Crystal Structure of Anti-polysialic Acid Antibody Single Chain Fv Fragment Complexed with Octasialic Acid: INSIGHT INTO THE BINDING PREFERENCE FOR POLYSIALIC ACID*

Polysialic acid is a linear homopolymer of α2–8-linked sialic acids attached mainly onto glycoproteins. Cell surface polysialic acid plays roles in cell adhesion and differentiation events in a manner that is often dependent on the degree of polymerization (DP). Anti-oligo/polysialic acid antibodies have DP-dependent antigenic specificity, and such antibodies are widely utilized in biological studies for detecting and distinguishing between different oligo/polysialic acids. A murine monoclonal antibody mAb735 has a unique preference for longer polymers of polysialic acid (DP >10), yet the mechanism of recognition at the atomic level remains unclear. Here, we report the crystal structure of mAb735 single chain variable fragment (scFv735) in complex with octasialic acid at 1.8 Å resolution. In the asymmetric unit, two scFv735 molecules associate with one octasialic acid. In both complexes of the unit, all the complementarity-determining regions except for L3 interact with three consecutive sialic acid residues out of the eight. A striking feature of the complex is that 11 ordered water molecules bridge the gap between antibody and ligand, whereas the direct antibody-ligand interaction is less extensive. The dihedral angles of the trisialic acid unit directly interacting with scFv735 are not uniform, indicating that mAb735 does not strictly favor the previously proposed helical conformation. Importantly, both reducing and nonreducing ends of the bound ligand are completely exposed to solvent. We suggest that mAb735 gains its apparent high affinity for a longer polysialic acid chain by recognizing every three sialic acid units in a paired manner.     

Glycomic strategy for efficient linkage analysis of di-, oligo- and polysialic acids

Sialic acid polymers of glycoproteins and glycolipids are characterized by a high diversity in nature and are involved in distinct biological processes depending inter alia on the glycosidic linkages between the present sialic acid residues. Though suitable protocols are available for chain length and sialic acid determination, sensitive methods for linkage analysis of di-, oligo-, and polysialic acids (di/oligo/polySia) are still pending. In this study, we have established a highly sensitive glycomic strategy for this purpose which is based on permethylation of di/oligo/polySia after tagging their reducing ends with the fluorescent dye 1,2-diamino-4,5-methylenedioxybenzene (DMB). Using DMB-labeled sialic acid di/oligo/polymers glycosidic linkages could be efficiently determined and, optionally, the established working procedure can be combined with HPLC for in depth characterization of distinct di/oligo/polySia chains. Moreover, the outlined approach can be directly applied to mammalian tissue samples and linkage analysis of sialic acid polymers present in biopsy samples of neuroblastoma tissue demonstrating the usefulness of the outlined work flow to screen, for example, cancer tissue for the presence of distinct variants of di/oligo/polySia as potentially novel biomarkers. Hence, the described strategy offers a highly sensitive and efficient strategy for identification of glycosidic linkages in sialic acid di/oligo/polymers of glycoproteins and glycolipids.     

Mapping of antigenic epitopes on the alpha 1 subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) is a ligand-gated chloride channel protein that occurs in developmentally regulated isoforms in the vertebrate central nervous system. Monoclonal antibodies (mAbs) against the GlyR distinguish neonatal and adult GlyR proteins by identifying distinct alpha subunit variants within these receptor isoforms. Here, bacterially expressed fusion proteins of the rat GlyR alpha 1 subunit were used to localize the major antigenic epitopes of this protein within its N-terminal 105 amino acids. Synthetic peptides allowed further fine mapping of two mAb binding domains. MAb 2b, specific for the adult alpha 1 subunit, bound to a peptide corresponding to amino acids 1-10, whereas mAb 4a, which recognizes both neonatal and adult GlyR isoforms, reacted with a peptide representing residues 96-105 of the alpha 1 polypeptide. These data define unique and common antigenic epitopes on GlyR alpha subunit variants.     

Purification and characterization of a differentiation-specific sialoglycoprotein of lactating-guinea-pig mammary tissue.

