Leb-active glycolipid in human plasma: Measurement by radioimmunoassay

A radioimmunoassay that measures Le^b-active glycolipids in human plasma has been developed using antiserum from a goat immunized with a Le^b blood group hapten, lacto-N-difucohexaose I, conjugated to polylysine. Binding by the antiserum of lacto-N-difucohexaose I conjugated to ¹²⁵I-labeled bovine serum albumin is specifically inhibited by Le^b-active ceramide hexasaccharide. Plasma levels of the glycolipid are quantitated by comparing the inhibitory activity of plasma with that of the purified Le^b-active glycolipid. Plasma samples from 35 blood group O Le(a ? b +) individuals contain Le^b-active ceramide hexasaccharide at an average concentration of 0.9 μg/ml (range: 0.2 to 2.5 μg/ml); no Le^b-active glycolipid (less than 0.02 μg/ml) could be detected in plasma from blood group O Le(a + b?) or O Le(a? b?) individuals. Plasma from A₁ Le(a ? b+) individuals contains less Le^b-active glycolipid than plasma from A₂ Le(a? b+) individuals: its level in 19 samples of A, Le(a? b+) plasma averages 0.2 μg/ml (range: 0.1 to 0.45 μg/ml), and its level in 9 samples of A₂ Le(a? b+) plasma averages 1.1 μg/ml (range 0.8 to 1.3 μg/ml). About one-third of the total Le^b-active glycolipid in whole blood is associated with erythrocytes and the rest is found in plasma.     

A monoclonal antibody that recognizes both leb and Y (Ley) antigens

A hemagglutinating monoclonal antibody has been obtained from a mouse/mouse hybridoma after immunisation with the le^b-active oligosaccharide, lacto-N-difucohexaose I, coupled to edestin. The antibody agglutinated human red cells regardless of Lewis phenotype. Blood group O cells were strongly, agglutinated, and progressively weaker agglutination was observed with A₂, B and A₂B cells. Blood group A₁ and A₁B cells were not agglutinated.By examining the binding of the antibody to glycolipids and oligosaccharides it was shown that the Le^b and Y (Le^y)-haptens bind to a similar extent. Full binding activity was dependent on the presence of, both fucosyl residues.Abbreviations LND l lacto-N-difucohexaose l - IV²Fuc,lll⁴FucLcOse₄ LND l-OL, lacto-N-difucohexaitol l     

Affinity purification of monoclonal antibodies,using a bifunctional oligosaccharide hapten

A bifunctional hapten was synthesized consisting of a blood group A active tetrasaccharide (A-tetra) and a blood group Le^a active pentasaccharide. lacto-N-fucopentaose II (LNF II), linked to each other with a phenylaminothiourea spacer connecting the reducing ends (A-tetra-LNF II). The hapten was demonstrated to retain both blood group A and Le^a activity and could be easily bound to both monoclonal anti-A and anti-Le^a affinity columns. Due to the strong temperature dependence of the two antibodies in their binding to oligosaccharides, the bifunctional hapten could be utilized to achieve easy desorption in the final step of affinity purification of either monoclonal anti-Le^a or anti-A. The system is postulated to have general applicability in affinity purification of any ligate that binds with an avidity too high to achieve non-denaturing desorption.To whom correspondence should be addressed.     

Affinity chromatography of carbohydrate-specific immunoglobulins: coupling of oligosaccharides to sepharose.

A simple method has been developed for the coupling of oligosaccharides to Sepharose. The sugars are reacted with β-(p-aminophenyl)-ethylamine to form N-alkylglycosides which are then reduced with sodium borohydride to stable secondary amines. The derivatives are then coupled to cyanogen bromide-activated Sepharose through their arylamino groups. Yields are essentially quantitative based on starting oligosaccharides. An affinity column containing lacto-N-difucohexaose I coupled to Sepharose by this method was used for the purification of an antibody directed against this oligosaccharide. The antibody is absorbed by the gel and is specifically eluted by the free sugar.     

