Glycosyl conjugates of biotinylated diaminopyridine applied for study of carbohydrate-to-carbohydrate interaction

Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of homotypic CCI between “Os Fr.B” having 5–6 GlcNAc termini, vs. absence of such homotypic CCI between “Os Fr.1” having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3 ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using BAP conjugates of glycosyl epitopes.     

Interaction of N-linked glycans, having multivalent GlcNAc termini, with GM3 ganglioside

GM3 ganglioside interacts specifically with complex-type N-linked glycans having multivalent GlcNAc termini, as shown for (1) and (2) below. (1) Oligosaccharides (OS) isolated from ConA-non-binding N-linked glycans of ovalbumin, whose structures were identified as penta-antennary complex-type with bisecting GlcNAc, having five or six GlcNAc termini (OS B1, B2), or bi-antennary complex-type having two GlcNAc termini (OS I). OS I is a structure not previously described. (2) Multi-antennary complex-type N-linked OS isolated from fetuin, treated by sialidase followed by β-galactosidase, having three or four GlcNAc termini exposed. These OS, conjugated to phosphatidylethanolamine (PE), showed clear interaction with ³H-labeled liposomes containing GM3, when various doses of OS-PE conjugate were adhered by drying to multi-well polystyrene plates. Interaction was clearly observed only with liposomes containing GM3, but not LacCer, Gb4, or GalNAcα1-3Gb4 (Forssman antigen). GM3 interaction with PE conjugate of OS B1 or B2 was stronger than that with PE conjugate of OS I. GM3 interacted clearly with PE conjugate of N-linked OS from desialylated and degalactosylated fetuin, but not native fetuin. No binding was observed to cellobiose-PE conjugate, or to OS-PE conjugate lacking GlcNAc terminus. Thus, GM3, but not other GSL liposomes, interacts with various N-linked OS having multiple GlcNAc termini, in general. These findings suggest that the concept of carbohydrate-to-carbohydrate interaction can be extended to interaction of specific types of N-linked glycans with specific GSLs. Natural occurrence of such interaction to define cell biological phenomena is under investigation. All solvent ratios are by volume. An erratum to this article can be found at     

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

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

Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.     

The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x

Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies against the carbohydrate antigens Lewisx (Lex) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDNF) inhibit binding of DC-SIGN to SEAs, suggesting that these glycan antigens may be critically involved in binding. In a solid-phase adhesion assay, DC-SIGN-Fc binds polyvalent neoglycoconjugates that contain the Lex antigen, whereas no binding was observed to Galbeta1-4GlcNAc, and binding to neoglycoconjugates containing only alpha-fucose or oligosaccharides with a terminal alpha1-2-linked fucose is low. These data indicate that binding of DC-SIGN to Lex antigen is fucose-dependent and that adjacent monosaccharides and/or the anomeric linkage of the fucose are important for binding activity. Previous studies have shown that DC-SIGN binds HIV gp120 that contains high-mannose-type N-glycans. Site-directed mutagenesis within the carbohydrate recognition domain (CRD) of DC-SIGN demonstrates that amino acids E324 and E347 are involved in binding to HIV gp120, Lex, and SEAs. By contrast, mutation of amino acid Val351 abrogates binding to SEAs and Lex but not HIV gp120. These data suggest that DC-SIGN recognizes these ligands through different (but overlapping) regions within its CRD. Our data imply that DC-SIGN not only is a pathogen receptor for HIV gp120 but may also function in pathogen recognition by interaction with the carbohydrate antigens Lex and possibly LDNF, which are found on important human pathogens, such as schistosomes and the bacterium Helicobacter pylori.     