A large acidic glycoprotein, PAS-I, was purified from the fat-globule membrane of guinea-pig milk. Threonine and serine accounted for over 30 mol% of the amino acids, and galactose, N-acetylgalactosamine, N-acetylglucosamine, mannose and sialic acid were the principal sugars detected. On a molar basis, sialic acid accounted for over 60% of the total sugar. Removal of sialic acid by treatment with neuraminidase revealed the presence of binding sites for peanut (Arachis hypogaea) agglutinin, a lectin specific for the sugar sequence beta-D-Gal-(beta 1----3)-D-GalNac (the T antigen). The distribution of PAS-I-related epitopes, defined by five monoclonal antibodies, was determined in the mammary gland and in other guinea-pig tissues. PAS-I was maximally expressed on the apical surfaces of secretory cells in lactating mammary tissue and was either absent, or present in much lower amounts, in the glands of virgin or pregnant animals. PAS-I epitopes were not detected in liver, heart, spleen, pancreas, ovary, uterus, lung or intestine, either by immunofluorescence microscopy or by immunoblotting techniques. Several of the PAS-I-specific antibodies bound to mucins of high Mr in human fat-globule membrane, and similarities and differences between PAS-I and the human mucins are discussed. PAS-I and epitopes of this glycoprotein will be useful as indicators of differentiation in mammary cells and of markers of the apical surface of these cells during lactation.     

The intracellular concentration of sialic acid regulates the polysialylation of the neural cell adhesion molecule

Sialic acids are expressed as terminal sugars in many glycoconjugates and play an important role during development and regeneration, as they are involved as polysialic acid in a variety of cell-cell interactions mediated by the neural cell adhesion molecule NCAM. The key enzyme for the biosynthesis of sialic acid is the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase (GNE). Mutations in the binding site of the feedback inhibitor CMP-sialic acid of the GNE leads to sialuria, a disease in which patients produce sialic acid in gram scale. Here, we report on the consequences after expression of a sialuria-mutated GNE. Expression of the sialuria-mutated GNE leads to a dramatic increase of both cellular sialic acid and polysialic acid on NCAM. This could also be achieved by application of the sialic acid precursor N-acetylmannosamine. Our data suggest that biosynthesis of sialic acid regulates and limits the synthesis of polysialic acid.     

Biochemical engineering of surface alpha 2-8 polysialic acid for immunotargeting tumor cells

To target tumor cells for immunotherapy, we evaluated the feasibility of altering the epitopes on the surface polysialic acid of tumor cells. A precursor (N-propionylmannosamine), when incubated with leukemic cells, RBL-2H3 and RMA, resulted in substitution of the N-acetyl groups of surface alpha2-8 polysialic acid with N-propionyl groups. Expression of the altered alpha2-8 N-propionylpolysialic acid on the surface of tumor cells induced their susceptibility to cell death mediated by monoclonal antibody 13D9 (mAb 13D9), which specifically recognizes alpha2-8 N-propionylated polysialic acid. The expression of alpha2-8 N-propionylated polysialic acid and the lysis of tumor cells by antibody-dependent cytotoxicity depended on the time and dose of incorporation of N-propionylated mannosamine. In vivo, mAb 13D9 effectively controlled metastasis of leukemic cells RMA when mice were administered the precursor N-propionylated mannosamine.     

Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli. The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation

The capsular polysaccharide of Escherichia coli K1 is a linear polymer of N-acetylneuraminic acid in alpha-2,8 linkage. Certain substrains of E. coli K1 (designated OAc+) modify the polysaccharide by O-acetylation of the sialic acids. We demonstrate here an acetyl-coenzyme A: polysialosyl O-acetyltransferase activity that is found only in E. coli K1 OAc+ substrains. When form variation between the O-acetyl-positive and -negative states occurred in strain D698:K1, the fluctuations were accompanied by appropriate changes in the expression of enzyme activity. Thus, expression of this enzyme can account for the OAc+ phenotype and for the form variation between OAc+ and OAc-. The enzyme was solubilized in nonionic detergent and freed of endogenous acceptor activity by DEAE-cellulose chromatography, and its general properties were determined. Analysis of the reaction product showed a highly preferential acetylation reaction that was confined to polysialosyl units of greater than 14 residues. Acetyl groups were shown to be transferred to both the 7- and the 9-positions of the sialic acid residues. The partially purified enzyme was stable even after prolonged incubation at 57 degrees C. In contrast, any further purification resulted in loss of activity, even at 4 degrees C. Treatment of the stable enzyme with a polysialic acid-specific endoneuraminidase caused a similar loss of enzyme stability. This effect of the endoneuraminidase could be protected against by the addition of exogenous polysialic acid. This indicates that the partially purified enzyme contains traces of endogenous polysialic acid substrate that are required for the stability of the enzyme. Finally, the enzyme can O-acetylate the polysialic acid chains on the eucaryotic protein neural cell adhesion molecule, suggesting that enzymatic recognition of the substrate requires only the polysialic acid sequence.     