Diffusion-controlled reaction kinetics of the binding of carbon monoxide to the heme undecapeptide of cytochrome c (microperoxidase 11) in high viscosity solvents

Glycosphingolipids from human plasma with Le^a, Le^b, and H-type 1 (Le^dH) Lewis-blood-group activity have been analyzed after permethylation by electron impact mass spectrometry using an indirectly heated direct insertion probe. The spectra obtained are compared with that of permethylated neo-lactotetraosyl ceramide (Gl-3) from human plasma. The fragmentation patterns presented show clearly, that Le^a and H-type 1 glycosphingolipids are ceramide pentasaccharides while Le^b is a ceramide hexasaccharide. All Lewis-blood-group-active compounds investigated produced ions specific for type 1 carbohydrate chains. It is therefore concluded, that all compounds are derivatives of lacto-N-tetraose. The obtained spectra support the following sequences: Hexose-1→3-hexosamine[4←1-deoxyhexose]-hexose-hexose ceramide for the Le^a derivatives; deoxyhexose-hexose-1→3-hexosamine4←1-deoxyhexose]-hexose-hexose ceramide for the Le^b derivatives; and deoxyhexose-hexose-1→3-hexosamine-hexose-hexose ceramide for all H-type 1 (Le^dH) derivatives. In the case of the H-type 1 glycosphingolipids four subfractions were analyzed separately. While all four fractions contained the same carbohydrate sequence, significant differences were observed in the ceramide residues. Specific fragmentation patterns indicate the presence of sphingosine, icosasphingosine, and 4-hydroxysphinganine besides normal, unsaturated, and hydroxylated fatty acids in all Lewis-blood-group-active glycolipids.     

The hapten structure of a developmentally regulated glycolipid antigen (SSEA-1) isolated from human erythrocytes and adenocarcinoma: a preliminary note

Glycolipid antigen reacting to the monoclonal antibody directed to the developmentally regulated antigen SSEA-1 was isolated from human erythrocytes and colonic adenocarcinoma. The antigens have the Le^x (Galβl→4[Fucα]→3]GlcNAcβl→R) or Le^y (Fucαl→2Galβl→4[Fucαl→3]GlcNAcβl→R) structure at the termini of the branched polylactosaminolipid. In addition, a novel polyfucosyl structure locating exclusively at the internal GlcNAc was detected in the tumor antigen. The antibody reacts with a simple monovalent Le^x glycolipid (Galβl→4[Fucαl→3]GlcNAcβl→3Galβl→4Glcβl→Cer) previously isolated from colonic carcinoma when presented at a high density on liposomes. The antibody therefore may react to the bivalent or multivalent Le^x or Le^y structure.     

Immunogenicity of synthetic conjugates of Lewisy oligosaccharide with proteins in mice: towards the design of anticancer vaccines

 Many human carcinomas overexpress the Lewis^y (Le^y) blood-group epitope [Fucα1→2Galβ1→4 (Fucα1→3)GlcNAcβ1→3Gal-]. With a view to developing Le^y based vaccines we have examined the immunogenicity of Le^y-protein conjugates in mice. Le^y pentasaccharide was synthesized as its allyl glycoside and coupled to keyhole limpet hemocyanin (KLH) by reductive amination or by a novel method utilizing a maleido-derivitized alkyl carboxyhydrazide as a bridging group to 2-iminothiolane-derivitized KLH. Le^y oligosaccharide was also coupled to bovine serum albumin by reductive amination. Immunization of groups of mice with the three conjugates, together with the immunological adjuvant QS21, showed that Le^y oligosaccharide directly coupled to KLH was the most efficient conjugate for eliciting IgG and IgM antibody responses to naturally occurring forms of Le^y epitopes carried on mucins and glycolipids. These antibodies were also reactive with and cytotoxic to a human breast cancer cell line expressing Le^y (MCF-7). These experiments suggest that Le^y-KLH antigen and QS21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma patients. Received: 28 March 1997 / Accepted: 2 September 1997     

Synthesis of a BSA-Lex glycoconjugate and recognition of Lex analogues by the anti-Lex monoclonal antibody SH1: The identification of a non-cross reactive analogue