Bisecting GlcNAc mediates the binding of annexin V to Hsp47

The bisecting N-acetylglucosamine (GlcNAc) structure, formed through catalysis by UDP-N-acetylglucosamine : beta-D-mannoside beta-1,4-N-acetylglucosaminyltansferase III (GnT-III), is responsible for a variety of biological functions. We have previously shown that annexin V, a member of the calcium/phospholipid-binding annexin family of proteins, has binding activity toward the bisecting GlcNAc structure. In this study, we reported on a search for potential target glycoproteins for annexin V in a rat hepatoma cell line, M31. Using a glutathione S-transferase (GST)-annexin V immobilized sepharose 4B affinity column to trap interacting proteins produced by the GnT-III-transfected M31 cells, we isolated a 47 kDa protein. It was identified as Hsp47 by an N-terminal sequence analysis. Immunoprecipitation experiments showed that annexin V interacted with Hsp47. The association of annexin V and Hsp47 was abolished by treatment with N-glycosidase F or preincubation with sugar chains containing bisecting GlcNAc, suggesting that the bisecting GlcNAc plays an important role in the interaction. An oligosaccharide analysis of Hsp47 purified from GnT-III-transfected M31 cells was shown to have the bisecting GlcNAc structure, as detected by erythroagglutinating phytohemagglutinin (E4-PHA) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) analysis. Surface plasmon resonance analysis showed that annexin V was bound to Hsp47, bearing a bisecting GlcNAc with a Kd of 5.5 microM, whereas no significant binding was observed in the case of Hsp47 without a bisecting GlcNAc. In addition, immunofluorescence microscopy revealed the colocalization of annexin V, Hsp47, and a bisecting GlcNAc sugar chain around the Golgi apparatus. Collectively, these results suggest that the binding of annexin V to Hsp47 is mediated by a bisecting GlcNAc oligosaccharide structure and that Hsp47 is an intracellular ligand glycoprotein for annexin V.     

Identification of carbohydrates binding to lectins by using surface plasmon resonance in combination with HPLC profiling

A new, powerful method is presented for screening the binding in real time and taking place under dynamic conditions of oligosaccharides to lectins. The approach combines an SPR biosensor and HPLC profiling with fluorescence detection, and is applicable to complex mixtures of oligosaccharides in terms of ligand-fishing. Labeling the oligosaccharides with 2-aminobenzamide ensures a detection level in the fmol range. In an explorative study the binding of RNase B-derived oligomannose-type N-glycans to biosensor-immobilized concanavalin A (Con A) was examined, and an affinity ranking could be established for Man(5)GlcNAc(2) to Man(9)GlcNAc(2), as monitored by HPLC. In subsequent experiments and using well-defined labeled as well as nonlabeled oligosaccharides, it was found that the fluorescent tag does not interfere with the binding and that the optimum epitope for the interaction with Con A comprises the tetramannoside unit Manalpha2Manalpha6(Manalpha3)Man[D(3)B(A)4'], rather than the generally accepted trimannoside Manalpha6 (Manalpha3)Man [B(A)4' or 4(4')3]. In a similar experimental setup, the interaction of various fucosylated human milk oligosaccharides with the fucose-binding lectin from Lotus tetragonolobus purpureaus was studied, and it appeared that oligosaccharides containing blood group H could selectively be retained and eluted from the lectin-coated surface. Finally, using the same lectin and a mixture of O-glycans derived from bovine submaxillary gland mucin, minor constituents but containing fucose could selectively be picked from the analyte solution as demonstrated by HPLC profiling.     

Carbohydrate-based interactions of oviductal sperm reservoir formation-studies in the pig.

