Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico , in vitro and in vivo Sabine Andre ´ 1 , Carlo Unverzagt 2, *, Shuji Kojima 3 , Martin Frank 4 , Joachim Seifert 2, †, Christian Fink 5 , Klaus Kayser 6 , Claus-Wilhelm von der Lieth 4 and Hans-Joachim Gabius 1 1 Institut fu ¨ r Physiologische Chemie, Tiera ¨ rztliche Fakulta ¨ t, Ludwig-Maximilians-Universita ¨ tMu ¨ nchen, Germany; 2 Institut fu ¨ r Organische Chemie und Biochemie, Technische Universita ¨ tMu ¨ nchen, Garching, Germany; 3 Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan; 4 Zentrale Spektroskopie, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 5 Radiologische Diagnostik und Therapie, Deutsches Krebsforschungszentrum, Heidelberg, Germany; 6 Institut fu ¨ r Pathologie, Charite ´ , Humboldt Universita ¨ t, Berlin, Germany We have investigated the consequences of introducing a bisecting GlcNAc moiety into biantennary N-glycans. Computational analysis of glycan conformation with pro- longed simulation periods in vacuo and in a solvent box revealed two main effects: backfolding of the a1–6 arm and stacking of the bisecting GlcNAc and the neighboring Man/ GlcNAc residues of both antennae. Chemoenzymatic syn- thesis produced the bisecting biantennary decasaccharide N-glycan and its a2–3(6)-sialylated variants. They were conjugated to BSA to probe the ligand properties of N-glycans with bisecting GlcNAc. To assess affinity altera- tions in glycan binding to receptors, testing was performed with purified lectins, cultured cells, tissue sections and ani- mals. The panel of lectins, including an adhesion/growth- regulatory galectin, revealed up to a sixfold difference in affinity constants for these neoglycoproteins relative to data on the unsubstituted glycans reported previously [Andre ´ ,S., Unverzagt,C.,Kojima,S.,Dong,X.,Fink,C.,Kayser,K.& Gabius, H J. (1997) Bioconjugate Chem. 8, 845–855]. The enhanced affinity for galectin-1 is in accord with the increased percentage of cell positivity in cytofluorimetric and histochemical analysis of carbohydrate-dependent binding of labeled neoglycoproteins to cultured tumor cells and routinely processed lung cancer sections. Intravenous injec- tion of iodinated neoglycoproteins carrying galactose-ter- minated N-glycans into mice revealed the highest uptake in liver and spleen for the bisecting compound compared with the unsubstituted or core-fucosylated N-glycans. Thus, this substitution modulates ligand properties in interactions with lectins, a key finding of this report. Synthetic glycan tailoring provides a versatile approach to the preparation of newly substituted glycans with favorable ligand properties for medical applications. Keywords: bisecting GlcNAc; drug targeting; lectin; neo- glycoprotein; tumor imaging. A major challenge in the post-genomic era is the functional analysis of post-translational protein modifica- tions leading to medical applications. With about two thirds of eukaryotic protein sequences reported to harbor the N-glycosylation sequon, this type of modification is typical of membrane and secretory proteins [1]. The complex enzymatic machinery located in the endoplasmic reticulum and Golgi network, representing a notable investment in terms of genomic coding capacity, is known to produce a large variety of N-glycans [2,3]. These two aspects, i.e. frequent protein glycosylation and large panel of glycosyltransferases, suggest a nonrandom, albeit not template-driven, synthesis with a strong impact on cellular activities [3,4]. The glycan chains produced, referred to as the glycomic profile, reflect cellular parameters such as differentiation and disease processes [3,5]. Current efforts are directed to relating distinct glycan sequence motifs to key effector mechanisms at the cellular level. In this context our study focuses on defining the role of the bisecting GlcNAc residue. A key regulatory step in N-glycan processing is the addition of a b1–4-linked GlcNAc residue to the central b-mannose unit of the core pentasaccharide. This reaction is catalyzed by N-acetylglucosaminyltransferase III (EC 2.4.1.144; GnT-III) placing this GlcNAc residue (Fig. 1, bottom panel) between the a1–3 and a1–6 arms, Correspondence to S. Andre ´ , Institut fu ¨ r Physiologische Chemie, Tiera ¨ rztliche Fakulta ¨ t, Ludwig-Maximilians-Universita ¨ tMu ¨ nchen, Veterina ¨ rstr. 13, 80539 Mu ¨ nchen, Germany. Fax: + 49 89 2180 2508, Tel.: + 49 89 2180 2290, E-mail: Sabine.Andre@lmu.de Abbreviations: E-PHA, erythroagglutinating phytohemagglutinin; GnT-III, N-acetylglucosaminyltransferase III; MD, molecular dynamics. Enzymes: N-acetylglucosaminyltransferase III (EC 2.4.1.144; GnT-III). *Present address: Bioorganische Chemie, Geba ¨ ude NW1, Universita ¨ t Bayreuth, 95440 Bayreuth, Germany. Present address: 3 Hi-Tech Court, Brisbane Technology Park, Eight Mile Plains, Brisbane, QLD 4113, Australia. Dedication: dedicated to and in thankful commemoration of Professor F. Cramer who died recently three months before his 80th birthday. (Received 25 September 2003, revised 28 October 2003, accepted 6 November 2003) Eur. J. Biochem. 271, 118–134 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03910.x which extend from the core pentasaccharide [2]. The introduction of a bisecting GlcNAc residue will probably perturb conformational aspects proximal and also distal to its position in the glycan chain. Evidence has been provided for a shift of the conformational equilibrium of the torsional angle x of the C5–C6 bond of the a1–6 linkage between the two positions around 180 ° and )60 ° to the 180 ° position for the biantennary complex-type N-glycan and to the )60 ° position for the hybrid-type N-glycan [6–11]. Next, in the presence of chain elongation beyond the core pentasaccha- ride (but not in its absence), the a1–3 linkage becomes considerably more restrained than in the N-glycan without a bisecting GlcNAc unit [12–14]. In summary, these reports of NMR experiments and molecular modeling intimate that the bisecting GlcNAc acts like a wedge with implications for the shape of the N-glycan. The occurrence of distinct alterations in shape induced by this substitution prompted us to examine the ligand properties of the substituted N-glycans in detail. Undoubtedly, this work would gain substantial relevance and importance, if biological effects were known associated with cellular expression of this substitution. Indeed, the second premise for our study was provided by respective reports. The applied methods included up-regulation of GnT-III