates and for their analytical separation. Solid-phase extraction cartridges containing graphitized carbon can be used for the purification of nucleotide sugars by using triethylammonium acetate buffer as a ion-pairing reagent for decreasing retention. After that they are separated by high-performance liquid chromatography using a C18 reversed-phase column and the same ion-pairing reagent for increasing retention. These new sample preparation and analysis methods enable good separation of structurally similar sugar nucleotides, compatibility with rapid evaporative concentration, and possibility to automation. Monitoring the production of GDP-deoxyhexoses in genetically engineered yeast and native bacterial cells are described here as specific applications.

We have evaluated a newly introduced immunoturbidimetric transferrin receptor assay (IdeA TfR-IT, Orion Diagnostica, Finland) in healthy subjects and in a study population consisting of patients with rheumatoid arthritis and juvenile chronic arthritis. The IdeA TfR-IT assay was found to provide reproducible results which were in good agreement with the ELISA assays from Orion Diagnostica (IDeA-ELISA, correlation R2=0.8, n=102) and R&D systems (Quantikine TfR ELISA assay, correlation R2=0.95, n=39). The analysis of the patient samples suggested that, on the basis of serum transferrin receptor and ferritin concentrations, in approximately one third of patients with rheumatoid arthritis anemia is due to the depletion of iron stores. Apparently, in all patients with rheumatoid arthritis iron deficiency must be considered as a potential cause of the anemia. Now, that assays which are suitable for automated analyzers have become available for the measurement of serum transferrin receptor, this analyte has the potential to become a part of the routine evaluation of iron status.

Multifucosylated sialo-polylactosamines are known to be high affinity ligands for E-selectin. PSGL-1, the physiological ligand of P-selectin, is decorated in HL-60 cells by a sialylated and triply fucosylated polylactosamine that is believed to be of functional importance. Mimicking some of these saccharide structures, we have synthesized enzymatically a bivalent [sialyl diLex]-glycan, Neu5Acalpha2-3'Lexbeta1-3'Lexbeta1-3'(Neu5Acalpha2-3'Lexbeta1-3Lexbe ta1-6')LN [where Neu5Ac is N-acetylneuraminic acid, Lex is the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc and LN is the disaccharide Galbeta1-4GlcNAc]. Several structurally related, novel polylactosamine glycans were also constructed. The inhibitory effects of these glycans on two L-selectin-dependent, lymphocyte-to-endothelium adhesion processes of rats were analysed in ex-vivo Stamper-Woodruff binding assays. The IC50 value of the bivalent [sialyl diLex]-glycan at lymph node high endothelium was 50 nm, but at the capillaries of rejecting cardiac allografts it was only 5 nm. At both adhesion sites, the inhibition was completely dependent on the presence of fucose units on the sialylated LN units of the inhibitor saccharide. These data show that the bivalent [sialyl diLex]-glycan is a high affinity ligand for L-selectin, and may reduce extravasation of lymphocytes at sites of inflammation in vivo without severely endangering the normal recirculation of lymphocytes via lymph nodes.

The polylactosamine sLex beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc beta1-3'(sLex beta1-6')LacNAc (7) (where sLex is Neu5Ac alpha2-3Gal beta1-4(Fuc alpha1-3)GlcNAc and LacNAc is Gal beta1-4GlcNAc) is a nanomolar L-selectin antagonist and therefore a potential anti-inflammatory agent (Renkonen et al. (1997) Glycobiology, 7, 453). Here we describe an improved synthesis of 7. The octasaccharide LacNAc beta1-3'LacNAc beta1-3'LacNAc beta1-3'LacNAc (4) was converted into the triply branched undecasaccharide LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc beta1-3'(GlcNAc beta1-6')LacNAc (5) by incubation with UDP-GlcNAc and the midchain beta1,6-GlcNAc transferase activity of rat serum. Glycan 5 was enzymatically beta1,4-galactosylated to LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc beta1-3'(LacNAc beta1-6')LacNAc (6). Combined with the enzymatic conversion of 6 to 7 (Renkonen et al., loc. cit.) and the available chemical synthesis of 4, our data improve the availability of 7 for full assessment of its anti-inflammatory properties.