Competitive inhibition of sperm to explants of the oviductal epithelium was used to study the complementary receptor system that may be involved in the establishment of the oviductal sperm reservoir in the pig. Sperm binding to the oviductal explants is expressed as Binding Index (BI = sperm cells/0.01 mm(2)). From a set of glycoproteins with known oligosaccharide structures, only asialofetuin and ovalbumin showed inhibitory activity, indicating that ovalbumin may block high affinity binding sites (IC(50) congruent with 1.3 microM) and asialofetuin low affinity sites (IC(50) congruent with 18 microM) of the complementary receptor systems, whereas fetuin carrying terminal sialic acid has no effect. Ovalbumin glycopeptides were isolated by Con A affinity chromatography and reverse-phase HPLC following tryptic digestion. Glycopeptides and enzymatically released glycans were analyzed by MS, and were shown to represent preferentially the two high mannose type glycans (Man)(5)(GlcNAc)(2) and (Man)(6)(GlcNAc)(2), and as a minor component the hybrid type glycan (Hex)(4)(GlcNAc)(5). Glycopeptides (84% inhibition) and glycans (81% inhibition) significantly reduced sperm-oviduct binding at a concentration of 3 microM, whereas the deglycosylated peptides showed no inhibitory activity. Mannopentaose (IC(50) congruent with 0.8 microM) representing the oligomannose residue of the high mannose glycans of ovalbumin was as effective as ovalbumin. These data indicate that the carbohydrate-based mechanisms underlying the formation of the oviductal sperm reservoir in the pig is the result of the concerted action of at least the high-affinity binding sites for oligomannose or nonreducing terminal mannose residues and low-affinity binding of galactose.     

Rapid determination of the binding affinity and specificity of the mushroom Polyporus squamosus lectin using frontal affinity chromatography coupled to electrospray mass spectrometry

The binding affinity and specificity of the mushroom Polyporus squamosus lectin has been determined by the recently developed method of frontal affinity chromatography coupled to electrospray mass spectrometry (FAC/MS). A micro-scale affinity column was prepared by immobilizing the lectin ( approximately 25 microg) onto porous glass beads in a tubing column (9.8 microl column volume). The column was then used to screen several oligosaccharide mixtures. The dissociation constants of 22 sialylated or sulfated oligosaccharides were evaluated against the immobilized lectin. The lectin was found to be highly specific for Neu5Acalpha2-6Galbeta1-4Glc/GlcNAc containing oligosaccharides with K(d) values near 10 microM. The FAC/MS assay permits the rapid determination of the dissociation constants of ligands as well as a higher throughput screening of compound mixtures, making it a valuable tool for affinity studies, especially for testing large numbers of compounds.     

Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: in vitro studies

Conjugates of three components, folic acid-poly(ethylene glycol)-distearoylphosphatidylethanolamine (FA-PEG-DSPE), derived from PEG with molecular masses of 2000 and 3350 Da were synthesized by a carbodiimide-mediated coupling of FA to H2N-PEG-DSPE. The conjugates were characterized by 1H NMR, MALDI-TOF, and HPLC analysis of enzymatic cleavage with carboxypeptidase G. As a prototype of a folate receptor (FR)-targeted system, the conjugates were formulated at 0.5 mol % phospholipid in hydrogenated phosphatidylcholine/cholesterol liposomes with or without additional methoxyPEG2000-DSPE. In vitro binding studies were performed with sublines of M109 (murine lung carcinoma) and KB (human epidermal carcinoma) cells each containing high and low densities of FR. FA-PEG-DSPE significantly enhanced liposome binding to tumor cells. The best binding was observed when FA-PEG liposomes contained no additional mPEG-lipid. In fact, our experiments showed that the presence of mPEG on liposomal surfaces significantly inhibited FA-PEG-liposome binding to FR. Increasing the molecular mass of the PEG tether from 2000 to 3350 Da improved the FR binding, particularly in the case of mPEG-coated liposomes. The FA-PEG liposomes bound to M109-HiFR cells very avidly as demonstrated by the inability of free FA (used in a 700-fold excess either at the beginning or at the end of the incubation) to prevent the cell binding. This is in contrast to the 5-10-fold lower cell binding activity of mPEG-FA compared to that of free FA, and likely to be related to the multivalent nature of the liposome-bound FA. Only 22% of FA-PEG3350 and 32% of FA-PEG3350/mPEG cell-associated liposomes could be removed by exposure to pH 3, conditions that dissociate FA-FR, suggesting that more than two-thirds of the bound liposomes were internalized during incubation for 24 h at 37 degrees C. FA-targeted liposomes also show enhanced nonspecific binding to extracellular tissue culture components, a phenomenon especially relevant in short incubation time experiments.     