Structure-function relationships of human apolipoprotein D an immunochemical analysis

Apolipoprotein D (apoD), a 169 amino acid member of the lipocalin family, is thought to be a transporter of small, hydrophobic ligands. A panel of 10 anti-apoD monoclonal antibodies (mAbs) was prepared and characterized in order to define apoD structure-function relationships. An apoD epitope map was constructed based on reactivity of the mAbs with apoD fragments. Three mAbs react with epitopes between apoD residues 7-78, seven mAbs with epitopes between residues 128-169, one mAb recognizes an epitope that straddles residues 99-102 and one mAb is specific for an epitope composed of non-contiguous apoD residues. Several pairs of mAbs whose respective epitopes are widely separated in apoD primary structure can compete for binding to immobilized apoD. This would be consistent with the compact beta-barrel tertiary structure that apoD is thought to adopt. None of the mAbs block the interaction of apoD with pregnenolone, a putative physiological ligand for apoD.     

Occurrence of oligosialic acids on integrin alpha 5 subunit and their involvement in cell adhesion to fibronectin.

Integrin alpha(5)beta(1), a major fibronectin receptor, functions in a wide variety of biological phenomena. We have found that alpha 2-8-linked oligosialic acids with 5 < or = degree of polymerization (DP) < or = 7 occur on integrin alpha(5) subunit of the human melanoma cell line G361. The integrin alpha(5) subunit immunoprecipitated with anti-integrin alpha(5) antibody reacted with the monoclonal antibody 12E3, which recognizes oligo/polysialic acid with DP > or = 5 but not with the polyclonal antibody H.46 recognizing oligo/polysialic acid with DP > or = 8. The occurrence of oligosialic acids was further demonstrated by fluorometric C(7)/C(9) analysis on the immunopurified integrin alpha(5) subunit. Oligosialic acids were also found in the alpha(5) subunit of several other human cells such as foreskin fibroblast and chronic erythroleukemia K562 cells. These results suggest the ubiquitous modification with unique oligosialic acids occurs on the alpha(5) subunit of integrin alpha(5)beta(1). The adhesion of human melanoma G361 cells to fibronectin was mainly mediated by integrin alpha(5)beta(1). Treatment of cells with sialidase from Arthrobacter ureafaciens cleaving alpha 2-3-, alpha 2-6-, and alpha 2-8-linked sialic acids inhibited adhesion to fibronectin. On the other hand, N-acetylneuraminidase II, which cleaves alpha 2-3 and alpha 2-6 but not alpha 2-8 linkages, showed no inhibitory activity. After the loss of oligosialic acids, integrin alpha(5)beta(1) failed to bind to fibronectin-conjugated Sepharose, indicating that the oligosialic acid on the alpha(5) subunit of integrin alpha(5)beta(1) plays important roles in cell adhesion to fibronectin.     

Differential effects of unnatural sialic acids on the polysialylation of the neural cell adhesion molecule and neuronal behavior.

In this study we have examined how unnatural sialic acids can alter polysialic acid expression and influence the adhesive properties of the neural cell adhesion molecule (NCAM). Unnatural sialic acids are generated by metabolic conversion of synthetic N-acyl mannosamines and are typically incorporated into cell-surface glycoconjugates. However, N-butanoylmannosamine and N-pentanoylmannosamine are effective inhibitors of polysialic acid (PSA) synthesis in stably transfected HeLa cells expressing NCAM and the polysialyltransferase STX. These cells were used as substrates to examine the effect of inhibiting PSA synthesis on the development of neurons derived from the chick dorsal root ganglion. N-butanoylmannosamine blocked polysialylation of NCAM and significantly reduced neurite outgrowth comparable with enzymatic removal of PSA by endoneuraminidases. As a result, neurite outgrowth was similar to that observed for non-polysialylated NCAM. In contrast, previous studies have shown that N-propanoyl sialic acid (SiaProp), generated from N-propanoylmannosamine, is readily accepted by polysialyltransferases and permits the extension of poly(SiaProp) on NCAM. Despite being immunologically distinct, poly(SiaProp) can promote neurite outgrowth similarly to natural polysialic acid. Thus, subtle structural differences in PSA resulting from the incorporation of SiaProp residues do not alter the antiadhesive properties of polysialylated NCAM.     