We find that the beta 1,6-GlcNAc transferase activity present in hog gastric mucosal microsomes converts the linear pentasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) in a site-specific way to the branch-bearing hexasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (2). The product is a positional isomer of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (3), reportedly formed from 1 by another polylactosamine beta 1,6-GlcNAc transferase activity present in human serum (Leppänen et al., Biochemistry, 30 (1991) 9287). Combined use of the two kinds of activities gave in the present experiments the heptasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), in which one of the branches occupies the position of the branch in 2 and the other the position of the branch in 3.

Lymphocyte infiltration is a hallmark of acute rejections in solid organ transplants, such as cardiac allograft. We have previously shown that lymphocyte extravasation to cardiac grafts undergoing rejection is largely due to interactions between lymphocyte L-selectin and its sialyl Lewis x (sLex) decorated ligands. Our previous work demonstrated further that an enzymatically synthetized tetravalent sLex glycan of a branched polylactosamine backbone is a highly efficient inhibitor of L-selectin-dependent lymphocyte adhesion to graft endothelium. To improve the availability of multivalent sLex glycans for anti-inflammatory indications, we now report enzymatic synthesis of another tetravalent sLex glycan that can be potentially produced on a large scale, and show that even the new saccharide is a nanomolar inhibitor of L-selectin-dependent lymphocyte adhesion. The novel antagonist is sLex beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc beta 1-3' (sLex beta 1-6') LacNAc (8) (where LacNAc is the disaccharide Gal beta 1-4GlcNac and sLex is the tetrasaccharide Neu5Ac alpha 2-3Gal beta 1-4 (Fuc alpha 1-3) GlcNAc). Its five-step synthesis was started from the octameric polylactosamine LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc beta 1-3' (GlcNAc beta 1-6') LacNAc (3), which in turn is accessible in one step from the hexasaccharide LacNAc beta 1-3'LacNAc beta 1-3'LacNAc. Importantly, the hexasaccharide primer has been synthesized chemically (Alais and Veyrieres, Tetrahedron Lett., 24, 5223, 1983). Hence, our data outline a route to glycan 8, consisting of a combination of chemical and enzymatic methods of oligosaccharide synthesis. In addition, our data show that polylactosamine backbones are able to present multiple sialyl Lewis x determinants to L-selectin in high-affinity mode, without a requirement for uniqueness in the backbone structure.

Lymphocyte homing is initiated by their tethering to and rolling on the high endothelium and is followed by extravasation into the lymph nodes. We show here that glycosylated cell adhesion molecule-1 (GlyCAM-1), CD34, and sialyl Lewis x (sLex) are present on rat lymph node high endothelium analyzed by using monoclonal antibodies. alpha (1,3)fucosyltransferase VII (Fuc-TVII), the last enzyme involved in the synthesis of the sLex sequence is also expressed on the rat lymph node high endothelium. We have synthesized a family of sLex-decorated oligosaccharide structures and used them to inhibit lymphocyte binding to high endothelium in the Stamper-Woodruff assay. Monovalent sLex, branched di- and tetravalent sLex, as well as a linear tetravalent sLex significantly reduce lymphocyte binding to endothelium. The branched and linear forms of tetravalent sLex were clearly superior inhibitors of the L-selectin-dependent lymphocyte adhesion, with IC50 values in low nanomolar range. In contrast, the fucose-free analogs having the same charge and approximately the same size as the corresponding sLex glycans had no effect on lymphocyte binding and served as negative controls. Taken together, these data show the crucial importance of sLex in the endothelial ligands for L-selectin. Furthermore, we suggest that L-selectin acts as an oligomer on the lymphocyte surface as it binds multivalent sLex glycans.