A Le^x trisaccharide functionalized with a cysteamine arm was prepared and this synthesis provided additional information on the reactivity of N-acetylglucosamine O-4 acceptors when they are glycosylated with trichloroacetimidate donors activated with excess BF₃·OEt₂. In turn, this trisaccharide was conjugated to BSA lysine side chains through a squarate–mediated coupling. This BSA-Le^x glycoconjugate displayed 35 Le^x haptens per BSA molecule. The relative affinity of the anti-Le^x monoclonal antibody SH1 for the Le^x antigen and analogues of Le^x in which the d-glucosamine, l-fucose or d-galactose residues were replaced with d-glucose, l-rhamnose and d-glucose, respectively, was measured by competitive ELISA experiments. While all analogues were weaker inhibitors than the Le^x antigen, only the analogue of Le^x in which the galactose residue was replaced by a glucose unit showed no binding to the SH1 mAb. To confirm that the reduced or loss of recognition of the Le^x analogues by the anti-Le^x mAb SH1 did not result from different conformations adopted by the analogues when compared to the native Le^x antigen, we assessed the conformational behavior of all trisaccharides by a combination of stochastic searches and NMR experiments. Our results showed that, indeed, the analogues adopted the same stacked conformation as that identified for the Le^x antigen. The identification of a trisaccharide analogue that does not cross-react with Le^x but still retains the same conformation as Le^x constitutes the first step to the design of a safe anti-cancer vaccine based on the dimeric Le^x tumor associated carbohydrate antigen.     

Monoclonal antibody LU-BCRU-G7 against a breast tumour-associated glycoprotein recognizes the disaccharide Gal{beta}1-3GlcNAc

The monoclonal antibody LU-BCRU-G7, that was generated by invitro immunization, shows clinical value as a prognostic markerin early stage breast carcinoma. It has now been characterizedwith regard to its binding epitope. Using a recently describedmethod based on the construction of N-substituted polyacrylamide(PAA) derivatives of carbohydrates (pseudopolysaccharides),the structure of the epitope for the monoclonal antibody LU-BCRU-G7has been determined. Competitive binding assays and inhibitoryenzyme-linked immunosorbent assays (ELISAs) using these pseudopolysaccharideshave shown the LU-BCRU-G7 epitope to be a disaccharide Galß1-3GlcNAc(Le^c; where Gal is D-galactose, Glc is D-glucose and GlcNAcis N-acetyl-D-glucosainine). Both galactose and N-acetyl glucosaminemoieties are essential for binding. Substitution on C-2 or C-3of the terminal galactose abolished binding, as did galactose-terminated oligosaccharides. The galactose moiety alone, asexpressed by the Galß-PAA conjugate, appeared to hea more important feature of the epitope than the GlcNAc-PAAconjugate, which failed to bind or inhibit the LU-BCRU-G7 antibody.In the N-acetyl glucosamine moiety, binding was decreased butnot eliminated by fucose substitution, as in Le^a, or changein configuration of C-4, as in Galß1-3GlcNAc. Omissionof the NAc group resulted in complete loss of activity. Thetetrasaccharide lacto-N-tetraose, although containing the terminalLe^c disaccharide, does not react with the antibody, suggestingconformational interference of the binding site. These findingsshow that the monoclonal antibody LU-BCRU-G7 recognizes a terminalisolactosamine fragment on a tumour-associated glycoprotein,which we have previously shown to be inversely related to survivalin breast cancer. breast cancer Galß1-3GlcNAc LU-BCRU-G7 monoclonal antibody pseudopolysaccharides     

Isolation and purification of Lea blood-group active and related glycolipids from human plasma of blood-group A Lea individuals

From 8 1 of human plasma of blood-group A Le^a nonsecretors three different Le^a blood-group active ceramide pentasaccharides (a total of 4.65 mg) have been isolated, all revealing glucose, galactose, N-acetylglucosamine and fucose in molar ratios of 1 : 2 : 1 : 1 as determined by gas liquid chromatography. A fourth blood-group active fraction (0.72 mg) represents a mixture of a Le^a active ceramide pentasaccharide and an A active ceramide hexasaccharide (molar ratio 7.7 : 2.3 as calculated from the content of different aminosugars). Additionally, two different globosides, two different hematosides and a new N-acetylglucosamine containing ceramide tetrasaccharide were obtained. All 9 glycolipid fractions demonstrated homogeneity in analytical high performance thin layer chromatography (HPTLC) using 4 different solvent systems. 0.2 μg of each Le^a active glycolipid completely inhibited the agglutination of O Le(a + b ?) erythrocytes by 50 μl of 4 hemagglutinating units of caprine anti Le^a serum. At least 0.04 μg of each Le^a antigen are sufficient for incubation to convert 9 × 10⁷ O Le(a?b?) erythrocytes into Le^a-positive cells. Mainly due to the relatively low content of the blood-group A glycolipid in plasma (0.17 mg/8 1), previously negative erythrocytes readily become agglutinable by anti Le^a sera and not by anti A sera after incubation with appropriate plasma.     