Separate pathways for O acetylation of polymeric and monomeric sialic acids and identification of sialyl O-acetyl esterase in Escherichia coli K1

O acetylation at carbon positions 7 or 9 of the sialic acid residues in the polysialic acid capsule of Escherichia coli K1 is catalyzed by a phase-variable contingency locus, neuO, carried by the K1-specific prophage, CUS-3. Here we describe a novel method for analyzing polymeric sialic acid O acetylation that involves the release of surface sialic acids by endo-N-acetylneuraminidase digestion, followed by fluorescent labeling and detection of quinoxalinone derivatives by chromatography. The results indicated that NeuO is responsible for the majority of capsule modification that takes place in vivo. However, a minor neuO-independent O acetylation pathway was detected that is dependent on the bifunctional polypeptide encoded by neuD. This pathway involves O acetylation of monomeric sialic acid and is regulated by another bifunctional enzyme, NeuA, which includes N-terminal synthetase and C-terminal sialyl O-esterase domains. A homologue of the NeuA C-terminal domain (Pm1710) in Pasteurella multocida was also shown to be an esterase, suggesting that it functions in the catabolism of acetylated environmental sialic acids. Our combined results indicate a previously unexpected complexity in the synthesis and catabolism of microbial sialic and polysialic acids. These findings are key to understanding the biological functions of modified sialic acids in E. coli K1 and other species and may provide new targets for drug or vaccine development.     

Inhibitory antibodies to human angiotensin-converting enzyme: fine epitope mapping and mechanism of action

Angiotensin I-converting enzyme (ACE), a key enzyme in cardiovascular pathophysiology, consists of two homologous domains (N and C), each bearing a Zn-dependent active site. We modeled the 3D-structure of the ACE N-domain using known structures of the C-domain of human ACE and the ACE homologue, ACE2, as templates. Two monoclonal antibodies (mAb), 3A5 and i2H5, developed against the human N-domain of ACE, demonstrated anticatalytic activity. N-domain modeling and mutagenesis of 21 amino acid residues allowed us to define the epitopes for these mAbs. Their epitopes partially overlap: amino acid residues K407, E403, Y521, E522, G523, P524, D529 are present in both epitopes. Mutation of 4 amino acid residues within the 3A5 epitope, N203E, R550A, D558L, and K557Q, increased the apparent binding of mAb 3A5 with the mutated N-domain 3-fold in plate precipitation assay, but abolished the inhibitory potency of this mAb. Moreover, mutation D558L dramatically decreased 3A5-induced ACE shedding from the surface of CHO cells expressing human somatic ACE. The inhibition of N-domain activity by mAbs 3A5 and i2H5 obeys similar kinetics. Both mAbs can bind to the free enzyme and enzyme-substrate complex, forming E.mAb and E.S.mAb complexes, respectively; however, only complex E.S can form a product. Kinetic analysis indicates that both mAbs bind better with the ACE N-domain in the presence of a substrate, which, in turn, implies that binding of a substrate causes conformational adjustments in the N-domain structure. Independent experiments with ELISA demonstrated better binding of mAbs 3A5 and i2H5 in the presence of the inhibitor lisinopril as well. This effect can be attributed to better binding of both mAbs with the "closed" conformation of ACE, therefore, disturbing the hinge-bending movement of the enzyme, which is necessary for catalysis.     

Use of a bacteriophage-derived endo-N-acetylneuraminidase and an equine antipolysialyl antibody to characterize the polysialyl residues in salmonid fish egg polysialoglycoproteins. Substrate and immunospecificity studies

Polysialoglycoproteins (PSGP), a class of glycoproteins containing oligo(poly)sialylglycan chains, are the major glycoprotein components in cortical alveoli of a number of Salmonidae fish eggs. Lake trout, Salvelinus namaycush, egg PSGP (PSGP(Sn)) differs from rainbow trout, Salmo gairdneri, egg PSGP (PSGP(Sg)) in its sialic acid composition; the former contains both N-acetyl- and N-glycolyl-D-neuraminic acid residues, designated Neu5Ac and Neu5Gc, while the latter contains only Neu5Gc residues. Fragmentation analysis of oligo(poly)sialyl chains in lake trout PSGP(Sn) has established that there are two distinct types of oligo(poly)sialyl structures in this PSGP molecule, namely alpha-2,8-linked oligo/poly(Neu5Ac) and alpha-2,8-linked oligo/poly(Neu5Gc). No hybrid structure having both Neu5Ac and Neu5Gc residues in the fragment oligosialic acids was detected. These two distinct PSGP preparations from eggs of lake trout and rainbow trout have been used to compare their immunoreactivity with anti-polysialyl antibodies (H.46) and sensitivity to a bacteriophage-derived (Escherichia coli K1F) endo-N-acetylneuraminidase (Endo-N). H.46 was found to cross-react only with lake trout PSGP(Sn) in immunodiffusion assays but not with rainbow trout PSGP(Sg), indicating that H.46 is a specific probe for alpha-2,8-linked poly(Neu5Ac) but not for poly(Neu5Gc). In contrast, Endo-N was found to catalyze the hydrolysis of both alpha-2,8-linked poly (Neu5Ac) and poly(Neu5Gc), so that this enzyme can be used as a diagnostic reagent for detecting both types of polysialic acids. H.46 was used in indirect immunofluorescence experiments to localize PSGP(Sn) in cortical alveoli isolated from lake trout eggs.     