activity, studies on normal or tumor cells with natural or increased GnT-III activity, ectopic GnT-III expression, and generation of GnT-III- deficient mice. In detail, the mutant phenotype of LEC10 CHO cells displaying resistance to ricin was attributed to induction of GnT-III activity [15,16]. Transcriptional up- regulation of GnT-III gene expression by forskolin and thus increased production of N-glycans with bisecting GlcNAc led to the decreased cell surface presence of lysosomal- associated membrane glycoprotein-1 and c-glutamyltrans- peptidase [17]. This exemplary result links the bisecting motif to intracellular routing. Regarding tumors, GnT-III was markedly increased in the preneoplastic stage of rat (but not mouse) liver carcinogenesis, the blast crisis of chronic myelogenous leukemia, and pediatric brain tumors [18]. The bisecting GlcNAc then makes its presence felt in tumor progression. This process can apparently be influenced nonuniformly, as attested by analysis of different cell types. GnT-III overexpression yielded suppression of lung meta- stasis for mouse B16 melanoma cells, increased adhesivity Fig. 1. Neoglycoproteins with BSA as carrier for the test panel of six complex-type bianten- nary N-glycans. The upper part shows the structures of the biantennary nonasaccharide (Bi9) and its a2–6(3)-sialylated undecasac- charides (Bi1126, Bi1123). The bottom panel illustrates the corresponding decasaccharides and dodecasaccharides with bisecting Glc- NAc. For expample, the abbreviation BiB1226-BSA stands for biantennary bisecting N-glycan 12-mer a2–6-sialylated BSA conju- gate. The linker arm and the attachment point to an e-amino group of lysine of the carrier protein are also shown. Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 119 and decreased migration activity was observed for human U373 glioma cells bearing mutual overexpression of GnT- III relative to GnT-V (shift from biantennary N-glycans with bisecting GlcNAc to highly branched N-glycans), and GnT-III-overexpressing human K562 erythroleukemic cells rather effectively colonized the spleen in athymic mice with acquisition of resistance to NK cell cytotoxicity [19–21]. Moreover, hemopoiesis supported by bone marrow stroma requires, in part, integrity of N-glycans, and hemopoietic dysfunction with lowered production of nonadherent cells in transgenic mice with GnT-III overexpression demonstrates that respective changes in the N-glycan profile account for suppression of proliferation and differentiation of hemopoi- etic cells [22,23]. Altered glycosylation appears to translate into cellular responses via biochemical pathways. Apart from having a bearing on the protein part’s folding or access to binding partners, the capacity of protein-bound glycan chains to Ôimpart a discrete recognitional role to the proteinÕ [24] deserves attention in this respect. Explicitly, the bisecting GlcNAc has potential either to be directly engaged in intermolecular interactions with receptors, e.g. endogenous lectins [4,25–30], or to modulate binding processes at other sites in the chain through the shape alterations induced by its presence. That bisecting GlcNAc constitutes a docking point for protein–carbohydrate interactions is known from the plant lectin erythroagglutinating phytohemagglutinin (E-PHA), whereas the presence of a bisecting GlcNAc in a core-fucosylated biantennary N-glycan did not affect bind- ing to the L -fucose-specific lectin from Aleuria aurantia [31–35]. Annexin V, a cell surface marker of apoptotic cells, has recently been reported to share this property with the plant lectin E-PHA [36]. To determine if and to what extent ligand properties are conveyed and/or affected by this natural substitution, and then to devise routes for potential medical application, we combined synthetic, biochemical, cell biological, histopatho- logical and in vivo procedures. The question of whether introduction of the bisecting GlcNAc changes the ligand properties (binding affinities) of biantennary N-glycans for branch-end-specific lectins as well as for cells and organs has so far not been tackled. To this end, it is desirable to obtain pure test substances, i.e. N-glycans free of natural micro- heterogeneity, by a convenient preparative route. After developing a chemoenzymatic synthesis for biantennary N-glycans with this distinct substitution, the preparation of the corresponding glycan-bearing neoglycoproteins made the required tools available. In a previous study, we prepared neoglycoproteins carrying unsubstituted biantennary N-glycans [37]. These compounds were examined for their properties in enzyme-linked lectin and cell binding assays, histopathological monitoring, and organ biodistribution [37]. The utility of our combined approach and the validity of the concept were then tested by assessing the properties of N-glycans modified with a1–6 core fucose. Changes in cell biological parameters could be attributed to the N-glycan modification [38]. Pursuing this line of research, we report results obtained with neoglycoproteins carrying biantennary N-glycans with the bisecting Glc- NAc motif. The chemoenzymatic synthesis of bisecting N-glycans with terminal galactose or sialic acid residues is presented followed by the generation of neoglycoproteins. To take advantage of the enormous strides made over recent years in acquiring, storing and handling increasing quantities of data from molecular modeling calculations, we devoted part of the study to scrutinizing the conformational properties of the substituted and unsub- stituted N-glycans. Their behavior patterns were compar- atively analyzed by refined computational protocols in vacuo and, compared with previous calculations, in the presence of a defined solvent. The simulation periods were significantly extended. Experimentally, the ligand characteristics of the N-glycans as part of the neoglyco- proteins (Fig. 1) were evaluated in biochemical, cell biological and histopathological assays including deter- mination of biodistribution in vivo relative to the control compounds (complex-type biantennary N-glycans lacking bisecting GlcNAc). It is shown that the bisecting substi- tution triggers alteration of ligand properties, implying functional significance of the bisecting GlcNAc as a modulator of N-glycan biorecognition. Materials and methods Synthetic and analytical procedures NMR spectra were recorded with a Bruker AMX 500 spectrometer. HPLC separations were performed on a Pharmacia LKB gradient system 2249 equipped with a Pharmacia LKB Detector