Application and limitations of the methyl imidate protection strategy of N-acetylglucosamine for glycosylations at O-4: synthesis of Lewis A and Lewis X trisaccharide analogues

We describe here the synthesis of the allyl Le^a trisaccharide antigen as well as that of an analogue of the Le^x trisaccharide antigen, in which the galactose residue has been replaced by a glucose unit. Although successful fucosylations at O-4 of N-acetylglucosamine acceptors have been reported using perbenzylated thioethyl fucosyl donors under MeOTf activation, such conditions led in our case to the conversion of our acceptor to the corresponding alkyl imidates. Indeed, in this synthesis of the Le^a analogue, we demonstrate that the temporary protection of the N-acetyl group as a methyl imidate is advantageous to fucosylate at O-4. In contrast, we report here that glucosylation at O-4 of an N-acetylglucosamine monosaccharide acceptor using the α-trichloroacetimidate of peracetylated glucopyranose as a donor proceeded in better yields under activation with excess BF₃·OEt₂ than that of the corresponding methyl imidate. Therefore, we conclude that activation of thioglycoside donors by MeOTf to glycosylate at O-4 of a glucosamine acceptor is best accomplished following the temporary protection of the N-acetyl group as a methyl imidate, especially when the donors are highly reactive and prone to degradation. In contrast, if donor and acceptor can withstand multiple equivalents of BF₃·OEt₂, glycosylations at O-4 of a glucosamine acceptor with a trichloroacetimidate donor does not benefit from the temporary protection of the N-acetyl group as a methyl imidate.     

Synthesis of sialyl Lewis<Superscript>a</Superscript> (sLe<Superscript>a</Superscript>, CA19-9) and construction of an immunogenic sLe<Superscript>a</Superscript> vaccine

Sialyl Lewis^a (sLe^a), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe^a is known to be the ligand for endothelial cell selectins suggesting a role for sLe^a in cancer metastases and adhesion. For these reasons, sLe^a may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe^a is structurally similar to sLe^x and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe^a will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe^a hexasaccharide and its conjugation to KLH to construct a sLe^a-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe^a-HSA by ELISA and against sLe^a positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized with unconjugated sLe^a plus GPI-0100 or unconjugated sLe^a mixed with KLH plus GPI-0100 failed to produce antibodies against sLe^a. However, mice immunized with sLe^a-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLe^a-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLe^a by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLe^a positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related antigens, including Le^y, Le^x, and sLe^x by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLe^a positive cancer in clinical settings. Govind Ragupathi and Philip O. Livingston are paid consultants and shareholders in MabVax Therapeutics, Inc., San Diego, CA 92121. The sLe^a vaccine is licensed to MabVax.     

Structural Complexity of Non-acid Glycosphingolipids in Human Embryonic Stem Cells Grown under Feeder-free Conditions

Due to their pluripotency and growth capability, there are great expectations for human embryonic stem cells, both as a resource for functional studies of early human development and as a renewable source of cells for use in regenerative medicine and transplantation. However, to bring human embryonic stem cells into clinical applications, their cell surface antigen expression and its chemical structural complexity have to be defined. In the present study, total non-acid glycosphingolipid fractions were isolated from two human embryonic stem cell lines (SA121 and SA181) originating from leftover in vitro fertilized human embryos, using large amounts of starting material (1 × 10⁹ cells/cell line). The total non-acid glycosphingolipid fractions were characterized by antibody and lectin binding, mass spectrometry, and proton NMR. In addition to the globo-series and type 1 core chain glycosphingolipids previously described in human embryonic stem cells, a number of type 2 core chain glycosphingolipids (neo-lactotetraosylceramide, the H type 2 pentaosylceramide, the Le^x pentaosylceramide, and the Le^y hexaosylceramide) were identified as well as the blood group A type 1 hexaosylceramide. Finally, the mono-, di-, and triglycosylceramides were characterized as galactosylceramide, glucosylceramide, lactosylceramide, galabiaosylceramide, globotriaosylceramide, and lactotriaosylceramide. Thus, the glycan diversity of human embryonic stem cells, including cell surface immune determinants, is more complex than previously appreciated.     