Kidney transplant rejection is an inflammatory process characterized by lymphocyte infiltration. Our earlier observations have shown that peritubular capillary endothelium (PTCE) is the site of lymphocyte entry into the rejecting renal allograft. During rejection, PTCE begins to express sialyl Lewis x de novo, and binds lymphocytes by a mechanism largely dependent on L-selectin. Hence, inhibiting the lymphocyte-endothelial interaction with oligosaccharide ligands of L-selectin offers an attractive possibility to prevent the inflammation and rejection. Here, we report enzyme-assisted synthesis of N-acetyllactosamine-based tetra-, deca-, and docosameric saccharides carrying one, two or four distally located sialyl Lewis x groups [Neu-NAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc] (sLex), respectively. When tested for their ability to inhibit lymphocyte-endothelial interaction during rat kidney transplant rejection, all sLex-saccharides were inhibitors in the Stamper-Woodruff binding assays; the analogues lacking fucose showed no inhibitory potency. The tetravalent sLex glycan proved to be a high-affinity adhesion inhibitor with an IC50 < 50 nM. While less powerful than the tetravalent glycan, also the divalent sLex saccharide was a much better inhibitor than the monovalent glycan. Hence, increasing multivalency and, possibly, increasing chain length of the polylactosamine backbone, enhances the inhibitory potency of sLex bearing glycans in the lymphocyte-endothelial adhesion assay. This suggests that L-selectin behaves as a "functional oligomer" on lymphocyte surfaces.

The recognition of cell-surface L-selectin by its carbohydrate ligands causes lymphocytes to roll on capillary endothelium at sites of inflammation. As this primary contact is a prerequisite for extravasation of the leukocytes to the tissue, its inhibition by free oligosaccharides capable of competing with the natural L-selectin ligands in an attractive therapeutic possibility. The exact structures of the biological ligands of L-selectin are not yet known, but the principal carbohydrate epitopes share some structural features: they are O-glycosidically linked mucin-type oligosaccharides with N-acetyllactosamine backbone, which is 3'-sialylated or 3'-sulfated, 3-fucosylated and sometimes 6- or 6'-sulfated at the distal N-acetyllactosamine termini. Multivalency of the ligand, which is believed to enhance the binding, is achieved by a branched polylactosamine backbone or by a clustered array of O-glycans. We report here enzymic synthesis of a large oligosaccharide fulfilling several of the features characteristic to the L-selectin ligands: it is a dodecameric O-glycosidic core-2-type oligosaccharide alditol with a branched polylactosamine backbone carrying two distal alpha-2,3'-sialylated and alpha-1,3-fucosylated N-acetyl-lactosamine groups (sialyl Lewis x, sialyl Le(x)). The structure of each saccharide on the synthesis route from disaccharide Gal beta 1-3GalNAc to the dodecasaccharide alditol was established by several methods including one- and two-dimensional 1H-NMR spectroscopy. The last step of the synthesis, the alpha-1,3-fucosylation of the 6-linked arm proceeded sluggishly, and was associated with a noticeable shift in H1 resonance of the GlcNAc residue of the branch-bearing N-acetyllactosamine unit. The final synthesis product and its analogs lacking one or both of the fucose residues were tested as inhibitors of L-selectin-mediated lymphocyte-endothelium interaction in vitro in rejecting rat kidney transplant. While the non-fucosylated O-glycosidic oligosaccharide alditol did not possess any inhibitory activity, the mono-fucosylated one (i.e. monovalent sialyl Le(x)) prevented the binding significantly and the difucosylated dodecasaccharide alditol (i.e. divalent sialyl Le(x)) was a very potent inhibitor (IC50, inhibitory concentration preventing 50% of binding = 0.15 microM). Besides the multivalency, also the Gal beta 1-3GalNAc-ol sequence of the O-glycosidic core appeared to increase the affinity of the glycan to L-selectin. This was indicated by parallel inhibition experiments, where a disialylated and difucosylated branched polylactosamine decasaccharide, similar to the divalent dodecasaccharide alditol, but lacking the reduced O-glycosidic core, was a less effective inhibitor (IC50 = 0.5 microM) than the O-glycosidic dodecasaccharide alditol.

Proposing to study the molecular mechanisms of mouse gamete adhesion with the aid of high affinity adhesion inhibitors of saccharide nature, we report here the enzymatic synthesis of a bivalent oligosaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (4), consisting of two long arms that link together two distal alpha 1,3-galactose residues. Binding data reported elsewhere (E. Litscher et al., Biochemistry, 1995, 34, 4662-4669) show that 4 is a high affinity inhibitor of mouse gamete adhesion in vitro (IC50 = 9 microM), while a related octasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, consisting of two short arms is of very low inhibitory activity. The data highlight the importance of the two alpha-galactose residues of 4, and the length of the sugar chains joining them.