VWM 2141 (Freiburg, Germany). For size-exclusion chromatography, a Pharmacia Hi Load Superdex 30 column (600 · 16 mm) was used. RP-HPLC was performed on a Macherey-Nagel Nucleogel RP 100-10 column (300 · 25 mm). BSA, b1–4-galactosyltransferase, a2–6-sialyltransferase and nucleotide sugars were purchased from Sigma (Munich, Germany), and alkaline phosphatase (calf intestine, molecular biology grade; EC 3.1.3.1) from Roche Diagnostics (Heidelberg, Germany). We are grateful to J. C. Paulson (Cytel Corp., San Diego, USA) for a sample of recombinant a2–3-sialyltransferase. ESI mass spectra were recorded on a Finnigan TSQ 700 in methanol/water. MALDI-TOF mass spectra were collected by D. Renauer at the Boehringer Mannheim research facility (Tutzing, Ger- many) on a Voyager Biospectrometry workstation (Vestec/ Perseptive) MALDI-TOF mass spectrometer, using 2,5- dihydroxybenzoic acid as a matrix. The structures of the synthetic N-glycans were invariably confirmed by the following 2D NMR experiments: TOCSY, NOESY, HMQC (heteronuclear multiple-quantum coherence), HMQC-COSY, HMQC-DEPT (distortionless enhance- ment by polarization transfer), HMQC-TOCSY. Signals of NMR spectra were assigned according to the following convention including the spacer. N 1 -(6-Benzyloxycarbonyl-6-aminohexanoylamido)-2-ace- tamido-2-deoxy-b- D -glucopyranosyl)-(1 fi 2)-a- D -mann- opyranosyl-(1 fi 3)-[2-acetamido-2-deoxy-b- D -glucopy- ranosyl-(1 fi 4)]-[2-acetamido-2-deoxy-b- D -glucopyran- 120 S. Andre ´ et al.(Eur. J. Biochem. 271) Ó FEBS 2003 osyl-(1 fi 2)-a- D -mannopyranosyl-(1 fi 6)]-b- D -mann- opyranosyl-(1 fi 4)-2-acetamido-3,6-di-O-benzyl-2-de- oxy-b- D -glucopyranosyl-(1 fi 4)-2-acetamido-3,6-di-O- benzyl-2-deoxy-b- D -glucopyranoside (3; Bzl3-BiB8AH- Z). To a portion of 61.1 mg (32.1 lmol) glycosylazide (1) dissolved in 2 mL methanol were added 65 lLtriethylam- ine. The flask was flushed with argon followed by addition of 200 lL propane-1,3-dithiol [39]. After completion of the reaction (3.5 h; TLC: R f amine ¼ 0.22; propan-2-ol/1 M ammonium acetate, 4 : 1, v/v), the solution was evaporated and dried in high vacuo for 15 min. The remainder was allowed to react with a solution of activated Z-aminohex- anoic acid (2) prepared as follows: 236 mg (0.89 mmol, 20 eq.) Z-aminohexanoic acid were dissolved in 3.5 mL N-methylpyrrolidone. Subsequently, 136.3 mg (0.89 mmol, 20 eq.) N-hydroxybenzotriazol (HOBT), 285.8 mg (0.89 mmol, 20 eq.) TBTU [(1H-benzotriazol-1-yl)-1,1,3,3- tetramethyluronium tetrafluoroborate] and 233 lL (1.32 mmol) di-isopropylethylamine were added, following a protocol for standard peptide chemistry activation [40]. The mixture was stirred until a clear solution was obtained and adjusted to pH ¼ 9 by adding 130 lL di-isopropyl- ethylamine. The dried glycosylamine was dissolved in 2.5 mL of the solution prepared as above, and the pH was adjusted to 9.0 with di-isopropylethylamine. After 30 min at ambient temperature (TLC: propan-2-ol/1 M ammonium acetate, 4 : 1, v/v), 0.5 mL of the solution was added and stirring was continued for 5 min. The reaction mixture was evaporated and dried in high vacuo. Purification of the remainder was performed by RP-HPLC [acetonitrile/water, gradient of 35–45% acetonitrile run in 40 min; flow rate ¼ 8mLÆmin )1 , Macherey–Nagel Nucleogel RP 100- 10 (300 · 25 mm)]. The yield was 36.8 mg (54.0%), and analytical data were as follows: R f (amine) ¼ 0.22 (propan- 2-ol/1 M ammonium acetate, 2 : 1) and R f (3) ¼ 0.47 (propan-2-ol/1 M ammonium acetate, 2 : 1); ½a 22 D ¼ ) 2.3° (0.7, CH 3 CN/H 2 O, 1 : 1) and C 100 H 139 N 7 O 43 (M ¼ 2127.22). ESI-MS: M calcd ¼ 2125.9, M found ¼ 1064.3 (M + 2 H) 2+ . (Complete set of 1 H/ 13 C-NMR data not shown.) N 1 -(6-Aminohexanoylamido)-2-acetamido-2-deoxy-b- D - glucopyranosyl)-(1 fi 2)-a- D -mannopyranosyl-(1 fi 3)- [2-acetamido-2-deoxy-b- D -glucopyranosyl-(1 fi 4)]-[2- acetamido-2-deoxy-b- D -glucopyranosyl-(1 fi 2)-a- D - mannopyranosyl-(1 fi 6)]-b- D -mannopyranosyl-(1 fi 4)- 2-acetamido-2-deoxy-b- D -glucopyranosyl-(1 fi 4)-2-ace- tamido-2-deoxy-b- D -glucopyranoside (4; BiB8AH). A 35.8 mg (16.8 lmol) portion of the protected octasaccha- ride (3) was dissolved in a mixture of 6.7 mL methanol and 330 lL acetic acid. After addition of 61 mg palladium-(II)- oxide hydrate (84.6%; Pd), the suspension was stirred under hydrogen at atmospheric pressure for 24 h (TLC: propan-2- ol/1 M ammonium acetate, 2 : 1, v/v). To drive the reaction to completion, 50 mg palladium-(II)-oxide hydrate and 300 mL acetic acid were added, and hydrogenation was continued for 6 h. The catalyst was removed by centrifu- gation and washed three times with 10% (v/v) acetic acid in methanol. The combined supernatants were concentrated, and the remainder (36.0 mg) was purified by gel filtration (column: Pharmacia Hi Load Superdex 30, 600 · 16 mm; mobile phase: 100 m M NH 4 HCO 3 ; flow rate: 750 lLÆmin )1 ; detection: 220 and 260 nm) and lyophilized. The yield was 26.1 mg (95.0%) and analytical data were as follows: R f ¼ 0.29 (propan-2-ol/1 M ammonium acetate, 2 : 1, v/v), ½a 22 D ¼ )1.0 ° (1.7, H 2 O) and C 64 H 109 N 7 O 41 (M ¼ 1632.59). ESI-MS: M calcd ¼ 1631.68, M found ¼ 816.9 (M + 2H) 2+ . (Complete set of 1 H/ 13 C-NMR data not shown.) N 1 -(6-Aminohexanoylamido)-b- D -galactopyranosyl-(1 fi 4)- 2-acetamido-2-deoxy-b- D -glucopyranosyl-(1 fi 2)-a- D -mannopyranosyl-(1 fi 3)-[2-acetamido-2-deoxy-b- D - glucopyranosyl-(1 fi 4)]-[b- D -galactopyranosyl-(1 fi 4)- 2-acetamido-2-deoxy-b- D -glucopyranosyl-(1 fi 2)-a- D - mannopyranosyl-(1 fi 6)]-b- D -mannopyranosyl-(1 fi 4)- 2-acetamido-2-deoxy-b- D -glucopyranosyl-(1 fi 4)-2-ace- tamido-2-deoxy-b- D -glucopyranoside (5; BiB10AH). A 6.8 mg portion (4.17 lmol) octasaccharide (4) was dissolved in 2.4 mL 20 m M sodium cacodylate buffer, pH 7.4, containing 1.7 mg BSA, 4.34 lmol NaN 3 ,2.43lmol MnCl 2 ,8.3 mg(12.5 lmol) UDP-galactose, 10.3 U alkaline phosphatase and 206 mU GlcNAc-b1–4-galactosyltrans- ferase (EC 2.4.1.22). The reaction mixture was incubated for 48 h at 37 °C. After complete reaction (TLC: propan-2-ol/ 1 M ammonium acetate, 2 : 1, v/v), the precipitate was removed by centrifugation. The supernatant was concen- trated to a volume of 450 lL, purified by gel filtration (column: Pharmacia Hi Load Superdex 30, 600 · 16 mm; mobile phase: 100 m M NH 4 HCO 3 ; flow rate: 750 lLÆmin )1 ; detection: 220 and 260 nm) and lyophilized. The yield was 7.3 mg (89.4%) and analytical data were as follows: R f ¼ 0.15 (propan-2-ol/1 M ammonium acetate, 2 : 1, v/v), ½a 22 D ¼ +2.6° (0.46; H 