Introduction of extended LEC14-type branching into core-fucosylated biantennary N-glycan Glycoengineering for enhanced cell binding and serum clearance of the neoglycoprotein ´ Sabine Andre1, Shuji Kojima2, Ingo Prahl3, Martin Lensch1, Carlo Unverzagt3 and Hans-Joachim Gabius1 Institut fur Physiologische Chemie, Tierarztliche Fakultat, Ludwig-Maximilians-Universitat Munchen, Germany ă ă ă ă ă Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan Bioorganische Chemie, Universitat Bayreuth, Germany ă Keywords drug targeting; galectin; glycosylation; lectin; tumor imaging Correspondence ´ S Andre, Institut fur Physiologische ă Chemie, Tierarztliche Fakultat, Ludwigă ă Maximilians-Universitat Munchen, ă ¨ Veterinarstr 13, 80539 Munchen, Germany ¨ ¨ Fax: +49 80 2180 2508 Tel: +49 89 2180 2290 E-mail: Sabine.Andre@lmu.de (Received 16 December 2004, revised 23 February 2005, accepted March 2005) doi:10.1111/j.1742-4658.2005.04637.x A series of enzymatic substitutions modifies the basic structure of complex-type biantennary N-glycans Among them, a b1,2-linked N-acetylglucosamine residue is introduced to the central mannose moiety of the core-fucosylated oligosaccharide by N-acetylglucosaminyltransferase VII This so-called LEC14 epitope can undergo galactosylation at the b1,2linked N-acetylglucosamine residue Guided by the hypothesis that structural modifications in the N-glycan alter its capacity to serve as ligand for lectins, we prepared a neoglycoprotein with the extended LEC14 N-glycan and tested its properties in three different assays In order to allow comparison to previous results on other types of biantennary N-glycans the functionalization of the glycans for coupling and assay conditions were deliberately kept constant Compared to the core-fucosylated N-glycan no significant change in affinity was seen when testing three galactoside-specific proteins However, cell positivity in flow cytofluorimetry was enhanced in six of eight human tumor lines Analysis of biodistribution in tumorbearing mice revealed an increase of blood clearance by about 40%, yielding a favorable tumor ⁄ blood ratio Thus, the extended LEC14 motif affects binding properties to cellular lectins on cell surfaces and organs when compared to the core-fucosylated biantennary N-glycan The results argue in favor of the concept of viewing substitutions as molecular switches for lectin-binding affinity Moreover, they have potential relevance for glycoengineering of reagents in tumor imaging N-Glycosylation is the most frequent and structurally most variegated form of post-translational modification [1–3] Ironically, it is exactly due to this unsurpassed molecular complexity that progress to assign functional significance to distinct glycan epitope lags behind the advances of work on other types of protein modifications Taking a step to change this situation was the driving force for our study At first glance, we consider it reasonable to interpret the enormous structural complexity of the carbohydrate part of glycoproteins as a wide array of signals; this concept provides direction for research [4,5] As documented already at the level of nascent glycoproteins, their N-glycan structure is relevant for quality control, underscoring the Abbreviations CHO, Chinese hamster ovary; GlcNAc, N-acetylglucosamine; GlcNAc-TVII, N-acetylglucosaminyltransferase VII; LacNAc, N-acetyllactosamine; LCA, Lens culinaris agglutinin; PSA, Pisum sativum agglutinin; TFA, trifluoroacetic acid 1986 FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS ´ S Andre et al notion that the glycan’s intimate interplay with specific lectins can be central to realization of its functional potential [6–8] Because glycans differing in structure by specific substitutions might react with cell lectins in a characteristic manner, a route of translating structural differences into effects, e.g leading to altered cell adhesion, growth control or endocytic uptake, is envisioned Fittingly, the complexity of glycan structures is matched by expression of lectin families [5,9–11] Our aim, in essence, is to systematically measure ligand properties of N-glycans with different substitutions In so doing we combine the emerging technology for chemoenzymatic tailoring of complex-type N-glycans on a preparative scale with biochemical ⁄ cell biological methods, starting with the preparation of neoglycoproteins with a homogeneous sugar part as suitable test substances Our initial studies with complex-type biantennary N-glycans bearing either a bisecting N-acetylglucosamine (GlcNAc) or a core-fucose (Fuc) residue have lent support to the validity of our hypothesis [12–14] These two substitutions act like switches on ligand properties With these data in hand, the description of the naturally occurring core-fucosylated N-glycan containing an additional b1,2-linked GlcNAc moiety attached to the central Man residue (LEC14) ([15,16]; for glycan structures see lower part of Fig 1) prompted us to take the next step in our program by examining its properties as ligand The approach to track down the LEC14 N-glycan variant actually exploited an impact of N-glycan substitutions on lectin binding Development of resistance to two agglutinins with dual affinity to the core-fucose unit and the trimannosyl core region, i.e Pisum sativum and Lens culinaris agglutinins (PSA, LCA) [17], led to the selection