Purification and immunochemical characterization of ascitic fluid glycoproteins containing certain tumor-associated and blood group antigen markers

Ascitic fluids from patients with various types of cancer were screened for the CA 19-9 and CA 125 tumor-associated antigenic activities. Two fluids exhibiting the highest activities were tested for their binding to various lectin-Sepharose columns resulting in both being bound best to wheat germ agglutinin (WGA) Sepharose. The WGA column eluate of one fluid was further chromatographed by HPLC and three peaks were obtained with approximate molecular weights of 3.65 MDa, 664 kDa and 330 kDa, of which only the largest fraction contained the CA 19-9 activity. The fluids were also fractionated on a Sephacryl S-400 column with most of the activity being present in or near the void volume.Monoclonal antibodies were used to demonstrate that the purified glycoproteins also contained the blood group A determinant, the four Lewis determinants Le^a, Le^b, Le^x and Le^y, and the sialylated-Le^x determinant, while other antibody analyses failed to detect other blood group and/or carbohydrate sequence determinants. Some of the blood group expressions could be separated from the CA 19-9 and CA 125 active glycoproteins by adsorption with various lectins other than the WGA.Abbreviations used NeuAc N-acetyl-D-neuraminic acid - Gal galactose,D-galactopyranose - Fuc fucose,L-fucopyranose - GlcNAc N-acetyl-D-glucosamine - GalNAc N-acetyl-D-galactosamine - WGA wheat germ agglutinin - PBS phosphate buffered saline     

Formation of heterophile antibodies by human tonsilar lymphocytes: II. In vitro stimulation with allogeneic cells

Short-term cultures of human tonsilar lymphocytes (HTL), 5 × 10⁶ cells/culture, in medium RPMI 1640 supplemented with human group AB serum were studied for the production of plaque-forming cells (PFC) against sheep (SRBC) and bovine (BRBC) red blood cells following in vitro stimulation by various allogeneic lymphoid cells. Of 55 HTL specimens examined, 48 produced a significant number (50–300/culture) of PFC against SRBC and/or BRBC following the in vitro stimulation. The optimal doses of the stimulator HTL and peripheral blood lymphocytes (PBL) were 10⁷ and 5 × 10⁶/culture, respectively. After the stimulation, PFC appeared in significant numbers on the third day, reached the peak number on the sixth day, and decreased sharply in number thereafter. Removal of E-rosetting cells from both stimulator and responder populations abolished the PFC formation. PFC formation against SRBC was inhibited by solubilized Forssman antigen, while PFC formation against BRBC was inhibited strongly by Hanganutziu-Deicher antigen, hardly by Paul-Bunnell antigen and not at all by Forssman antigen. Supernatants of mixed lymphocyte culture of PBL were shown to enhance PFC formation of HTL cultures stimulated by allogeneic lymphocytes. The results of this study indicated that in vivo primed B cells of the HTL were triggered in vitro by allogeneic stimulation for the heterophile antibody formation. Since these antibodies are apparently directed against Forssman and Hanganutziu-Deicher antigens, the “allo” nature of these antigens as well as their relationship to the previously described heterophile transplantation antigens have to be clarified.     

Structures of the oligosaccharides isolated from milk of the platypus

Eight major oligosaccharides were isolated from platypus milk. By sequential exoglycosidase digestion and methylation study, their structures were elucidated as shown in Fig. 9 of this paper. The characteristics feature of the platypus milk oligosaccharides is that lacto-N-neotetraose and lacto-N-neohexaose are the major cores in contrast to human milk oligosaccharides in which lacto-N-tetraose and lacto-N-hexaose are found as the major core.     

Immunochemical studies on blood groups. 53. A study of various conditions of alkaline borohydride degradation on human and hog blood group substances and on known oligosaccharides

Blood group A, B, H, Le^a, Le^b, and I substances, their products of periodate oxidation and Smith degradation, and disaccharides containing 3-O-substituted reducing N-acetylhexosamines were treated with base-borohydride under three defined sets of conditions. Procedures for the assay and quantitation of the possible reduced base-degradation products, including hexenetetrol(s), 3-deoxygalactitol, galactitol, reduced chromogens, N-acetylglucosaminitol, and N-acetylgalactosaminitol are described. Extensive degradation occurred by two methods. 1 m NaBH₄ in 0.05 n NaOH at 50 ° cleaves the glycosidic linkage of the oligosaccharide chains from serine and threonine with reduction of the terminal-reducing N-acetylgalactosamine with minimal base degradation. The method is useful for isolation of complete reduced oligosaccharides from blood group substances; the structural implications of the free and oligosaccharide-bound N-acetylgalactosaminitol released are discussed.     