During fertilization in mice, free-swimming sperm bind to mZP3, an 83-kDa glycoprotein present in the egg extracellular coat, the zona pellucida [Wassarman, P. M. (1990) Development 108, 1-17]. Mouse sperm recognize and bind to a specific class of serine/threonine-linked (O-linked) oligosaccharides present on mZP3. After binding to mZP3, sperm undergo a form of cellular exocytosis, the acrosome reaction, thereby enabling them to penetrate the zona pellucida and fertilize the egg. Thus, gamete interactions in mice are carbohydrate-mediated. In this context, we tested 15 O-linked-related oligosaccharide constructs with defined structures for their ability to inhibit binding of mouse sperm to ovulated eggs and to induce sperm to undergo the acrosome reaction in vitro. Thirteen of the oligosaccharides were constructed and characterized in our laboratory [Seppo, A., Pentillä, L., Niemelä, R., Maaheimo, H., Renkonen, O., & Keane, A. (1995) Biochemistry 34, 4655-4661]; two were obtained commercially. We found that, while none of the oligosaccharides induced sperm to undergo the acrosome reaction, a few of them inhibited binding of sperm to eggs at relatively low concentrations (ID50 < 5 microM). In certain cases, sperm formed head-to-head aggregates in the presence of the oligosaccharides. The results suggest that the ability of oligosaccharides to inhibit binding of sperm to eggs is dependent on several parameters, including the size and branching pattern of the oligosaccharide, as well as on the nature of the sugar residue at the nonreducing end of the oligosaccharide.

Radiolabeled oligosaccharide constructs were prepared to evaluate carbohydrate determinants involved in gamete adhesion in mice. The octasaccharide primer GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc (1) was incubated with UDP-GlcNAc and beta 1,6-GlcNAc-transferase of hog gastric microsomes, producing the tetraantennary decasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (2). The decasaccharide was then incubated with UDP-Gal and beta 1,4-galactosyltransferase from bovine milk, yielding the tetradecasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (3). Incubation of the tetradecasaccharide 3 with UDP-Gal and alpha 1,3-galactosyltransferase from bovine thymus gave the octadecameric glycan Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-3[Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4GlcNAc (4).(ABSTRACT TRUNCATED AT 250 WORDS)

Radiolabelled lacto-N-neohexaose was fucosylated with partially purified alpha (1,3)fucosyltransferase(s) from human milk. Structural analysis of the monofucosylated products obtained at an early stage of the reaction revealed that both distal branches of the acceptor had reacted equally well, generating Lewis x determinants, while the reducing end glucose had not reacted. The two isomeric Lewis x glycans were readily separated from each other by chromatography on immobilized wheat germ agglutinin (WGA), because alpha (1,3)fucosylation of the (1 --> 6)-linked branch of lacto-N-neohexaose was associated with a dramatic loss of WGA affinity. The fucosylation mixture of lacto-N-neohexaose also contained a difucosylderivative that carried Lewis x determinants at both distal branches. Attempted refucosylation of this octasaccharide failed to transfer fucose to the glucose unit.

We have constructed by enzyme-aided in vitro synthesis a hexasaccharide alditol Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3) GalNAc-ol and shown that it resists the action of endo-beta-galactosidase from Bacteroides fragilis under conditions where a related pentasaccharide alditol, GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-6(Gal beta 1-3)GalNAc-ol, was completely cleaved. Together with earlier results from this laboratory, our present data imply that endo-beta-galactosidase from B. fragilis, apparently, can be used to distinguish between GlcNAc beta 1-6Gal and GlcNAc beta 1-3Gal units within linear backbone sequences of all known types of oligo-(N-acetyllactosamino)glycans.

alpha 1,3-Galactosylation of radiolabelled bi-antennary acceptors Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal-R (R = 1-OH, beta 1-4GlcNAc or beta 1-4Glc) with bovine thymus alpha 1,3-galactosyltransferase was studied. At all stages of the reactions the three acceptors reacted faster at the 1-->6 linked arm than at the 1-->3 linked branch. Hence, in addition to the doubly alpha 1,3-galactosylated products, practically pure Gal beta 1-4GlcNAc beta 1-3(Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-6)Gal-R could be obtained from the three acceptors in reactions that had proceeded to near completion. The isomeric mono-alpha 1,3-galactosylated products were identified by using exoglycosidases to remove the branches unprotected by alpha 1,3-galactoses and by subsequently identifying the resulting linear glycans chromatographically.