of the LEC14 mutant of Chinese hamster ovary cells (CHO) [15,16] Besides substantial branching in poly(N-acetyllactosamine) chains implied by the occurrence of 3,4-disubstituted GlcNAc in methylation analysis the presence of a b1,2-linked GlcNAc moiety attached to the central Man residue in the trimannoside core was defined [18] It apparently perturbs binding to PSA ⁄ LCA, the likely cause for the enhanced lectin resistance, and contributes to the complex changes in the glycoproteomic profile of the LEC14 mutant vs wild-type cells [18] The enzymatic introduction of the b1,2-linked GlcNAc moiety into the biantennary N-glycan by N-acetylglucosaminyltransferase VII (GlcNAc-TVII) depends critically on the presence of the core-fucose unit so that the LEC14 type of glycan will invariably harbor two core substitutions [19] An immediate question concerns the possibility of further processing FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS Extended LEC14-type N-glycan as lectin ligand While the bisecting GlcNAc residue in b1,4-linkage to the central mannose unit has only been described as an acceptor for chain elongation in glycans of Mgat2null mice [20,21], the question on branch elongation of the LEC14-specific b1,2-linked GlcNAc could initially not be answered unequivocally The extensive treatment of glycopeptides by b-galactosidase and N-acetylb-d-glucosaminidase to trim the glycan antennae prior to structural analysis of the core may well have also impaired this branch elongation [18] This situation was resolved by total synthesis of a complete LEC14 N-glycan The availability of material derived from chemical synthesis not only unambiguously confirmed this particular core structure but also provided sufficient quantities for glycosyltransferase assays [22] Galactosyltransferase was found to elongate the unusual b1,2-linked GlcNAc residue ([23]; for structure of the glycan with the new branch see Fig 1) In accordance with the high level of resistance to glycosidase treatment of this GlcNAc residue (an indication for rather poor spatial accessibility compared with the GlcNAc moieties in the a1,3- and a1,6-arms) its reactivity towards galactosyltransferase was lower than for terminal GlcNAc residues in the antennae [23] Thus, the demonstration of substrate properties of the LEC14 core for galactosylation suggests, but does not prove the presence of this type of triantennary N-glycan in the complex profile observed for CHO cell glycans Its occurrence might account for enhanced glycopeptide binding to immobilized Ricinus communis agglutinin I relative to wild-type glycans [18] Consequently, we addressed the ensuing question whether the addition of an N-acetyllactosamine (LacNAc) unit to a core-fucosylated biantennary N-glycan at the central Man residue in b1,2-linkage will alter the ligand properties especially for endogenous receptors Because chemical conjugation of the synthetic oligosaccharides to a carrier protein is feasible for a spacered N-glycan [23], we prepared a neoglycoprotein from an extended LEC14 N-glycan after suitable linker design The ligand properties of the resulting neoglycoprotein (A) were tested in three different systems: (a) tested as ligand immobilized to a plastic surface with five sugar receptors, among them growth-regulatory galectins [24]; (b) tested as ligand for surface receptors of different types of tumor cells; and (c) injected into circulation with monitoring of the time course of biodistribution The data set on the neoglycoprotein prepared from a core-fucosylated N-glycan devoid of the b1,2-substitution (C) and tested previously under identical conditions [13] allowed direct comparison to pinpoint any detectable influence of the new b1,2-branch 1987 Extended LEC14-type N-glycan as lectin ligand 1988 ´ S Andre et al FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS ´ S Andre et al Results Extended LEC14-type N-glycan as lectin ligand with: (a) purified sugar receptors; (b) tumor cell surfaces in vitro; and (c) organ lectins in vivo Synthetic background We have set ourselves the task of systematically determining ligand properties of N-glycans, here the pergalactosylated LEC14 dodecasaccharide (Fig 1, compound 7), by combining chemoenzymatic synthesis with biochemical ⁄ cell biological techniques In order to imitate the natural presentation of N-glycans on a glycoprotein, conjugation of the synthetic product to the carrier protein, which is otherwise free of ligand properties, was necessary Toward this end we started from the protected nonasaccharide [22] to reach the pergalactosylated LEC14 epitope [23] as strategically outlined in Fig The resulting spacered dodecasaccharide was conjugated to BSA after the spacer’s amino function was activated by reaction with thiophosgene to its isothiocyanate N-glycan attachment to the carrier protein was visualized by gel electrophoretic analysis revealing the N-glycan-dependent shift of the molecular mass (lower part of Fig 1) The protein was turned totally into a glycan carrier, because no staining was visible at the position of unsubstituted albumin To determine the incorporation yield, additional analytical procedures were performed MS gave evidence for a spectrum of neoglycoproteins with one to four attached N-glycan chains, and the colorimetric assay determined an average of 3.2 N-glycans per carrier molecule [23] Of note for the intended comparison to the other so far tested complex-type biantennary N-glycans without ⁄ with substitutions, the linker design could thus be kept constant Even more important, the incorporation yield of this reaction was only slightly lower than for the N-glycan with a bisecting GlcNAc moiety (B) or the unsubstituted form (D) at 3.6 N-glycans per carrier protein and the core-fucosylated N-glycan at 3.9 oligosaccharide chains (C) [12–14] This result, ensuring rather similar glycan density, was the prerequisite to proceed to testing the ligand properties of the extended LEC14 dodecasaccharide using neoglycoprotein A in three different assay systems Affinity to purified lectins/antibodies The first assay system was designed to simulate properties of glycoproteins presented on a cell surface by adsorbing the neoglycoprotein to the plastic surface of microtiter plate wells The homogeneity of the structure of the synthetic N-glycan, rigorously controlled by our analytical procedures (see Experimental procedures) and definitively excluding the presence of contaminating glycoforms, will account for a clear-cut correlation between structure and ligand properties Also, the assay deliberately avoided surface immobilization of the carbohydrate-binding proteins, which might affect their binding properties Under these conditions, carbohydrate-dependent and saturable binding of toxic mistletoe lectin, the growth ⁄ invasion-regulatory galectin-1 and the natural autoantibody was invariably detected (Fig 2) The calculated Scatchard plots gave straight lines in all cases, evidence for a single class of binding sites Although the different sugar receptors home in on the same basic unit, i.e terminal galactose, their affinity is clearly disparate (Fig 2A–C) The IgG subfraction bound with the highest affinity, followed by the plant agglutinin with two binding sites per B-subunit in the (AB)2-tetramer and the homodimeric endogenous lectin with its two binding sites at opposing sides of the protein Galectins afford the opportunity to further examine the relationship between the spatial presentation of carbohydrate recognition domains and ligand affinity We thus tested two further members of the galectin family, i.e galectins-3 and -5 These two monomeric proteins share ligand specificity with galectin-1 but not its homodimeric cross-linking design Due to their monomeric constitution in solution no affinity enhancement by ligand clustering through a bivalent module is expected Indeed, these two lectins were inferior in terms of binding affinity to galectin-1, their KD values at 820 ± 71 nm (galectin-3) and 734 ± 157 nm (galectin5; Fig 2D) with about three to fivefold increases in Bmax Fig Chemical and enzymatic steps to produce the LEC14-type N-glycan with the LacNAc branch in b1,2-linkage at the central Man unit starting from the protected nonasaccharide [34,35] (a) (NH4)2Ce(NO3)6, CH3CN-H2O, 80 °C (71%); (b) Ethylenediamine, n-BuOH, 80 °C; Ac2O, pyridine; MeNH2 (41%) in H2O (96% for steps 1–3); (c) Propanedithiol, NEt3, MeOH; N-carbobenzoxy-6-amino hexanoic acid 4, TBTU, HOBt, N-methylpyrrolidone (31% for steps 1–2 after RP-HPLC); (d) PdO-H2O ⁄ H2, MeOH-AcOH (95%); (e) UDP-Gal (4 eq.), b1,4-galactosyltransferase, alkaline phosphatase (75%); (f) Thiophosgene, CH2Cl2-H2O, NaHCO3; BSA, H2O, NaHCO3; days The last two schemes for N-glycan sequences allow structural comparison between the N-glycan of neoglycoprotein A and the previously studied N-glycans in neoglycoproteins B–D (upper panel) Gel documentation is added in the bottom panel for visualization of the gel electrophoretic mobility of the carbohydrate-free carrier protein BSA (lanes a, c, e; 0.15 lgỈlane)1) relative to that of the neoglycoprotein A (lanes b, d, f; 0.2 lgỈper lane, 0.15 lgỈper lane and 0.1 lgỈper lane, respectively) Positions of two marker proteins for molecular mass designation (in kDa) are indicated by arrowheads FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS 1989 ´ S Andre et al Extended LEC14-type N-glycan as lectin ligand Fig Illustration of Scatchard plot analysis of carbohydrate-dependent interaction between the carrier-immobilized N-glycan (A) and the mistletoe lectin (VAA; A), human galectin-1 (B), the lactoside-binding IgG subfraction (C) and rat galectin-5 (D) in a representative experimental series KD values (mean ± SD) are given for the complete set of analytical data with at least four different experimental series for each sugar receptor The extent of total binding (s) was reduced by that of binding which was not inhibitable by glycoinhibitors (h, 75 mM lactose and mg asialofetuinỈmL)1) to calculate the level of carbohydrate-dependent binding (n) (see inset) values These results confirm a striking effect of receptor topology at constant spatial features of the ligand They also enable us to set the ligand properties of the extended LEC14 dodecasaccharide in relation to the so far studied N-glycans, especially the N-glycans without any substitution or with core-fucosylation The presence of the new glycan branch in the extended LEC14 neoglycoprotein appeared to enhance affinities toward human proteins and reduce affinity toward the plant lectin relative to the properties of the complex-type biantennary N-glycan lacking a substitution However, the strict dependence of GlcNAc-TVII activity on core-fucosylation in