Identification of an inducible catabolic system for sialic acids (nan) in Escherichia coli.

Escherichia coli K-12 and K-12 hybrid strains constructed to express a polysialic acid capsule, the K1 antigen, were able to efficiently use sialic acid as a sole carbon source. This ability was dependent on induction of at least two activities: a sialic acid-specific transport activity, and an aldolase activity specific for cleaving sialic acids. Induction over basal levels required sialic acid as the apparent inducer, and induction of both activities was repressed by glucose. Induction also required the intracellular accumulation of sialic acid, which could be either added exogenously to the medium or accumulated intracellularly through biosynthesis. Exogenous sialic acid appeared to be transported by an active mechanism that did not involve covalent modification of the sugar. Mutations affecting either the transport or degradation of sialic acid prevented its use as a carbon source and have been designated nanT and nanA, respectively. These mutations were located by transduction near min 69 on the E. coli K-12 genetic map, between argG and glnF. In addition to being unable to use sialic acid as a carbon source, aldolase-negative mutants were growth-inhibited by this sugar. Therefore, the intracellularly accumulated sialic acid was toxic in aldolase-deficient E. coli strains. The dual role of aldolase in dissimilating and detoxifying sialic acids is consistent with the apparent multiple controls on expression of this enzyme.     

Structure-function study of the p55 subunit of murine IL-2 receptor by epitope mapping.

Using a cell-free translation system we have expressed the Mr 55,000 subunit of the murine IL-2R (p55 IL-2R), which binds IL-2 with low affinity (Kd = 10 nM). Mutants and truncated forms of p55 IL-2R have been used to map the epitopes recognized by three anti-p55 IL-2R mAb: 135D5, 7D4, and 2E4. The mAb 135D5 inhibits IL-2 binding to p55 IL-2R and recognizes an epitope located between amino acids 64 to 125. This epitope can be mimicked by a synthetic peptide corresponding to the region defined by residues 72 to 88. However, the mAb 7D4 and 2E4 do not affect the IL-2 binding to p55 IL-2R. These mAb recognize an epitope of p55 IL-2R lying between residues 125 to 212 that can be mimicked with a peptide corresponding to amino acids 188 to 208. A strong correlation emerged between the experimental results on epitope mapping and predictions of potential antigenicity of murine p55 IL-2R. In addition, we described two internal initiation sites of p55 IL-2R mRNA under the in vitro conditions used leading to the production of significant amounts of N-terminal truncated p55 IL-2R proteins.     

Analytical methods for identifying and quantitating deamidated sialic acid (2-keto-3-deoxy-D-glycero-D-galactonononic acid) and alpha 2----8-linked poly(oligo)nonulosonate residues in glycoconjugates.

In 1986 we reported the natural occurrence of deaminated neuraminic acid (2-keto-3-deoxy-D-glycero-D-galactonononic acid, KDN) in fish egg glycoprotein. Subsequently, we have shown that many types of sialic acid as well as KDN occur in polymeric chains, poly(oligo)-Sia and poly(oligo)KDN in nature. In this study we demonstrate that the conventional colorimetric and gas-liquid chromatographic methods used in the analysis of sialic acid can be applied to analysis of these new nonulosonate and poly(oligo)nonulosonates. We report that the thiobarbituric acid reaction can be used to analyze both free and bound KDN, but gives lower extinction values when applied to poly(oligo)KDN without prior hydrolysis. Further, the published hydrolytic and/or methanolytic procedures are suitable to release the terminal sialic acid residues, but are not appropriate for quantitative release of the nonulosonic acids from poly(oligo)nonulosonates. A new gas-liquid chromatographic procedure for the identification-quantitation of nonulosonates in poly(oligo)meric forms is described.     