Hog gastric mucosal microsomes contain beta-N-acetylglucosaminidase activity which cleaves GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc at the terminal GlcNAc beta 1-3Gal linkage faster than at the GlcNAc beta 1-6Gal bond, producing mainly GlcNAc beta 1-6Gal beta 1-4GlcNAc. In a marked contrast, GlcNAc beta 1-3(GlcNAc beta 1-6)Gal is cleaved primarily at the GlcNAc beta 1-6Gal bond, while partial hydrolysis of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc reveals similar rates of cleavage for the (1-3) and (1-6) linkages. Our data support the notion that the terminal beta 1,6-linked GlcNAc unit of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc may interact with the reducing end GlcNAc unit intramolecularly in water solution.

Partial reactions catalyzed by a (1----3)-N-acetyl-beta-D- glucosaminyltransferase (EC2.4.1.149), known to be present in human serum, were studied by use of biantennary "backbone" saccharides of oligo-N-acetyllactosamine-type as acceptors. Incubation of the radiolabeled blood-group I-active hexasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp- (1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-GlcNAc (1) and UDP-GlcNAc with serum gave first a transient 1:1 mixture of two isomeric heptasaccharides, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D- GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D- Galp-(1----4)-D-GlcNAc (2) and beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----3)- beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Galp-(1----4)-D-Glc NAc (3), showing that both branches of 1 react equally well. The two heptasaccharides reacted further in the incubation mixture to form the radiolabeled octasaccharide, beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[be ta-D- GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----6)]-beta-D-Ga lp- (1----4)-D-GlcNAc (4); during this second reaction, the composition of the heptasaccharide mixture remained unchanged, indicating that 2 and 3 reacted at approximately equal rates. The heptasaccharides 2 and 3 could not be separated from each other, but they could be detected, identified, and quantitatively determined by stepwise enzymic degradations. Partial (1----3)-N-acetyl-beta-D-glucosaminylation reactions, carried out with another acceptor, the branched pentasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-[beta-D-Galp-(1----4)-beta- D- GlcpNAc-(1----6)]-beta-D-Gal (11), revealed that it reacted also equally well at both branches.(ABSTRACT TRUNCATED AT 400 WORDS)

Four radiolabeled pentasaccharides, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4Glc, and Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc, were prepared in virtually pure form. They were obtained by partial enzymic beta 1,4-galactosylations of the appropriate tetrasaccharide acceptors or by partial enzymic degalactosylations of the appropriate hexasaccharides, followed by paper chromatographic separations. All four pentasaccharides contain two nonidentical distal branches, making them valuable primers for enzymatic in vitro synthesis of larger oligo(N-acetyllactosaminoglycans).