its substrate [19], and therefore the presence of this substitution in the LEC14 epitope, must not be neglected The ensuing comparison between the properties of the LEC14 dodecasaccharide epitope and the core-fucosylated decasaccharide clearly revealed that the b1,2-linked glycan branch did not significantly affect affinity to the three tested types of sugar receptor in this assay system In contrast, the consideration of the data for the N-glycan with bisecting GlcNAc (B) with reductions in affinity underscores the sensitivity of this parameter to other structural alterations in the N-glycan Because the nature of the carbohydrate-binding protein matters notably in this respect, it is mandatory to proceed to test the pergalactosylated LEC14 N-glycan against a 1990 complex panel of binding partners In order to extend mapping the ligand properties of the LEC14 dodecasaccharide, we thus moved in our analysis from a test system with purified sugar receptors to cell surfaces with an array of lectins To add potential clinical relevance we selected tumor cells of different histogenesis representing common cancer types In the same way as isolated lectins these established cell models also offer the advantage for comparative analysis when adequately controlled for constant surface properties Affinity to tumor cell surfaces The first step in the cytofluorimetric analysis was dedicated to documenting the carbohydrate-dependent and saturable binding of the labeled neoglycoprotein to cell surfaces (Fig 3) Biotinylated carrier protein without the N-glycan failed to produce a signal above background, excluding interactions by the protein part or its label Mimicking the situation when a glycoprotein encounters a cell surface, the neoglycoprotein A reacted with cell surfaces in a cell type-specific manner (Fig 4) As highlighted by these results, distinct preference of binding was determined for the B- and T-lymphoblastoid cells among the set of leukemia ⁄ lymphoma lines and to the mammary carcinoma cells among the carcinoma lines, when measuring staining FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS ´ S Andre et al 128 100 101 102 103 128 128 102 103 104 101 102 103 104 D 104 101 102 103 104 100 55.0 %/45.1 100 128 104 F 100 101 D 100 G 100 101 102 103 101 102 103 104 H 64.4 %/62.3 88.4 %/56.1 104 104 104 C 103 128 101 102 103 16.3 %/15.3 103 102 74.4 %/29.4 101 102 104 128 100 128 128 104 E 100 101 98.2 %/177.8 83.3 %/420.6 number of events 103 103 C B 70.3 %/252.2 101 102 102 A 100 101 B 0 number of events 100 100 128 128 Fig Semilogarithmic representation of the fluorescent surface staining of cells of the human T-lymphoblastoid line CCRF-CEM in the absence of incubation with the biotinylated neoglycoprotein (negative control; shaded) and after incubation with increasing concentrations of neoglycoprotein in two steps: up to lgỈmL)1 (lines with 0.5 lgỈmL)1, lgỈmL)1 and lgỈmL)1 from left to right); (A) and up to 50 lgỈmL)1 (lines with lgỈmL)1, lgỈmL)1, 10 lgỈmL)1, 25 lgỈmL)1 and 50 lgỈmL)1 from left to right); (B) Controls with an incubation step using biotinylated carrier protein (25 lgỈmL)1); (C) instead of the neoglycoprotein and an inhibition of staining using glycoinhibitors (75 mM lactose and mg asialofetuinỈmL)1); (D) document lack of label ⁄ carrier protein-dependent binding and the carbohydrate dependence of binding A 128 128 Extended LEC14-type N-glycan as lectin ligand 100 101 102 103 104 Fig Semilogarithmic representation of the binding of the fluorescent indicator (streptavidin ⁄ R-phycoerythrin conjugate) in the absence of the probe during processing (negative control; shaded) and after the incubation step with the biotinylated neoglycoprotein (25 lgỈmL)1; black line) for the B-lymphoblastoid line Croco II (A), the T-lymphoblastoid line CCRF-CEM (B), the erythroleukemia line K-562 (C), the acute myelogenous leukemia line KG-1 (D), the mammary carcinoma line DU4475 (E) and the colon adenocarcinoma lines C205 (F), SW480 (G) and SW620 (H) Quantitative data on percentage of positive cells (%) and mean channel fluorescence are given in each panel intensity Extending the data of the solid-phase assay, the LEC14 dodecasaccharide is a ligand for cellular lectins To give potential reason to the presence of the GlcNAc-TVII, as detected in the LEC14 mutant [19], FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS the comparison between binding data of this neoglycoprotein (A) and that presenting the core-fucosylated N-glycan (C) without the b1,2-branch is expedient In general, comparison to the other, so far tested 1991 ´ S Andre et al Extended LEC14-type N-glycan as lectin ligand Biodistribution in vivo Fig Comparison of the percentage of positive cells (upper panel) and mean channel fluorescence (bottom panel) in flow cytofluorimetric analysis using the LEC14-dodecasaccharide-bearing neoglycoprotein A and neoglycoproteins with complex-type biantennary N-glycan ligand parts substituted by bisecting GlcNAc (B) or corefucosylation (C) or without any substitution (D) (see Fig for structural comparison) Data for neoglycoproteins B, C, D have previously been published [12–14] and are shown for comparison The standard deviations within experimental series are generally below 7.5% carrier-immobilized N-glycans revealed rather favorable ligand properties (Fig 5) A tendency for enhanced binding relative