Monoclonal antibodies directed against the sugar sequence of lacto-N-fucopentaose III are obtained from mice immunized with human tumors

Four hybridomas obtained from mice immunized with human adenocarcinomas of colon or stomach produce antibodies that bind specifically in solid-phase radioimmunoassay to the ceramide pentasaccharide that contains the lacto-N-fucopentaose III sequence of sugars. Binding of the antibodies to the glycolipid is inhibited by lacto-N-fucopentaose III,

but not by structurally related oligosaccharides. The antibodies bind to glycolipids of erythrocytes, granulocytes, and certain normal and malignant tissues.     

Immunochemical studies on the combining site of the d-galactose/N-acetyl-d-galactosamine specific lectin from Erythrina cristagalli seeds

The combining site of the Erythrina cristagalli lectin was studied by quantitative precipitin and precipitin inhibition assays. The lectin precipitated best with two fractions of a precursor human ovarian cyst blood group substance with I and i activities. A₁, A₂, B, H, Le^a, and Le^b blood group substances precipitated poorly to moderately and substances of the same blood group activity precipitated to varying extents. These differences are attributable to heterogeneity resulting from incomplete biosynthesis of carbohydrate chains. Specific precipitates with the poorly reactive blood group substances were found to be more soluble than those reacting strongly. Precipitation was minimally affected by EDTA or divalent cations. Among the monosaccharides and glycosides tested for inhibition of precipitation, p-nitrophenyl βdGal was most active and was 10 times more active than methyl βdGal, indicating involvement of hydrophobic contacts in site specificity. Methyl αdGalNAc, p-nitrophenyl αdGalNAc, methyl αdGal, N-acetyl-d-galactosamine, p-nitrophenyl αdGal, methyl βdGal, and p-nitrophenyl βdGalNAc were progressively less active than p-nitrophenyl βdGal. The best disaccharide inhibitor dGalβ1 → 4dGlcNAc was 7.5 times more potent than p-nitrophenyl βdGal. A tetraantennary and triantennary oligosaccharide containing four and three dGalβ1 → 4dGlcNAcβ1 → branches, respectively, were, because of cooperative binding effects, 1.6 and 2.5 times more active than the bi- and monoantennary oligosaccharides, respectively. dGalβ1 → 4dGlcNAcβ1 → 6dGal and dGalβ1 → 4dGlcNAcβ1 → 2dMan had the same activity, being 1.5 times more active than dGalβ1 → 4dGlcNAc, which was 2.6 and 8.5 times more active than dGalβ1 → 3dGlcNAc and dGalβ1 → 3dGlc, respectively. Substitutions by N-acetyl-d-galactos-amine or l-fucose on the d-galactose of inhibitory compounds blocked activity. These results suggest that a hydrophobic interaction with the subterminal sugar is important in the binding and that the specificity of the lectin combining site involves a terminal dGalβ1 → 4dGlcNAc and the β linkage of a third sugar.     

A Possible Role for Metallic Ions in the Carbohydrate Cluster Recognition Displayed by a Lewis Y Specific Antibody

Background

Lewis Y (Le^y) is a blood group-related carbohydrate that is expressed at high surface densities on the majority of epithelial carcinomas and is a promising target for antibody-based immunotherapy. A humanized Le^y-specific antibody (hu3S193) has shown encouraging safety, pharmacokinetic and tumor-targeting properties in recently completed Phase I clinical trials.

Methodology/Principal Findings

We report the three-dimensional structures for both the free (unliganded) and bound (Le^y tetrasaccharide) hu3S193 Fab from the same crystal grown in the presence of divalent zinc ions. There is no evidence of significant conformational changes occurring in either the Le^y carbohydrate antigen or the hu3S193 binding site, which suggests a rigid fit binding mechanism. In the crystal, the hu3S193 Fab molecules are coordinated at their protein-protein interface by two zinc ions and in solution aggregation of Fab can be initiated by zinc, but not magnesium ions. Dynamic light scattering revealed that zinc ions could initiate a sharp transition from hu3S193 Fab monomers to large multimeric aggregates in solution.

Conclusions/Significance

Zinc ions can mediate interactions between hu3S193 Fab in crystals and in solution. Whether metallic ion mediated aggregation of antibody occurs in vivo is not known, but the present results suggest that similar clustering mechanisms could occur when hu3S193 binds to Le^y on cells, particularly given the high surface densities of antigen on the target tumor cells.