Quantification of indole-3-acetic acid and amino acid conjugates in rice by liquid chromatography-electrospray ionization-tandem mass spectrometry

A method for quantifying indole-3-acetic acid (IAA) and its conjugates with the six amino acids, Ala, -Asp, -Ile, -Glu, -Phe and -Val, in rice (Oryza sativa) by using high-performance liquid chromatography coupled with electrospray ionization and tandem mass spectrometry (HPLC-ESI-MS/MS) is described. Samples from the rice plant or callus were treated with 80% acetone in water containing 2.5 mM diethyl dithiocarbamate. Each extract was partially purified in C18 cartridge column for solid-phase extraction (SPE) and subjected to HPLC-ESI-MS/MS without converting the product. The detection limit was 3.8 fmol for IAA, and 0.4-2.9 fmol for the IAA amino acid conjugates. The method was applied to the analysis of IAA and its conjugates in rice seedlings, dehulled rice and calli, using 20-100 mg tissue samples.     

Mimetic ligand-based affinity purification of immune complexes and immunoconjugates

We developed a simple purification method to purify alkaline phosphatase/anti-alkaline phosphatase IgG as immune complexes using mimetic affinity chromatography wherein the antibody was either a monospecific antibody, a bispecific antibody or a commercial polyclonal IgG conjugated with alkaline phosphatase (AP–IgG) covalently. The immune complexes or conjugates were efficiently bound on the mimetic Blue A6XL column and eluted under mild conditions (5–20 mM phosphate buffer). A similar strategy of purifying peroxidase/anti-peroxidase antibody complexes was also successfully demonstrated using the mimetic Red 3 column. Mimetic affinity chromatography thus appears to be a simple method to purify the desired monospecific or bispecific antibodies from the respective hybridomas and quadromas.     

Caenorhabditis elegans N-glycans containing a Gal-Fuc disaccharide unit linked to the innermost GlcNAc residue are recognized by C. elegans galectin LEC-6

We report a detailed structural analysis of the N-glycans of Caenorhabditis elegans recognized by C. elegans galectin LEC-6. Glycoproteins of C. elegans captured by an immobilized LEC-6 affinity adsorbent were isolated. The N-glycans of these glycoproteins were then liberated by hydrazinolysis and labeled with the fluorophore 2-aminopyridine (PA). The derived pyridylaminated (PA)-sugars were further fractionated by rechromatography on immobilized LEC-6 adsorbent and by reversed-phase high-performance liquid chromatography (HPLC). The structures of the PA-sugars thus obtained were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS) in conjunction with glycosidase digestion. We confirmed that all PA-sugars having affinity for LEC-6 contain a Gal-Fuc disaccharide unit, and that this unit is bound to the innermost GlcNAc residue of the N-glycan chain. The dissociation constants of LEC-6 for these glycans were measured by frontal affinity chromatography. LEC-6 exhibited higher affinity for oligosaccharides having a Gal-Fuc unit linked to position 6 of the innermost GlcNAc residue than for those having Galbeta1-4GlcNAc units. Affinity for the former disappeared, however, following treatment with beta-galactosidase. If the glycan contained a Hex-Fuc disaccharide linked to the penultimate GlcNAc residue, the affinity would be diminished. We propose, therefore, that the galectins of C. elegans utilize the Gal-Fuc disaccharide unit for recognition instead of the Gal-GlcNAc unit that is common in vertebrates.     

Use of heavy-metal clusters in the design of N-succinimidyl ester acylation reagents for side-chain-specific labeling of proteins.

New heavy transition metal carbonyl markers for protein labeling, containing an "Mn(CO)11" (M = Ru, Os, n = 3; M = Ir, n = 4) moiety, were prepared by reaction of "lightly stabilized" clusters with an N-succinimidyl ester functionalized phosphine, namely N-succinimidyl 3-diphenylphosphine-propionate (DPPS). The reaction of Os3(CO)11(DPPS) with the model amino acid beta-alanine was performed and led to the expected amide. From the reaction of Mn(CO)11(DPPS) with bovine serum albumin (BSA) in mixed organic/aqueous medium, conjugates bearing a fairly high number of metal carbonyl fragments were obtained, thus demonstrating the usefulness of this class of reagents for the selective and covalent graft of heavy metal clusters to side chain of proteins.     