Incubation of UDP-GlcNAc and radiolabeled GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (1) with human serum resulted in the formation of the branched hexasaccharide GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (2) in yields of up to 22.2%. The novel reaction represents midchain branching of the linear acceptor; the previously known branching reactions of oligo-(N-acetyllactosaminoglycans) involve the nonreducing end of the growing saccharide chains. The structure of 2 was established by use of appropriate isotopic isomers of it for degradative experiments. The hexasaccharide 2 was cleaved by an exhaustive treatment with jack bean beta-N-acetylhexosaminidase, liberating two GlcNAc units and the tetrasaccharide Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (3). Endo-beta-galactosidase from Bacteroides fragilis cleaved 2 at one site only, yielding the disaccharide GlcNAc beta 1-3Gal (4) and the branched tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (5). The structure of 5 was established by partial acid hydrolysis and subsequent identification of the disaccharide GlcNAc beta 1-6Gal (6), together with the trisaccharides GlcNAc beta 1-6Gal beta 1-4GlcNAc (7) and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (8) among the cleavage products. Galactosylation of 2 with bovine milk beta 1,4-galactosyltransferase and UDP-[6-3H]Gal gave the octasaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3 Gal beta 1-4GlcNAc beta 1-3([6-3H]-Gal beta 1-4GlcNAc beta 1-6)[U-14C] Gal beta 1-4GlcNAc (17), which could be cleaved with endo-beta-galactosidase into the trisaccharide [6-3H]Gal beta 1-4GlcNAc beta 1-3Gal (18) and the branched pentasaccharide GlcNAc beta 1-3-([6-3H]Gal beta 1-4GlcNAc beta 1-6) [U-14C]Gal beta 1-4GlcNAc (19). Partial hydrolysis of 2 with jack-bean beta-N-acetylhexosaminidase gave the linear pentasaccharide 1 and the branched pentasaccharide Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (20). The serum beta 1,6-GlcNAc transferase catalyzed also the formation of GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (11) from UDP-GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc (10). The pentasaccharide Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc (16), too, served as an acceptor for the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Endo-beta-galactosidase (EC 3.2.1.103) of Bacteroides fragilis, at 250 mU ml-1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc beta 1-6Gal beta 1-4GlcNAc, or those of tetrasaccharides Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc and Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4Glc. The isomeric glycans which contained the GlcNAc beta 1-3Gal beta 1-4GlcNAc/Glc sequence were readily cleaved.

Relative affinities of several fucosylated and nonfucosylated oligo-N-acetyllactosaminoglycans for immobilized wheat germ agglutinin (WGA) were studied using a chromatographic technique. alpha(1-3) Fucosylation of the N-acetylglucosamine unit(s) in mono- and biantennary saccharides of the Gal beta 1-4GlcNAc-R type strongly reduced the WGA-affinity. In contrast, alpha(1-2) fucosylation of the nonreducing galactose unit(s) of the saccharides did not reduce the affinity.

Radiolabelled GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (1), GlcNAc beta 1-3)GlcNAc beta 1-6)Gal beta 1-OCH3 (4), GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (7), and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (10) were cleaved partially with jack bean beta-N-acetylhexosaminidase (EC 3.2.1.30), and the digests were analysed chromatographically. All four oligosaccharides were hydrolysed faster at the (1-6) branch, than at the (1-3) branch, but a high branch specificity was observed only with the glycan 4. The saccharides 1 and 7 resembled each other in the kinetics of the enzyme-catalysed release of their two non-reducing N-acetylglucosamine units, but the glycan 10 was rather different. The partial digestions made it possible to obtain radiolabelled GlcNAc beta 1-6Gal, GlcNAc beta 1-6Gal beta 1-OCH3, GlcNAc beta 1-6Gal beta 1-4Glc, and, in particular, GlcNAc beta 1-6Gal beta 1-4GlcNAc.

Affinity chromatography of unreduced oligosaccharides on a small column of immobilized wheat germ agglutinin (WGA) revealed high-binding affinities for several radiolabeled molecules containing at the reducing end either beta-D-GlcpNAc-(1----6)-D-Gal, beta-D-GlcpNAc-(1----6)-beta- D-Galp-(1----4)-D-GlcNAc, beta-D-GlcpNAc-(1----6)-beta-D-Galp-(1----4)DGlc, D-GlcpNAc-(1----3)-[beta-D-GlcpNAc-(1----6)]-D-Gal, beta-D-GlcpNAc-(1----6)- D-GalNAc, or beta-D-Galp-(1----3)-[beta-D-GlcpNAc-(1----6)]-D-GalNAc sequences. Reduction changed the binding affinities remarkably: The sequences carrying a D-galactose or 2-acetamido-2-deoxy-D-galactose residue at the reducing end lost most of their affinities, but the sequences containing a D-glucose or 2-acetamido-2-deoxy-D-glucose residue at the reducing end gained additional affinity upon reduction. These findings emphasize the role of the unreduced, 6-o-substituted D-galactose and 2-acetamido-2-deoxy-D-galactose residues for the binding of saccharides to WGA, which has been recognized previously as a lectin specific for oligosaccharides containing a 2-acetamido-2-deoxy-D-glucose or sialic acid unit. The results suggested also that WGA-agarose chromatography of alditols may become a valuable method for the fractionation of oligo-N-acetyllactosaminoglycans and related saccharides.