Interaction of rat peritoneal macrophages with homologous, sialidase-treated lymphocytes in vitro

The interaction in vitro between rat peritoneal macrophages and homologous, sialidase-treated lymphocytes was investigated. Lymphocytes were isolated from blood, thymus, and spleen on a density gradient. Total sialic acids obtained by acid hydrolysis were 10 nmol/10(8) lymphocytes, composed of 29% N-acetyl-neuraminic acid and 71% N-glycoloylneuraminic acid. Sialidase treatment released maximally 33% of membrane sialic acids. Lymphocytes were bound to peritoneal macrophages to an extent which increased in parallel with the amount of sialic acids released, whereas binding of untreated lymphocytes was not significant. This interaction was inhibited by free galactose and substances containing terminal galactose residues. Asialoorosomucoid with its oligoantennary sugar chains proved to be a 10(5) times more potent inhibitor of the interaction than lactose. The addition of homologous serum had no influence on binding. Electron microscopy revealed that vital lymphocytes were tightly bound to macrophages and only damaged lymphocytes appeared to be phagocytozed. The experiments demonstrate that the interaction between rat peritoneal macrophages and sialidase-treated lymphocytes is mediated by a macrophage receptor specific for galactose. This sugar is demasked on the surface of lymphocytes after the removal of terminal sialic acids. The role of this mechanism in cell recognition, elimination and homing of lymphocytes is discussed.     

alpha-2,8-Polysialic acid is the neuronal surface receptor of antennapedia homeobox peptide.

A synthetic peptide that is 60 amino acids in length and corresponds to the homeobox sequence of antennapedia protein (pAntp) is specifically and efficiently captured by neurons in culture and conveyed to their nuclei. The internalization process is followed by a strong induction of neuronal morphological differentiation. In the study described here, all treatments masking or removing the alpha-2,8-polysialic acid (PSA) chains specific to the neuronal cell adhesion molecule (NCAM) were found to block the penetration of pAntp and abolish its morphogenetic effects. Structural comparison between PSA and double-stranded DNA suggests that a sequence of eight sialic acid residues can mimic one large groove of the DNA. We propose that this structural similarity is the basis for the property of NCAM polysialic acid to participate in the internalization of the homebox polypeptide.     

Identification of amino acid residues at the active site of endosialidase that dissociate the polysialic acid binding and cleaving activities in Escherichia coli K1 bacteriophages

Endosialidase (endo-N-acetylneuraminidase) is a tailspike enzyme of bacteriophages specific for human pathogenic Escherichia coli K1, which specifically recognizes and degrades polySia (polysialic acid). polySia is also a polysaccharide of the capsules of other meningitis- and sepsis-causing bacteria, and a post-translational modification of the NCAM (neural cell-adhesion molecule). We have cloned and sequenced three spontaneously mutated endosialidases of the PK1A bacteriophage and one of the PK1E bacteriophage which display lost or residual enzyme activity but retain the binding activity to polySia. Single to triple amino acid substitutions were identified, and back-mutation constructs indicated that single substitutions accounted for only partial reduction of enzymic activity. A homology-based structural model of endosialidase revealed that all substituted amino acid residues localize to the active site of the enzyme. The results reveal the importance of non-catalytic amino acid residues for the enzymatic activity. The results reveal the molecular background for the dissociation of the polySia binding and cleaving activities of endosialidase and for the evolvement of 'host range' mutants of E. coli K1 bacteriophages.     

Membrane potential-dependent binding of polysialic acid to lipid monolayers and bilayers

Polysialic acids are linear polysaccharides composed of sialic acid monomers. These polyanionic chains are usually membrane-bound, and are expressed on the surfaces of neural, tumor and neuroinvasive bacterial cells. We used toluidine blue spectroscopy, the Langmuir monolayer technique and fluorescence spectroscopy to study the effects of membrane surface potential and transmembrane potential on the binding of polysialic acids to lipid bilayers and monolayers. Polysialic acid free in solution was added to the bathing solution to assess the metachromatic shift in the absorption spectra of toluidine blue, the temperature dependence of the fluorescence anisotropy of DPH in liposomes, the limiting molecular area in lipid monolayers, and the fluorescence spectroscopy of oxonol V in liposomes. Our results show that both a positive surface potential and a positive transmembrane potential inside the vesicles can facilitate the binding of polysialic acid chains to model lipid membranes. These observations suggest that these membrane potentials can also affect the polysialic acid-mediated interaction between cells.