2 O) and C 76 H 129 N 7 O 51 (M ¼ 1956.87). ESI-MS: M calcd ¼ 1955.79, M found ¼ 978.9 (M + 2H) 2+ . (Complete set of 1 H/ 13 C-NMR data not shown.) N 1 -(6-Aminohexanoylamido)-(5-acetamido-3,5-dideoxy- a- D -glycero- D -galacto-2-nonulopyranulosonic acid)- (2 fi 6)-b- D -galactopyranosyl-(1 fi 4)-2-acetamido-2- deoxy-b- D -glucopyranosyl-(1 fi 2)-a- D -mannopyranosyl- (1 fi 3)-[2-acetamido-2-deoxy-b- D -glucopyranosyl- (1 fi 4)]-[(5-acetamido-3,5-dideoxy-a- D -glycero- D -gal- acto-2-nonulopyranulosonic acid)-(2 fi 6)b- D -galactopy- ranosyl-(1 fi 4)-2-acetamido-2-deoxy-b- D -glucopyrano- syl-(1 fi 2)-a- D -mannopyranosyl-(1 fi 6)]-b- D -man- nopyranosyl-(1 fi 4)-2-acetamido-2-deoxy-b- D -glucopy- ranosyl-(1 fi 4)-2-acetamido-2-deoxy-b- D -glucopyrano- side (6; BiB1226AH). A 6.8 mg portion (4.17 lmol) of octasaccharide (4) was dissolved in 2.4 mL 20 m M sodium cacodylate buffer, pH 7.4, containing 1.7 mg BSA, 4.34 lmol NaN 3 ,2.43lmol MnCl 2 , 8.3 mg (12.5 lmol) UDP-galactose, 10.3 U alkaline phosphatase and 206 mU GlcNAc-b1–4-galactosyltransferase. The reaction mixture was incubated for 48 h at 37 °C. After complete reaction (TLC: propan-2-ol/1 M ammonium acetate, 2 : 1, v/v), 7.0 mg (9.1 lmol) CMP-N-acetylneuraminic acid and 75 mU b-galactoside-a2–6-sialyltransferase (EC 2.4.99.1) were added. After the pH had been adjusted to 6.0, incubation at 37 °C was continued for 24 h. A second portion of 7.0 mg (9.1 lmol) CMP-N-acetylneuraminic acid and 75 mU b-galactoside-a2–6-sialyltransferase was Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 121 added (pH 6.0). Incubation at 37 °C for 24 h was followed by removal of the precipitate by centrifugation. The supernatant was concentrated to a volume of 450 lL, purified by gel filtration (column: Pharmacia Hi Load Superdex 30, 600 · 16 mm; mobile phase: 100 m M NH 4 HCO 3 ; flow rate: 750 lLÆmin )1 ; detection: 220 and 260 nm) and lyophilized. The yield was 8.4 mg (79.5%), and analytical data were as follows: R f ¼ 0.11 (propan-2-ol/ 1 M ammonium acetate, 2 : 1, v/v), ½a 22 D ¼ +8.1° (0.28; H 2 O) and C 98 H 163 N 9 O 67 (M ¼ 2539.39). ESI-MS: M calcd ¼ 2537.96, M found ¼ 1270.3 (M + 2H) 2+ . (Com- plete set of 1 H/ 13 C-NMR data not shown.) N 1 -(6-Aminohexanoylamido)-(5-acetamido-3,5-dideoxy- a- D -glycero- D -galacto-2-nonulopyranulosonic acid)- (2 fi 3)-b- D -galactopyranosyl-(1 fi 4)-2-acetamido-2- deoxy-b- D -glucopyranosyl-(1 fi 2)-a- D -mannopyranosyl- (1 fi 3)-[2-acetamido-2-deoxy-b- D -glucopyranosyl- (1 fi 4)]-[(5-acetamido-3,5-dideoxy-a- D -glycero- D -gal- acto-2-nonulopyranulosonic acid)-(2 fi 3)-b- D -galacto- pyranosyl-(1 fi 4)-2-acetamido-2-deoxy-b- D -glucopyra- nosyl-(1 fi 2)-a- D -mannopyranosyl-(1 fi ]-b- D -manno- pyranosyl-(1 fi 4)-2-acetamido-2-deoxy-b- D -glucopyran- osyl-(1 fi 4)-2-acetamido-2-deoxy-b- D -glucopyranoside (7; BiB1223AH). A 6.8 mg portion (4.17 lmol) of octasaccharide (4) was dissolved in 2.4 mL 20 m M sodium cacodylate buffer, pH 7.4, containing 1.7 mg BSA, 4.34 lmol NaN 3 ,2.43lmol MnCl 2 , 8.3 mg (12.5 lmol) UDP-galactose, 10.3 U alkaline phosphatase and 206 mU GlcNAc-b1–4-galactosyltransferase. The reaction mixture was incubated for 48 h at 37 °C. After complete reaction (TLC: propan-2-ol/1 M ammonium acetate, 2 : 1, v/v), 7.0 mg (9.1 lmol) CMP-N-acetylneuraminic acid and 103 mU b-galactoside-a2–3-sialyltransferase (EC 2.4.99.6) were added. After the pH had been adjusted to 6.0, incubation at 37 °C was continued for 24 h. A second portion of 7.0 mg (9.1 lmol) CMP-N-acetylneuraminic acid and 103 mU b-galactoside-a2–3-sialyltransferase were added (pH 6.0). Incubation at 37 °C for 24 h was followed by removal of the precipitate by centrifugation. The supernatant was concentrated to a volume of 450 lL, purified by gel filtration (column: Pharmacia Hi Load Superdex 30, 600 · 16 mm; mobile phase: 100 m M NH 4 HCO 3 ; flow rate: 750 lLÆmin )1 ; detection: 220 and 260 nm) and lyophilized. The yield was 6.5 mg (61.4%) and analytical data were as follows: R f ¼ 0.14 (propan-2-ol/1 M ammonium acetate, 2 : 1, v/v), ½a 22 D ¼ +11.2° (0.1; H 2 O) and C 98 H 163 N 9 O 67 (M ¼ 2539.39). ESI-MS: M calcd ¼ 2537.96, M found ¼ 1270.3 (M + 2H) 2+ . (Com- plete set of 1 H/ 13 C-NMR data not shown.) To obtain the neoglycoproteins from the spacered N-gly- cans, the terminal amino group was converted into a reactive isothiocyanate [41,42]. In detail, a 0.34 lmol portion of each 6-aminohexanoyl-N-glycan (5–7) was dissolved in 200 lL dilute NaHCO 3 (100 mg Na 2 CO 3 /10 mL H 2 O) in a 1.5 mL plastic vessel. A solution of 1 lL(13.1 lmol) thiophosgene in 200 lL dichloromethane was added, and the biphasic mixture was vigorously stirred. After the amine had been consumed (1.5 h; TLC: 2-propanol/1 M ammonium acetate 2 : 1, v/v; R f of the decasaccharide derivative ¼ 0.45; R f of the a2–3-disialylated derivative ¼ 0.35; R f of the a2–6- disialylated derivative ¼ 0.28), the phases were separated by centrifugation, and the aqueous phase was collected. The organic phase was extracted twice with 100 mL dilute NaHCO 3 . To remove traces of thiophosgene, the combined aqueous phases were extracted twice with dichloromethane. A 2 mg portion of carbohydrate-free BSA was dissolved in the aqueous solution containing the isothiocyanate deriv- ative. The pH was adjusted to 9.0 by addition of 1 M NaOH. After 6 days at ambient temperature, the neoglycoprotein was purified by gel filtration (column: Pharmacia Hi Load Superdex 30, 600 · 16 mm; mobile phase: 100 m M NH 4 HCO 3 ; flow rate: 750 lLÆmin )1 ; detection: 220 and 260 nm). The product-containing solution was lyophilized. To calculate the extent of oligosaccharide incorporation into the protein carrier, a colorimetric assay was employed [43]. Gel electrophoretic analysis under denaturing conditions combined with silver staining of the neoglycoproteins was performed as described [37,38]. In addition to these three neoglycoproteins, lactosylated albumin was produced as control by the diazonium and phenylisothiocyanate reac- tions with p-aminophenyl b-lactoside [41,42]. Molecular modeling The simulations were performed on an IBM-SP2 parallel machine using the program DISCOVER on four or eight processors in parallel. Typically, they took several days of CPU time until completed and produced history files with a size ranging from several hundred megabytes to about 2 GB. In detail, molecular dynamics calculations of the N-glycans using parametrization by the CFF91 force field and automatic procedures of INSIGHT II (Molecular Simulations, San Diego, CA, USA) were