The branch specificity of Escherichia coli beta-galactosidase (EC 3.2.1.23) was studied by analyzing the cleavage of the branched hexasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc (1). This hexasaccharide was cleaved to pentasaccharides Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (3) and GlcNAc beta 1-3(Gal-beta 1-4GlcNAc beta 1-6) [14C(U)]Gal beta 1-4GlcNAc (4) without any appreciable branch specificity. Even the further conversions of the pentasaccharides 3 and 4 into the tetrasaccharide GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc seemed to proceed at similar rates, without any appreciable branch specificity. In marked contrast to the hexasaccharide 1, the pentasaccharide Gal beta 1-4GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)[14C(U)]Gal (2), missing the reducing end GlcNAc, is known to be cleaved selectively at the 6-branch; this finding was confirmed in the present study. The different behaviour of hexasaccharide 1 and pentasaccharide 2 reflects differences in the reactivity of their 6-branches; the preferred conformations of these closely related molecules may be quite different.

GlcNAc beta 1-3(GlcNAc beta 1-6) [14C(U)]Gal and GlcNAc beta 1-3(GlcNAc beta 1-6)[14C(U)]Gal beta 1-4GlcNAc were prepared by in vitro synthesis. They were characterized by enzymatic sequencing, by partial acid hydrolysis, and by periodate oxidation experiments. The two saccharides were isolated also from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine embryonal carcinoma cells (line PC 13). The tetrasaccharide was retarded in a column of agarose-linked wheat germ agglutinin; the trisaccharide was strongly bound. Chromatography in this column separated the trisaccharide into two distinct peaks, which represented interconvertible molecules. Together with our previous data on linear teratocarcinoma saccharides, these findings show that affinity chromatography with immobilized wheat germ agglutinin can be advantageously used in fractionating radiolabeled oligo-N-acetyllactosaminoglycans and saccharides related to them.

A novel linear tetrasaccharide, Gal beta 1-4GlcNAc beta 1-6Gal beta 1-4GlcNAc, was isolated from partial acid hydrolysates of metabolically labeled poly-N-acetyllactosaminoglycans of murine teratocarcinoma cells. It was characterized by exo-glycosidase sequencing and by mild acid hydrolysis followed by identification of all partial cleavage products. The tetrasaccharide, and likewise labelled GlcNAc beta 1-6Gal beta 1-4GlcNAc, resisted the action of endo-beta-galactosidase (EC 3.2.1.103) from E. freundii at a concentration of 125 mU/ml, while the isomeric, radioactive teratocarcinoma saccharides Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4GlcNAc and GlcNAc beta 1-3Gal beta 1-4GlcNAc were cleaved in the expected manner.

A spoilage organism isolated from turbid beer is described. The bacterium was gram negative, catalase negative, strictly anaerobic, and rod shaped, having flagella only on one side of the cell. The main metabolic product was propionic acid. In addition acetic, succinic, and lactic acids and acetoin were formed. Malonate inhibited the production of propionic acid by the strain studied and by both Pectinatus and Propionibacterium strains. The guanine-plus-cytosine content of deoxyribonucleic acid was 36 mol%. Differences between this strain and Pectinatus strains were 2 to 5 percentage points. Immunofluorescent staining and gel diffusion precipitin tests revealed that the antigenic structure differed from those of Pectinatus strains. The isolated organism can, despite some differences, be regarded as belonging to the genus Pectinatus.

Five pure oligosaccharides derived from poly-N-acetyllactosaminoglycans of teratocarcinoma cells were chromatographed on immobilized wheat germ agglutinin (WGA). Three of them, Gal beta 1-4GlcNAc, GlcNAc beta 1-3Gal, and GlcNAc beta 1-3Gal beta 1-4GlcNAc, revealed only weak binding, but GlcNAc beta 1-6Gal beta 1-4GlcNAc was bound moderately and GlcNAc beta 1-6Gal was bound quite strongly. The differences in the binding affinities were large enough to allow chromatographic separation of the five oligosaccharides into three distinct fractions in a small column of WGA-agarose.