to the core-fucosylated N-glycan indicated that the new branch is not an inert modification at this level of testing The decrease of cell positivity for KG-1 cells and rather constant results for K-562 cells can be judged as internal controls for cell-type specificity in the direct comparison to the core-fucosylated N-glycan Thus, this assay system revealed several cases with an improvement of ligand properties with cell type-dependent characteristics Because the N-glycan profile of glycoproteins not only governs cell surface binding in vitro but also serum survival in circulation, a parameter of interest for prolonging bioavailability of pharmaproteins, we next tested the influence of this N-glycan in biodistribution analysis in vivo 1992 Organ retention and blood content of the iodinated neoglycoprotein (A) were monitored after intravenous injection Six time points from 15 to 12 h were set to determine the time course of these parameters Hepatic uptake was rapid and the major route of blood clearance (Table 1) When comparing blood ⁄ organ retention of this neoglycoprotein for the four major sites to the cases of the so far tested N-glycans (B–D), blood clearance was best for the neoglycoprotein bearing the LEC14 dodecasaccharide (A) (Fig 6) We have deliberately run these experiments in tumor-bearing mice to look at tumor uptake relative to blood background, a factor with impact on sensitivity of tumor imaging The tumor ⁄ blood ratio after h was 0.7 for neoglycoprotein (A) but 0.53 for C bearing a corefucosylated N-glycan with 3.11 ± 0.17% in blood and 1.65 ± 0.06% in the tumor The detected difference adds to the evidence for modulation of ligand properties by the extended LEC14 motif Placing this glycan at best position in this respect, the ratio for the unsubstituted nonasaccharide N-glycan (D) was 0.38 (tumor: 1.16 ± 0.09; blood: 3.07 ± 0.06%) and 0.47 (tumor: 1.47 ± 0.09; blood: 3.11 ± 0.14%) for the decasaccharide with the bisecting GlcNAc moiety (B) Regarding the individual organ sites no major alteration of uptake and retention after h was detectable except for the N-glycan with bisecting GlcNAc (Fig 6) Discussion The basic complex-type biantennary N-glycan is subject to enzymatic substitutions Structural aspects have been mostly clarified but the functional significance of their presence is still a matter of debate Our hypothesis interprets occurrence of substitutions as a means to modulate ligand properties in interactions with endogenous lectins The versatile potential for fine-tuning a respective information transfer would then clearly outweigh the required investment in coding for the diversity of glycosyltransferases and regulation of their activity In other words, glycosyltransferase activities produce lectin-binding epitopes By virtue of adding substitutions, which may not even directly participate in binding, they might also affect the affinity of the binding sites To demonstrate that a structural alteration in an N-glycan changes its binding parameters requires experimental evidence difficult to collect with natural glycoproteins They generally present more than one type of glycan chain and exhibit microheterogeneity, confounding efforts to establish a direct structure ⁄ activity profile To address this issue, we turned FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS ´ S Andre et al Extended LEC14-type N-glycan as lectin ligand Table Biodistribution of LEC14-dodecasaccharide-bearing neoglycoprotein A in Ehrlich-solid-tumor-bearing mice (% injected dosg)1 tissue) Each value indicates the mean ± SD for four to five mice Time (h) 1⁄4 h Blood Liver Kidneys Spleen Heart Lungs Thymus Pancreas Lymph node Muscle Brain Vertebrae Tumor 5.15 34.45 1.53 0.73 0.24 0.55 0.08 0.36 0.09 0.28 0.07 0.21 0.36 1⁄2 h ± ± ± ± ± ± ± ± ± ± ± ± ± 0.36 4.49 0.23 0.10 0.02 0.07 0.01 0.07 0.02 0.02 0.01 0.03 0.02 4.49 6.51 4.60 2.37 1.50 1.99 1.71 1.87 1.25 0.59 0.18 1.04 1.97 ± ± ± ± ± ± ± ± ± ± ± ± ± 1h 0.22 0.74 0.56 0.27 0.11 0.14 0.14 0.21 0.12 0.06 0.02 0.09 0.22 2.19 2.52 2.96 1.23 0.69 1.10 1.01 1.05 0.69 0.36 0.10 0.67 1.54 Fig Comparison of aspects of the biodistribution patterns of iodinated neoglycoproteins with the following complex-type N-glycan ligand parts h afer injection: LEC14 dodecasaccharide (A) and complex-type biantennary N-glycans substituted by bisecting GlcNAc (B) or core-fucosylation (C) or without any substitution (D) (see Fig for structural comparison) Data for neoglycoproteins B, C, D have previously been published [12–14] and are shown for comparison The range of the standard deviation shown for each result by bars was between 1.95 and 21.6% to the synthesis of neoglycoproteins In contrast to free N-glycans they harbor a homogeneous sugar part and favorably maintain a local density akin to natural glycoproteins In fact, affinity to galectins is sensitive to changes in local density of glycan chains and improved by certain modes of clustering [25–27] As stated above, it is our aim to delineate structure ⁄ activity profiles for N-glycans Toward this end, we have so far tested three types of biantennary N-glycans in different assay systems [12–14] They were confronted with different situations in vitro and in vivo, i.e the Nglycan as ligand in a matrix simulating a cell membrane or in solution ⁄ serum confronted with lectin-presenting FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS 3h ± ± ± ± ± ± ± ± ± ± ± ± ± 0.33 0.14 0.64 0.20 0.13 0.17 0.16 0.20 0.13 0.04 0.01 0.15 0.21 0.92 1.29 1.45 0.58 0.29 0.48 0.47 0.40 0.38 0.16 0.04 0.32 0.58 6h ± ± ± ± ± ± ± ± ± ± ± ± ± 0.13 0.17 0.33 0.10 0.04 0.09 0.16 0.06 0.09 0.04 0.01 0.10 0.07 0.62 0.95 0.81 0.33 0.18 0.38 0.23 0.20 0.18 0.11 0.02 0.16 0.37 12 h ± ± ± ± ± ± ± ± ± ± ± ± ± 0.06 0.07 0.28 0.04 0.02 0.08 0.07 0.04 0.07 0.01 0.01 0.03 0.08 0.45 0.71 0.51 0.25 0.15 0.26 0.27 0.17 0.19 0.07 0.03 0.16 0.31 ± ± ± ± ± ± ± ± ± ± ± ± ± 0.10 0.03 0.22 0.05 0.01 0.01 0.12 0.12 0.03 0.01 0.00 0.03 0.02 cell surfaces Evidently, monitoring tumor cells and biodistribution has relevance to the glycan’s suitability for drug targeting or imaging [28–30] Our previous results with the neoglycoprotein probes, which were kept rather constant in all relevant features (nature of carrier protein, linker chemistry, yield of glycan incorporation), supported the hypothesis given above [12–14] In this report, we examined the impact of the pergalactosylated LEC14 motif, a b1,2-linked LacNAc disaccharide emerging from the central bMan unit of a core-fucosylated N-glycan [7] (Fig 1) The bMan moiety of a LEC14 N-glycan is substituted in positions 2, and 6, a remarkable illustration of the capacity of glycan units to engender branching, in contrast with amino acids and nucleotides As outlined in the introduction, a distinct N-acetylglucosaminyltransferase (GlcNAc-TVII) is responsible for the introduction of the b1,2-GlcNAc into mammalian N-glycans [19] Of note, a b1,2-substitution at this site of the core is also found in nonmammalian N-glycan chains, here with xylose as added sugar unit [31–33] This position is thus apparently predisposed for enzymatic modification Immunologically, this residue is relevant due to its allergenic activity, an indication for accessibility to interactions with immunoglobulin E [33] Likewise, the b1,2-GlcNAc residue at this position of a mammalian-type core-fucosylated N-glycan is a contact point, as shown by its acceptor capacity in enzymatic galactosylation [23] Moreover, the selection process to isolate the LEC14 mutant cells exploited the detrimental effect of this core substitution on an interaction with a receptor protein, i.e reduction of binding of the plant agglutinins PSA ⁄ LCA [15,16] Our results with purified lectins ⁄ antibodies reveal no major influence of this enzymatically elongated branch on ligand properties, when compared with the core-fucosylated N-glycan lacking this branch 1993 ´ S Andre et al Extended LEC14-type N-glycan as lectin ligand Looking at the growth ⁄ invasion-regulatory galectin-1, it might be that the necessary contact to the subterminal GlcNAc residue during binding, a factor contributing to ligand selection [34], is spatially hindered No affinity enhancement relative to the core-fucosylated decasaccharide was detectable The affinity of binding of the monomeric galectins-3 and -5 was considerably reduced, arguing in favor of an influence of the strong cross-linking activity of galectin-1 as a clue for the functional divergence noted in a tumor cell system [35,36] No indication for positive cooperativity of galectin-3 binding was observed This binding mode was operative with laminin as substratum for this generally monomeric lectin which can form a small extent of pentamer in solution [37,38] When testing cell surfaces with their full array of carbohydrate-binding proteins, a clear impact of presence of the new branch was determined This effect hinged on the cell type, preferentially leading to increased binding relative to the core-fucosylated decasaccharide as ligand In addition to its principal value to delineate evidence for a structure ⁄ activity correlation this result signifies that cell-presented lectins in most of these tumor lines not share the core specificity with the plant agglutinins PSA ⁄ LCA which would have been impaired by introducing the b1,2-branch Our result underscores differences between plant and mammalian lectins and recommends using endogenous lectins for functional glycoproteomic profiling of clinical samples [39] The evidence for a contribution of this b1,2-branch in the LEC14-type dodecasaccharide to overall ligand properties was supported by the biodistribution analysis, revealing rapid clearance elicited by pergalactosylated LEC14 epitope In contrast to galectins the C-type endocytic receptor of hepatocytes accommodates galactose as central contact point [40] This result can be relevant for an application Actually, tailoring of the glycan part of pharmaproteins (glycoengineering) has become a fertile field of research in order to manipulate cellular uptake and serum half-life [41–47] The measured rapid clearance of the respective neoglycoprotein bearing a b1,2-branch constituted by a LacNAc disaccharide can be advantageous when using an iodinated glycoprotein for imaging, as it lowers the background The detection of this property immediately raises the question of how this parameter will be altered when the b1,2-branch is shifted away from the central Man unit to the Man residues in the branch extensions by GlcNAc-TIV or GlcNAc-TV Indeed, the consequences of hereby generating the two natural versions of triantennary N-glycans as part of neoglycoproteins have not yet been rigorously determined using 1994 our panel of assays Thus, it is our next