run at 1000 K in vacuum using a dielectric constant of four and at 300 K, 400 K and 450 K in a solvent box with explicit water molecules in the program frame DISCOVER version 2.98 (Accelrys Inc., San Diego, USA). An additional force was applied to the pyranose rings at 1000 K to avoid ring inversions. After an equilibration period of 100 ps, simulations proceeded for 50–100 ns in the gas phase and for 1–25 ns in the solvent box. To be able to describe the conformational space, which is occupied during the simulation, several characteristic distances between pseudo atoms were evaluated. The pseudo atom co-ordinates of each sugar moiety are defined by the arithmetic mean value of the co-ordinates of its heavy atoms. Conformational analysis of the data obtained used suitable software tools, as described previously [44,45]. Lectin-binding assay Purification of galactoside-specific lectins from dried leaves of mistletoe (Viscum album L) and bovine heart and of the b-galactoside-binding IgG fraction from human serum, the purity controls, biotinylation of the sugar receptors under activity-preserving conditions, and quantitation of carbo- hydrate-specific binding to surface-immobilized neoglyco- proteins have been described in detail previously [37,38,46]. The experimental series with increasing concentrations of labeled markers and duplicates at each concentration step were performed at least four times up to saturation of binding, and each data set was algebraically transformed to obtain the K d value and the number of bound sugar receptor molecules at saturation. 122 S. Andre ´ et al.(Eur. J. Biochem. 271) Ó FEBS 2003 Flow cytofluorimetry Automated flow cytofluorimetric analysis of carbohydrate- dependent binding of biotinylated marker to the surface of a panel of human tumor cells of different histogenetic origin [BLIN-1, pre-B cell line; Croco II, B-lymphoblastoid cell line; CCRF-CEM, T-lymphoblastoid cell line; K-562, erythroleukemia cell line; KG-1, acute myelogenous leukemia cell line; HL-60, promyelocytic cell line; DU4475, mammary carcinoma cell line; NIH:OVCAR-3, ovarian carcinoma cell line; C205, SW480, and SW620, colon adenocarcinoma cell lines; Hs-294T, melanoma cell line; HS-24, nonsmall cell (epidermoid) lung carcinoma cell line] using the streptavidin–R-phycoerythrin conjugate as fluorescent indicator (1 : 40 dilution; Sigma, Munich, Germany) was performed on a FACScan instrument (Becton-Dickinson, Heidelberg, Germany) equipped with the software CONSORT 30, as described previously [37,38]. To reduce nonspecific binding by protein–protein interac- tions, cells were incubated with 100 lg ligand-free carrier protein (BSA) per mL for 30 min at 4 °C before incubation with the biotinylated neoglycoprotein in Dulbecco’s phosphate-buffered saline containing 0.1% (w/v) ligand- free BSA. The extent of noncarbohydrate-dependent fluorescence intensity was subtracted in each case from the total binding. Glycohistochemical processing Following an optimized procedure for visualizing carbohy- drate-ligand-dependent binding of the neoglycoproteins to sections of bronchial tumour [small cell (18 cases) and the three types of nonsmall cell lung cancer (total number of cases 60; 20 for each type), mesothelioma (20 cases) and carcinoid (20 cases)], the specimens were processed under identical conditions with ABC kit reagents and the substrates diaminobenzidine/H 2 O 2 for development of the colored, water-insoluble product [42,47,48]. A case was considered to be positive when at least clusters of tumor cells were specifically stained and the controls excluded binding of the labeled neoglycoprotein via the protein, the spacer or the biotin moieties [42,47,48]. Also, control experiments without the incubation step with the marker ruled out staining by binding of kit reagents, i.e. the glycoprotein avidin or horseradish peroxidase [49]. Biodistribution of radioiodinated neoglycoproteins Incorporation of 125 I into the neoglycoproteins to a specific activity of 11.5 MBqÆ(mg protein) )1 was achieved by the chloramine-T method using limiting amounts of reagents [50]. The retention of radioactivity in organs of Ehrlich- solid-tumor-bearing ddY mice (7 weeks old; Nihon Clea Co., Tokyo, Japan) after injection of 28.75 kBq per animal into the tail vein was determined by a c-counter (Aloka ARC 300, Tokyo, Japan) and expressed as percentage of the injected dose per gram of wet tissue or per milliliter of blood for a group of four mice for each type of neoglycoprotein and for each time point, as described [37,51,52]. The mice were treated and/or handled according to the Guide Principles for the Care and Use of Laboratory Animals of the Japanese Pharmacological Society and with the approval of Tokyo University of Science’s Institutional Animal Care and Use Committee. Results Synthetic chemistry A synthetic biantennary heptasaccharide N-glycan with a single unprotected hydroxy group at the 4¢ position of the b-linked mannose moiety was used to introduce the bisecting GlcNAc residue in a yield of 56%. This reaction pathway was planned as an extension of the basic chemo- enzymatic strategy of N-glycan synthesis and required no change in protecting group manipulations [53,54]. After completion of the synthesis of the bisecting octasaccharide, the removal of the base-labile protective groups was straightforward and led to compound 1 (Fig. 2). Selective reduction of the azido function at the anomeric center was achieved by the propanedithiol method. After removal of the volatile compounds in high vacuo, the intermediate glycosylamine was coupled to the 6-aminohexanoic acid derivative 2 using standard peptide chemistry activation with TBTU/HOBt which generates the intermediate active ester of 2 (Fig. 2). An excess of the activated spacer 2 was required in the coupling step, suggesting that the remaining traces of propane-1,3-dithiol were scavenging the active ester. The purified octasaccharide spacer conjugate 3 was obtained in 54% yield after preparative HPLC. In the final deprotection step, catalytic hydrogenation was used to simultaneously remove the four permanent benzyl groups from the chitobiosyl core and liberate the primary amino function in the spacer part. Compound 4 was easily purified by gel filtration and processed further to the final carbo- hydrate derivatives 5–7 by enzymatic elongation of the carbohydrate chain using glycosyltransferases. The presence of alkaline phosphatase ensured removal of inhibitors [55]. In the first enzymatic step, bovine milk