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

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

Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG(2000)) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG(2000). At this proportion of DSPE-PEG(2000), the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG(2000) in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.     

Relative accessibility of N-acetylglucosamine in trimers of the adenovirus types 2 and 5 fiber proteins.

Fiber is an adenovirus capsid protein responsible for virus attachment to the cell surface and contains O-linked N-acetylglucosamine (GlcNAc). Results of both amino acid analysis and Dionex chromatography indicated that 3 to 4 and 1.7 to 2.5 mol of GlcNAc are attached per mol of affinity-purified adenovirus type 2 (Ad2) and Ad5 fibers, respectively. Fiber shares an epitope with nuclear pore proteins containing O-linked GlcNAc, as shown by reactivity to monoclonal antibody RL2 directed against these pore proteins. GlcNAc on fiber was found to serve as an acceptor for the transfer of galactose from UDP-galactose by 4 beta-galactosyl-transferase in Ad2 and Ad5 but not in Ad7; quantitation by labeling with UDP-[U-14C]galactose in this reaction gave a 100-fold-lower estimate of the GlcNAc content of fiber, suggesting that these monosaccharides are buried within fiber trimers and are not accessible to the transferase. Affinity chromatography on lectin-bound Sepharose beads showed that Ad2 and Ad5 fibers bound weakly to wheat germ agglutinin and did not bind to ricin or concanavalin A; weak binding to wheat germ agglutinin suggests either that GlcNAc is not easily accessible or that there are not sufficient GlcNAcs for efficient binding. These data suggest that O-linked GlcNAc might be important for Ad2 and Ad5 fiber assembly or stabilization.     

Immunochemical characterization of antisera reactivities toN-acetyl-d-glucosamine oligosaccharides with theβ(1–4)-glycosidic linkage

The (1–4)-linked oligosaccharides ofN-acetyl-d-glucosamine (GlcNAc) isolated from chitin were used to prepare synthetic immunogens and antigens by reductive amination of (GicNAc)_n to bovine serum albumin (BSA). The rabbit antisera produced to the (GlcNAc)_n-BSA conjugates were characterized using an enzyme-linked immunosorbent assay (ELISA) system under conditions that, only the antibodies with carbohydrate specificity were reactive with the solid-phase adsorbed (GlcNAc)_n-BSA antigens. Inhibition assays using the (GlcNAc)_n-BSA, (GlcNAc)_n oligosaccharides, and the reduced oligosaccharides showed a relative specificity of the antisera for the chain length of the (GlcNAc)_n sequences. For example, the anti-(GlcNAc)₅-and anti-(GlcNAc)₄-sera were inhibited best by the longer chain (GlcNAc)_n ologosaccharides with the antibody combining sites directed mainly to the cyclic GlcNAc residues of the (GlcNAc)_n-BSA conjugates. The antibody combining sites were in part directed to the acyclic moiety of the reducing end of the oligosaccharides as shown by the increased inhibitory activities of the reduced (GlcNAc)_n oligosaccharides particularly, with the anti-(GlcNAc)₂-and anti-(GlcNAc)₃-sera. The best hapten inhibitors for the anti-(GlcNAc)₂-BSA and anti-(GlcNAc)₁-BSA sera were theN-butylamine derivatives of (GlcNAc)₂ and (GlcNAc)₁, respectively, indicating that the antibodies were also reactive with the secondary amine formed between the reducing end of the oligosaccharides and the -amino groups of lysine.Abbreviations ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - GicNAc N-acetyl-d-glucosamine - (GlcNAc)_n Oligosaccharides containing GlcNAc in 1–4 linkages - (GlcNAc)₂ DGlcNAc(1–4)-d-GlcNAc - (GlcNAc)₃ (GlcNAc)₄ and (GlcNAc)₅, the homologous oligosaccharides of (GlcNAc)₂ - PBS phosphate buffered saline (0.01 M sodium phosphate, pH73 containing 0.15%M (NaCl) - PBSA PBS containing 1% BSA and 0.1% Tween-20 - ONPG o-nitrophenyl--d-galactopyranoside     

Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: Relating plasma circulation lifetimes to protein binding

The incorporation of poly(ethylene glycol) (PEG)-conjugated lipids in lipid-based carriers substantially prolongs the circulation lifetime of liposomes. However, the mechanism(s) by which PEG-lipids achieve this have not been fully elucidated. It is believed that PEG-lipids mediate steric stabilization, ultimately reducing surface-surface interactions including the aggregation of liposomes and/or adsorption of plasma proteins. The purpose of the studies described here was to compare the effects of PEG-lipid incorporation in liposomes on protein binding, liposome-liposome aggregation and pharmacokinetics in mice. Cholesterol-free liposomes were chosen because of their increasing importance as liposomal delivery systems and their marked sensitivity to protein binding and aggregation. Specifically, liposomes containing various molecular weight PEG-lipids at a variety of molar proportions were analyzed for in vivo clearance, aggregation state (size exclusion chromatography, quasi-elastic light scattering, cryo-transmission and freeze fracture electron microscopy) as well as in vitro and in vivo protein binding. The results indicated that as little as 0.5 mol% of 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE) modified with PEG having a mean molecular weight of 2000 (DSPE-PEG₂₀₀₀) substantially increased plasma circulation longevity of liposomes prepared of 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC). Optimal plasma circulation lifetimes could be achieved with 2 mol% DSPE-PEG₂₀₀₀. At this proportion of DSPE-PEG₂₀₀₀, the aggregation of DSPC-based liposomes was completely precluded. However, the total protein adsorption and the protein profile was not influenced by the level of DSPE-PEG₂₀₀₀ in the membrane. These studies suggest that PEG-lipids reduce the in vivo clearance of cholesterol-free liposomal formulations primarily by inhibition of surface interactions, particularly liposome-liposome aggregation.     

Biochemical characterization of inner sugar chains of acrosome reaction-inducing substance in jelly coat of starfish eggs

The inception of the acrosome reaction (AR) in the starfish Asterias amurensis is perceived to be strongly associated with sulfated polysaccharide chains derived from an extremely large proteoglycan-like molecule called AR-inducing substance (ARIS), in which one of the sugar fragments, named fragment 1 (Fr. 1), was composed of the repeating units of [-->4]-beta-D-Xylp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Fucp-4 (SO3-)-(1-->3)- alpha-L-Fucp-4(SO3-)-(1-->4)-alpha-L-Fucp-(1-->)n. In the current study, this sugar chain is inferred to link to the peptide part by O-glycosidic linkage through a sugar chain with different structural features from Fr. 1. This inner sugar portion of ARIS was isolated as Fr. 2 from the sonicated products of pronase digest of ARIS. Fr. 2, which retains AR-inducing activity to an admirable extent and has an apparent molecular size of 400 kDa, is composed of Gal, Xyl, Fuc, GalNAc, and GlcNAc in a molar ratio of 5:1:5:4:2 with O-sulfate substitutions at Gal-4, Gal-2, Gal-2,3 and Gal-2,4 (disulfated), Fuc-4, and GlcNAc-6. The study of Fr. 2 revealed that the major portion of the inner sugar chain of ARIS is composed of the heptasaccharide units of -->3)-Galp-(1-->3)-Fucp-(1-->3)-Galp-(1-->4)-GalNAcp-(1-->4)-GlcNAcp-6(SO3-)-(1-->6)-Galp-4(SO3-)-(1-->4)-GalNAcp-(1-->. This new structure of inner sugar chains of ARIS is elucidated by using electrospray ionization MS along with tandem mass analysis, sugar composition analysis, and methylation analysis of the sugar fragments obtained by acid-catalyzed resin-based partial hydrolysis of Fr. 2. Furthermore, this study corroborates that the sulfate groups are solely liable to the anionic character of ARIS, which ought to be present in the sugar chains of ARIS for its biological activity.