challenge to address this issue Experimental procedures Synthetic and analytical procedures NMR spectra were recorded on a Bruker AMX 500 spectrometer (Karlsruhe, Germany) 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 (Duren, Germany, 300 Ã 25 mm) ă Carbohydrate-free BSA and bovine b1,4-galactosyltransferase were purchased from Sigma (Munich, Germany), alkaline phosphatase (calf intestine, molecular biology grade) from Roche Diagnostics (Heidelberg, Germany) UDPgalactose was a generous donation from Roche Diagnostics ESI-TOF mass spectra were recorded with methanol ⁄ water as solvent on a Micromass LCT spectrometer connected to an Agilent HP 1100 HPLC apparatus MALDI-TOF mass spectra were recorded on a Bruker Reflex III using linear mode and an acceleration voltage of 20 kV For sample preparation in MALDI-TOF-MS a solution of the neoglycoprotein (1 lL, mgỈmL)1) in 0.1% (v ⁄ v) trifluoroacetic acid (TFA) was mixed with 1.5 lL of 33% acetonitrile in 0.1% (v ⁄ v) TFA and 2.5 lL of a saturated solution of sinapinic acid in 0.1% (v ⁄ v) TFA and dried in high vacuum The structures of the synthetic N-glycans were routinely confirmed by the following 2D-NMR-experiments: TOCSY, NOESY, HMQC, HMQC-COSY, HMQC-DEPT, and HMQC-TOCSY Signals of NMR spectra were assigned according to the following convention including designation of spacer atoms illustrated for compound in Fig Preparation of neoglycoprotein A For conjugation of the derivatized dodecasaccharide to the carrier protein the amino group was transformed into its isothiocyanate In a 1.5 mL plastic vial 6-aminohexanoylN-glycan (0.77 mg, 0.34 lmol) was dissolved in sodium hydrogencarbonate (200 lL, 10 mgỈmL)1) followed by addition of dichloromethane (200 lL) and thiophosgene (5 lL, 19.7 lmol) The biphasic mixture was vigorously stirred After h (TLC: isopropanol ⁄ m ammonium acetate, : 1) the mixture was centrifuged and the aqueous phase was separated Subsequently, the organic phase was extracted twice with sodium hydrogencarbonate (100 lL, 10 mgỈmL)1) The combined aqueous phases were extracted twice with dichloromethane (500 lL) Carbohydrate-free BSA (2 mg) was dissolved in the aqueous solution of the isothiocyanate, and the reaction vial was kept for days at ambient temperature FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS ´ S Andre et al The reaction mixture was centrifuged, and the clear supernatant was fractionated by gel filtration (Pharmacia Hi Load Superdex 30 (600 · 16 mm); eluent: 0.1 m ammonium hydrogencarbonate; flow rate 0.75 mLỈmin)1; detection: 214 and 254 nm) Further quality controls were performed by gel electrophoretic analysis and colorimetric determination of the average glycan content as described previously [12–14]: yield, 2.07 mg; Rf amine ¼ 0.14 (i-propanol ⁄ m ammonium acetate, : 1); Rf isothiocyanate ¼ 0.50 (i-propanol ⁄ m ammonium acetate, : 1) MALDI-MS: Mcalcd ¼ 68694, 70957, 73220, 75484 (1, 2, 3, N-glycans per BSA molecule); Mfound ¼ 68649, 70971, 73242, 75475 (1, 2, 3, N-glycans per BSA molecule) The neoglycoprotein A was then used in solid-phase and cell-binding assays either free of label or for labeling with the N-hydroxysuccinimide ester derivative of biotin under conditions identical to the preparation of the other N-glycan-bearing probes tested previously [12–14] Solid-phase assay The matrix for the assay was established by adsorption of neoglycoprotein to the surface of plastic microtiter plate wells under conditions used previously [12–14] Controls for standardizing coating density were performed with biotinylated neoglycoprotein using streptavidin–peroxidase conjugate as indicator Ligand properties of the N-glycan were probed with different types of carbohydrate-binding proteins The galactoside-specific agglutinin from mistletoe (Viscum album L agglutinin, VAA), human galectin-1, murine galectin-3 and rat galectin-5 as well as the immunoglobulin G subfraction with preferential affinity to b-galactosides from human serum were isolated and checked for purity and quaternary structure by gel electrophoresis and filtration, electrospray ionization MS, ultracentrifugation and haemagglutination [48–54] Biotinylation was carried out under activity-preserving conditions, and label incorporation was assessed by binding assays with streptavidin– peroxidase conjugate or a proteomics protocol [48,55] Binding studies of the sugar receptors to the glycan-presenting matrix were performed by stepwise increases of probe concentration up to saturation with duplicates at each concentration and at least four independent series including controls to determine extent of carbohydrate-dependent binding by its inhibition using a mixture of 75 mm lactose and mg asialofetuinỈmL)1, and the data sets were algebraically transformed to obtain KD values and the number of bound sugar receptor molecules at saturation (Bmax), following the protocol of our previous reports on neoglycoproteins with synthetic N-glycans [12–14] Cell-binding assay Using the biotinylated neoglycoprotein as probe, automated flow cytofluorimetric analysis of carbohydrate-dependent FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS Extended LEC14-type N-glycan as lectin ligand cell surface binding was performed with the following human tumor lines: Croco II (B-lymphoblastoid cell line), CCRF-CEM (T-lymphoblastoid cell line), K-562 (erythroleukemia cell