b1–4-galactosyl- transferase attached galactosyl moieties to each of the terminal GlcNAc residues of the a1–3 and a1–6 arms. As expected, the bisecting GlcNAc residue was not a substrate, presumably because of sterically blocked access for the enzyme exerted by the two antennae [56]. The resulting digalactosylated dodecasaccharide 5 (Fig. 2) was purified (89% yield after gel filtration), and portions were subse- quently incubated in a one-pot reaction with CMP-sialic acid and either a2–6 or a2–3-sialyltransferase. After puri- fication, the desired sialylated dodecasaccharides 6 and 7 were obtained in a yield of 80% and 61%, respectively. To ensure the structural identity of all reaction products shown in Fig. 2, compounds 1–7 were routinely analyzed by electrospray ionization MS (detailed in Materials and methods) and by complete assignment of the ring carbons and hydrogens by state-of-the-art 2D NMR techniques (not shown). TOCSY, NOESY, HMQC, HMQC-COSY, HMQC-DEPT and HMQC-TOCSY experiments were employed. Conjugation of the three N-glycans 5–7 to BSA as cytochemically and histochemically inert carrier was acomplished by selective activation of the terminal amino group, as illustrated in Fig. 3. The free amines were converted into isothiocyanates by thiophosgene in a bipha- sic reaction, which was followed conveniently by TLC. The isothiocyanates [10 eq.Æ(mol BSA) )1 ] were added directly to Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 123 carbohydrate-free BSA and allowed to react for 6 days at pH 9.0. After purification of the neoglycoproteins, success- ful conjugation was first visualized by the shift in electro- phoretic mobility in standard SDS/PAGE (Fig. 4). The mean N-glycan incorporation into the carriers (measured by a colorimetric assay) for decasaccharide (5) was 3.6 N-glycan molecules per BSA molecule. The reactions with the sialylated dodecasaccharides 6 and 7 resulted in 4.9 and 3.1 carrier-conjugated glycan chains, respectively. Two main effects of the bisecting GlcNAc unit on the conformation of the biantennary glycan were pinpointed graphically by molecular modeling. In comparison with previous work [6–11], we (a) extended the simulation periods considerably, (b) added calculations in a solvent box, and (c) assessed probabilities of population density in the conformational space with improved statistical reliability and without the strict dependence on time-averaged distance constraints. Computational chemistry Computational methods were used to analyze the dynamic behavior of the a1–3/a1–6 branches in the absence and presence of the bisecting GlcNAc residue. The calculated xyz population densities of all monosaccharide building blocks were translated into a strict free-energy grading using the Boltzmann equation. Isocontour plots at a constant free energy level of 1.5 kcalÆmol )1 were drawn to visualize the inherent flexibility at each point of the branches and the relation of individual flexibility to structural changes (Fig. 5). Interestingly, the molecular dynamics (MD) calcu- lations set to vacuum or a solvent box with water mole- cules gave very similar results. A prevailing influence of van der Waals dispersive and repulsive forces on the conformational properties of this system is consistent with this outcome. Relative to the absence of solvent, the simulated presence of water molecules had a dampening effect on the extent of conformational fluctuations and dynamics of intramolecular mobility. The way in which the bisecting substituent shapes the population density of the biantennary nonasaccharide and decasaccharide is shown in Fig. 5B,C. As part of the sugar chain, the bisecting GlcNAc induced separation of the accessed space of the a1–6 arm into two sections (Fig. 5C). In comparison with the behavior of the unsubstituted N-glycan, the vicinity of the core trisaccharide becomes fairly accessible for terminal residues of the a1–6 arm, a process referred to as backfold- Fig. 2. Chemical and enzymatic steps to pro- duce galactosylated and sialylated N-glycans substituted with bisecting GlcNAc. (a) 1,pro- panedithiol, Et 3 N, MeOH; 2, N-benzyloxy- carbonyl-6-aminohexanoic acid, TBTU, 1- hydroxybenzotriazole (HOBT) (1–2: 54%). (b) Pd-H 2 , AcOH, MeOH (95%). (c) b1–4- galactosyltransferase, UDP-Gal, alkaline phosphatase (89%). (d) a2–6-sialyltransferase, CMP-NeuNAc, alkaline phosphatase (c + d: 80%). (e) a2–3-sialyltransferase, CMP-Neu- NAc, alkaline phosphatase (c + e: 61%). 124 S. Andre ´ et al.(Eur. J. Biochem. 271) Ó FEBS 2003 ing. The wedge-like central GlcNAc moiety accounts for other changes presented in Fig. 5. The inherent flexibility of the a1–3 arm is clearly restricted in the bisecting compound (Fig. 5B,C). Backfolding and restrained fluctuations have been confirmed by experimental NMR analysis with model oligosaccharides [6–11]. The contribution of carbohydrate stacking to chain flexibility becomes apparent when two energetically favored conformations are scrutinized (Fig. 6). Regions I and II in Fig. 6A,B comprise conformations from the MD trajectories, with distances between pseudo atoms characteristic of stacking. Examples of the topological arrangements of the chain constituents from these regions are illustrated in Fig. 6C,D. To compare major aspects of the conformational ensembles of the N-glycans, including the sialylated compounds without/with bisecting GlcNAc, representative snapshots from the MD trajectories are presented in Fig. 7. A topological constellation showing how the pronounced flexibility of the a1–6 branch can lead to a dramatic reduction in intramolecular contact with the bisecting GlcNAc, a situation especially encountered in region III, is depicted in Fig. 7C,D. At this point, the frequent occurrence of N-glycosylation should be recalled. It is still difficult to gauge the influence of the protein backbone on glycan flexibility in individual instances. In this context, it is reassuring to note that oligosaccharides from N-glycoproteins, for example the Man 5)8 N-glycans from RNase B [57,58], can exhibit conformational behavior similar to that when attached to the protein. Therefore, it can be assumed that constant dynamics or only slightly changed level of mobility will also be encountered in other cases, especially for neoglycopro- teins with spacer-bound glycans, where spatial constraints exerted by the protein are reduced by adding a linker. However, emerging evidence for the varying influence of a bisecting GlcNAc on the processing of branch ends for several glycoproteins precludes general apriorideductions being drawn [59]. Nonetheless, Figs 5–7, combined with the experimental evidence reviewed in the Introduction, show how the addition of the bisecting GlcNAc modifies the conformational behavior of the N-glycan. To determine whether binding of sugar receptors to the branches is affected by the bisecting modification, we performed solid- phase assays with the same set of lectins and antibodies, as described previously [37]. With galectin-1 as a test substance, we selected an endogenous lectin that mediates tumor cell adhesion to extracellular matrix glycoproteins, a key step in the metastatic process, invasion of the parenchyma, and growth regulation after ligand cross-linking [27,28,60–67]. Solid-phase assay The incorporation of 3.1–4.9 N-glycan chains per albumin molecule was comparable to the yields in our previous studies [37,38]. The neoglycoproteins obtained were thus Fig. 3. Activation of the amino group of the spacer at the reducing end of the synthetic N-glycans represented by dodecasaccharide (6) and the coupling to BSA. (a) Thiophosgene, CH 2 Cl 2 /H 2 O, NaHCO 3 , pH 8.5 (quant.); (b) BSA, H 2 O, NaHCO 3 ,pH9.0. Fig. 4. Visualization of the gel electrophoretic mobility under denaturing conditions of carbohydrate-free BSA (A) and the neoglycoproteins car- rying the decasaccharide with bisecting GlcNAc (5) (B) and the a2–3- sialylated (C) and a2–6-sialylated (D) derivatives (substances 6 and 7 in Fig. 2). Positions of marker proteins for molecular mass designation are indicated by arrowheads. Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 125 expected to have sufficient ligand density for interaction. In contrast with natural glycoproteins, the problem of micro- heterogeneity of the N-glycans is not encountered with these synthetic products. To monitor the ligand properties comparatively, we maintained the test profile with a dimeric plant lectin, mammalian homodimeric galectin-1 and b-galactoside-specific IgG fraction from human serum. Similar to the interaction of a lectin with a cell surface and following the protocol of our two previous studies, the neoglycoproteins were first bound to the surface and the glycan-binding proteins assayed in solution. Thereby, any distortion of the lectin/antibody structures by adsorption to plastic was avoided. In the solid-phase assay, saturable and carbohydrate-dependent binding was measured, and the resulting Scatchard plots gave straight lines indicating the presence of a single class of binding sites in each case (not shown). To allow convenient comparisons, we summarize our data together with previous results on unsubstituted Fig. 5. Illustration of the nomenclature system for the N-glycan constituents (A) and of their inherent flexibility by isocontour plots (derived from analysis of xyz population densities of each monosaccharide unit) at a constant energy level of 1.5 kcalÆmol -1 for the biantennary nonasaccharide (B; first structure in Fig. 1) and the decasaccharide containing the bisecting GlcNAc (C; structure 4 in Fig. 1 and substance 5 in Fig. 2). For convenient comparison, the conformations were positioned in space in the same way by superimposing the ring atoms of mannose units of the pentasaccharide core. Access to the conformational space in the vicinity of the linear part of the core for the terminal galactose moiety of the a1–6 arm is emphasized by introducing the term ÔbackfoldedÕ into the figure (C; also Fig. 7C,D). Fig. 6. Analysis of the involvement of the bisecting GlcNAc unit in stacking interactions by measuring pseudo atom distances between this residue (Fig. 5A, 9) and spatially neighbouring residues 4, 5 and 4¢,5¢ of the two branches, respectively, in MD simulations. The pseudo atom co-ordinates of each sugar moiety are defined by the arithmetic mean of the co-ordinates of its heavy atoms. Two energetically favored conformational families with glycosidic torsion angle sets of the GlcNAcb1–4Man linkage of F ¼ 50 °/Y ¼ 20 ° (A; global minimum) or F ¼ )30 °/Y ¼ )30 ° (B; side minimum) were separately analyzed. The two illustrated conformations representing examples from regions I and II were taken from the MD trajectories in explicit solvent (for clarity, water molecules are not shown). Distance values between residues 9 and 5 (5¢) located in region I (A) are characteristic of occurrence of stacking indicated by arrows (C). Spatial proximity to the 4 (4¢) residues can still be maintained for the bisecting GlcNAc in region II (A, B, D). In region III, populated by the flexible a1–6 branch, the intramolecular contact with the bisecting GlcNAc is clearly diminished. 126 S. Andre ´ et al.(Eur. J. Biochem. 271) Ó FEBS 2003 N-glycans [37] in Table 1. The general conclusion is that the presence of a bisecting GlcNAc affects lectin affinity. In the case of mistletoe lectin, the introduction of a bisecting GlcNAc into the glycan was unfavorable for binding. The affinity of the galactose-dependent interaction was about sixfold lower (Bi9-BSA vs. BiB10-BSA, Table 1). A similar result was obtained for the a2–6-sialylated derivative, which is a ligand with even greater binding affinity than the sialic-acid-free N-glycan (Bi1126-BSA vs. BiB1226-BSA, Table 1). a2–3-Sialylation produced an un- favorable docking site. The introduction of the glycan substituent at a distance from the actual contact site for the lectin can thus indeed modulate ligand properties, without being directly involved in binding. Remarkably, the plant Fig. 7. Illustration of major aspects of the conformational ensembles of sialylated derivatives of the biantennary nonasaccharide Bi9 (A) and of the decasaccharide BiB10 (for nomenclature, see Fig. 1) with the bisecting GlcNAc (B–D). The conformations presented were taken from the MD trajectories in explicit solvent (for clarity, water molecules are not shown). The terminal sialic acid moieties of the branches are designated as N and N¢ to allow easy visualization in the extended (A, B) and backfolded structures (C, D). A description of the populated conformational space has been given in Fig. 5B,C and that the conformations shown in Fig. 6C,D complement the illustration of the extended structure of the biantennary N-glycan with bisecting GlcNAc. Stacking interactions with this moiety (indicated by an arrow between residue 4 in the a1–3 branch as the main contact point and the bisecting GlcNAc) are also possible in backfolded structures (D). Table 1. Apparent dissociation constants (K d ) for the interaction of (neo)glycoproteins with sugar receptors and