line), KG-1 (acute myelogenous leukemia cell line), DU4475 (mammary carcinoma cell line) as well as C205, SW480 and SW620 (colon adenocarcinoma cell lines) Except for the Croco II line established in our laboratory [56] the cells were commercially available (American Type Culture Collection, Rockville, MD, USA) and routinely cultured under the recommended conditions The adherent colon carcinoma cells were detached by exposing them to NaCl ⁄ Pi containing mm EDTA Prior to the analysis cells were routinely washed carefully with Dulbeccos’s NaCl ⁄ Pi solution containing 0.1% (w ⁄ v) carbohydrate-free BSA to remove any inhibitory serum glycoproteins and to saturate nonspecific protein-binding sites For this purpose, an incubation step with ligand-free carrier protein for 30 at °C was added prior to the incubation with the labeled neoglycoprotein at this temperature to minimize uptake by endocytosis Carbohydrate-dependent binding of the neoglycoprotein (25 lgỈmL)1) to the cells (8 · 106 cellsỈmL)1) was assessed in a FACScan instrument (BectonDickinson, Heidelberg, Germany) with the fluorescent indicator conjugate streptavidin ⁄ R-phycoerythrin (1 : 40; Sigma) Controls to assess carbohydrate-independent binding of the carrier via its protein part or label and to document sugar inhibition were run in each series, as previously described [12–14] Analysis of in vivo biodistribution Radiolabeling of the neoglycoprotein was performed by the chloramine-T method [57] Briefly, fresh chloramine-T and sodium metabisulfite solutions were prepared, and the neoglycoprotein was dissolved in NaCl ⁄ Pi (pH 7.2) at a concentration of mg proteinỈmL)1 A 10 lL portion of 125 I-labelled NaI (74 MBqỈmL)1 NaCl ⁄ Pi) solution was added to 50 lL of the neoglycoprotein-containing solution, subsequently 10 lL of chloramine-T (3 mgỈmL)1 H2O) solution were added, and the mixture was incubated at room temperature for Thereafter, chloramine-T solution was pipetted to the above mixture in two further portions at intervals of min, and then the reaction was stopped by adding 30 lL of freshly prepared sodium metabisulfite solution (5 mgỈmL)1) Label-free neoglycoprotein (50 lg) was added as a carrier prior to the separation step by Sephadex G-50 (Pharmacia Biotech, Freiburg, Germany) gel permeation chromatography to remove any reagents from the radioiodinated product The specific radioactivity of batches of 125 I-labeled neoglycoprotein was in the range between and 10 MBqỈmg)1 protein To monitor biodistribution of the iodinated product tumor-bearing mice were used [58,59] Approximately · 106 Ehrlich ascites tumor (EAT) cells had been injected subcutaneously into the right rear leg of male ddY mice of the age of weeks for tumor inoculation On 1995 Extended LEC14-type N-glycan as lectin ligand the sixth to eighth day after inoculation, when the tumor had grown to 0.3–0.6 g in weight, radioiodinated neoglycoprotein was injected intravenously at a dose of 80–100 kBq (equivalent to 10 lg of protein) via the tail vein Tissues including blood samples were obtained after the indicated periods, weighed, and the radioactivity level was assessed with an Auto Well gamma System (Aloka ARC 300, Tokyo, Japan) The percentage of injected dose per gram of wet tissue or per ml of blood was calculated in each case as described previously [12] Acknowledgements We express our gratitude to B Hofer and L Mantel for skillful technical assistance, to Dr S Namirha for helpful discussion and to the Deutsche Forschungsgemeinschaft, the Dr M.-Scheel-Stiftung fur Krebsă forschung, the Fonds der Deutschen Chemischen Industrie, Roche Diagnostics and the Mizutani Foundation for Glycoscience for generous financial support References Brockhausen I & Schachter H (1997) Glycosyltransferases involved in N- and O-glycan biosynthesis In Glycosciences: Status and Perspectives (Gabius H-J & Gabius S, eds), pp 79–113 Chapman & Hall, Weinheim Reuter G & Gabius H-J (1999) 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determination of cell surface sugar receptor (lectin) expression by neoglycoenzymes of a human myeloid-marker-expressing B-lymphoblastoid cell line Anticancer Res 11, 793–800 57 Kojima S, Shimura N, Kubodera A, Takahashi T & Oyamada H (1991) Radioimmunodetection of human colon cancer in nude mice by a new monoclonal antibody A7 against human colorectal cancer Nucl Med Biol 18, 847–853 58 Kojima S & Gabius H-J (1988) Biodistribution of neoglycoproteins in mice bearing solid Ehrlich tumor J Cancer Res Clin Oncol 114, 468–472 ´ 59 Kojima S, Andre S, Korchagina EY, Bovin NV & Gabius H-J (1997) Tyramine-containing poly (4-nitrophenylacrylate) as iodinatable ligand carrier in biodistribution analysis Pharmaceut Res 14, 879–886 FEBS Journal 272 (2005) 1986–1998 ª 2005 FEBS ... of testing The decrease of cell positivity for KG-1 cells and rather constant results for K-562 cells can be judged as internal controls for cell- type specificity in the direct comparison to the. .. [12–14] and are shown for comparison The range of the standard deviation shown for each result by bars was between 1.95 and 21.6% to the synthesis of neoglycoproteins In contrast to free N-glycans they... highlighted by these results, distinct preference of binding was determined for the B- and T-lymphoblastoid cells among the set of leukemia ⁄ lymphoma lines and to the mammary carcinoma cells among the