the number of bound probe molecules at saturation for Viscum album agglutinin (VAA), bovine galectin-1 and the human b-galactoside-binding IgG subfraction from human serum in a solid- phase assay. K d isgiveninn M ; B max is expressed as bound probe molecules per well. The assays with VAA and the neoglycoprotein BiB1226-BSA were performed with 0.25 lg as matrix. For Lac-BSA (diazo) and Lac-BSA (thio) BSA was glycosylated by covalent attachment of either the diazophenyl derivative (diazo) or the p-isothiocyanatophenyl derivative (thio) of p-aminophenyl b- D -lactoside. Each value is the mean ± SD from at least four independent experimental series, the quantity of (neo)glycoprotein for coating in lg/well being given for each type of substance. Matrix VAA Galectin-1 IgG K d B max K d B max K d B max BiB10-BSA (0.5 lg) 163.3 ± 79.4 1.9 ± 0.4 · 10 10 518.6 ± 118 2.1 ± 0.6 · 10 10 73.9 ± 31.6 2.3 ± 1.9 · 10 10 BiB1223-BSA (0.5 lg) 1063 ± 347 1.4 ± 0.6 · 10 10 817.9 ± 493 1.6 ± 0.6 · 10 10 36.1 ± 26.5 2.9 ± 1.1 · 10 10 BiB1226-BSA (0.5 lg) 49.8 ± 19.9 4.2 ± 2.3 · 10 10 829.6 ± 50.6 2.2 ± 0.6 · 10 10 71.8 ± 48.9 2.5 ± 1.1 · 10 10 Bi9-BSA (0.5 lg) a 26.7 ± 11.6 4.6 ± 1.9 · 10 10 900.1 ± 176 42.8 ± 12.5 · 10 10 32.9 ± 19.6 0.35 ± 0.1 · 10 10 Bi1123-BSA (0.5 lg) a 938.4 ± 661 8.2 ± 4.4 · 10 10 829.5 ± 501 42.0 ± 16.5 · 10 10 87.3 ± 62.7 0.38 ± 0.1 · 10 10 Bi1126-BSA (0.5 lg) a 8.7 ± 4.5 6.1 ± 1.4 · 10 10 1025.5 ± 619 48.7 ± 18.4 · 10 10 33.9 ± 4.6 0.46 ± 0.1 · 10 10 Lac-BSA (diazo) (3 lg) 312.4 ± 190 4.7 ± 2.3 · 10 10 1127.2 ± 53.3 34.6 ± 17.6 · 10 10 139.0 ± 87.6 0.70 ± 0.1 · 10 10 Lac-BSA (thio) (0.5 lg) 13.4 ± 7.3 5.1 ± 0.2 · 10 10 516.0 ± 20.3 83.3 ± 6.5 · 10 10 7.6 ± 5.2 0.65 ± 0.1 · 10 10 Asialofetuin (1 lg) 7.4 ± 2.6 4.9 ± 0.5 · 10 10 819.0 ± 268 37.5 ± 10.7 · 10 10 69.2 ± 40.2 0.43 ± 0.1 · 10 10 a From [37]. Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur. J. Biochem. 271) 127 [...]... Probing the cons and pros of lectin-induced immunomodulation: case studies for the mistletoe lectin and galectin-1 Biochimie 83, 659–666 Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur J Biochem 271) 133 28 Danguy, A., Camby, I & Kiss, R (2002) Galectins and cancer Biochim Biophys Acta 1572, 285–293 29 Rabinovich, G.A., Rubinstein, N & Toscano, M.A (2002) Role of galectins in inflammatory and. .. carrier protein was labeled (by biotinylation or iodination) without affecting the glycan part Relative to the solid-phase assays with purified lectins, quantitation of cell binding comprises the complex display of carbohydrate-binding activities including the occurrence of E-PHA-like human lectins We monitored the ligand properties of the carrier-immobilized N-glycans by FACScan analysis, by staining tissue... modification Binding affinities to the IgG fraction did not reflect the structural difference between unsubstituted N-glycans and their bisecting derivatives To further examine the ligand properties of the neoglycoproteins, we determined their binding to cells and to tissue sections as well as their biodistribution in mice after injection of radioiodinated material Binding to cells and organs In these experiments,... ligand can alter the affinity for distinct lectins, notably including a mammalian lectin This message broadens our view on the levels of affinity modulation for carbohydrate ligands Besides alterations in sequence and local density by clustering, the presence of a substitution is to be reckoned with, making complex glycans study objects of choice to define the binding profile of a lectin, e.g a galectin... translate into changes in biological properties The occurrence of ricin resistance in CHO cell mutants being attributed to GnT-III activity [15,16] is an example in which an ensuing decrease in affinity in plant lectin binding can help to explain the new cell feature In the same way, it is reasonable to suggest galectin-1 and galectin-3 as candidates to rationalize the increase in Ó FEBS 2003 ´ 132 S Andre... studies of cell binding allowed determination of the ligand properties of biantennary N-glycans [37] with either bisecting GlcNAc (this study) Fig 8 Comparison of the percentage of positive cells in cytofluorimetric analysis using the biotinylated neoglycoproteins presenting as cytochemical ligand part the biantennary N-glycan without substitution (nonasaccharide Bi9) and its derivatives with core fucosylation... 2003 N-glycans with bisecting GlcNAc as ligands (Eur J Biochem 271) 129 design, we collected binding data to address two issues: (a) to examine staining with respect to cellular parameters and (b) to define any effect of the introduction of the substitution The results, expressed as median fluorescence intensity and percentage of positive cells, reflected (a) the histogenetic origin of the cell type and. .. were examined using the two neoglycoproteins with/without bisecting GlcNAc, staining intensity was significantly higher for the pre-B cells when using the unsubstituted N-glycan-bearing neoglycoprotein (Bi9; 53.4 [37,38]) instead of the bisecting congener BiB10-BSA (7.8; for nomenclature, see Fig 1) The nonuniform ligand properties of BiB10/Bi9 were underscored by measuring the percentage of positive cells... 0.01 0.16 Ó FEBS 2003 N-glycans with bisecting GlcNAc as ligands (Eur J Biochem 271) 131 Discussion Fig 11 Comparison of the numerical values for the percentage of injected dose per g of tissue (or ml of blood) of 125I-neoglycoproteins presenting as bioactive ligand part the biantennary N-glycans without substitution (nonasaccharides and undecasaccharides Bi9, Bi1123, Bi1126) and their derivatives... arises relating changes in this aspect of tumor glycan expression to bioaffinity modulation in the interaction with tissue lectins and thus to the pattern of expressed lectins (lectinome) Combined studies on glycan and lectin expression are thus proposed to define new functional correlations The path to the presented results and this perspective has been paved by a combination of synthetic and computational . Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico , in vitro and in vivo Sabine Andre ´ 1 ,. histopatho- logical and in vivo procedures. The question of whether introduction of the bisecting GlcNAc changes the ligand properties (binding affinities) of biantennary N-glycans for branch-end-specific lectins. the presence of a bisecting GlcNAc affects lectin affinity. In the case of mistletoe lectin, the introduction of a bisecting GlcNAc into the glycan was unfavorable for binding. The affinity of the galactose-dependent