Polysialic acid can mediate membrane interactions by interacting with phospholipids

Polysialic acid (polySia) is expressed on the surface of neural cells, neuroinvasive bacterial cells and several tumor cells. PolySia chains attached to NCAM can influence both trans interactions between membranes of two cells and cis interactions. Here, we report on the involvement of phospholipids in regulation of membrane interactions by polySia. The pH at the surface of liposomes, specific molecular area of phosphatidylcholine molecules, phase transition of DPPC bilayers, cyclic voltammograms of BLMs, and electron micrographs of phosphatidylcholine vesicles were studied after addition of polysialic acid free in solution. The results indicate that polySia chains can associate with phosphatidylcholine bilayers, incorporate into the polar part of a phospholipid monolayer, modulate cis interactions between phosphatidylcholine molecules, and facilitate trans interactions between apposing phospholipid vesicles. These observations imply that polySia attached to NCAM or to lipids can behave similarly.     

Biosynthesis of the polysialic acid capsule in Escherichia coli K1. The endogenous acceptor of polysialic acid is a membrane protein of 20 kDa

The nature of endogenous acceptor molecules implicated in the membrane-directed synthesis of the polysialic acid (polySia) capsule in Escherichia coli K1 serotypes is not known. The capsule contains at least 200 sialic acid (Sia) residues that are elongated by the addition of new Sia residues to the nonreducing termini of growing nascent chains (Rohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342). Presumably, chain growth starts when activated Sia residues are transferred to acceptors that are not already sialylated. In the present study, we used an acapsular mutant defective in synthesis of CMP-NeuAc to label acceptors with [14C]NeuAc and an anti-polySia-specific antibody (H.46) to identify the molecules to which the polySia was attached. [14C]Sia-labeled acceptors were solubilized with 2% Triton X-100, immunoprecipitated with H.46, and partially depolymerized with poly-alpha-2,8-endo-N-acetylneuraminidase. Approximately 5% of the [14C]Sia incorporated remained attached to endogenous acceptors. Double-labeling experiments were used to show that the non-Sia moiety of the acceptor was labeled in vivo with [14C]leucine and elongated in vitro with CMP-[3H]NeuAc. Concomitant with desialylation of the [3H]polySia-[14C]Leu acceptor was the appearance of a new [14C]Leu-labeled protein at 20 kDa. After strong acid hydrolysis, the 20-kDa labeled protein was shown to contain [14C]Leu. The acceptor molecules were not labeled metabolically with D-[3H]GlcN, 35SO4, or 32PO4, indicating that they do not appear to contain lipopolysaccharide, peptidoglycan, phosphatidic acid, or phospholipid. Based on these results, we conclude that the endogenous acceptor molecule is a membrane protein of about 20 kDa. The nature of attachment of polySia to acceptor is unknown. There are only 400-500 acceptor molecules/cell, which is about 100-fold fewer than the 50,000 polySia chains/cell. This suggests that each acceptor molecule may participate in the shuttling of about 100 polySia chains/cell. We hypothesize that the acceptor protein may function to translocate polySia chains from their site of synthesis on the cytoplasmic surface of the inner membrane to the periplasm.     

Membrane transport of polysialic acid chains: modulation of transmembrane potential

Polysialic acid (polySia) is a long polyanionic polymer (with the degree of polymerization, DP, up to 200) of negatively charged sialic acid monomers. PolySia chains are bound to the external surface of some neuroinvasive bacterial cells and neural cells. PolySia serves as a potent regulator of cell interactions via its unusual biophysical properties. In the present paper, the analysis, based on the Goldman-Hodgkin-Katz equation, of transmembrane potential changes resulting from transmembrane translocation of polySia is performed. The relationships between the transmembrane potential and the polySia DP (up to 200), the temperature, the cation/ anion permeability ratio, and the inner/outer concentration ratio of polySia has been plotted and discussed. The maximal membrane potential changes, up to 118 mV, were found for a permeability ratio greater than one. The increase of the polySia chain length resulted in the diminution of this effect. The temperature-dependent changes in membrane potential were less than 7 mV in the range 0-50 degrees C. The change in the concentration ratio (into its reciprocal) resulted in a mirror reflection of the membrane potential curves. The results show that the expression of polySia chains in bacterial cells can be responsible for the modulation of the transmembrane potential of the bacterial inner membrane. We suggest that the polySia chains can influence the transmembrane potential of neural cell membranes in a similar way. This analysis also describes the effect of the transmembrane translocation of negatively charged polyanionic polynucleotydes on the cell membrane potential.     

Translocation of polysialic acid across model membranes: kinetic analysis and dynamic studies.

Transmembrane translocation of polyion homopolymers takes place in the case of polyanionic polysialic acid (polySia), polyanionic polynucleotides and polycationic polypeptides. The purpose of this work was to determine the role of membrane electrical parameters on the kinetics of polyion translocation, the influence of polysialic acid on ion adsorption on positively charged membrane surface and the dynamics of the phospholipid hydrocarbon chains and choline group by using 1H-NMR. The analysis of polyion translocation was performed by using the electrical equivalent circuit of the membrane for the initial membrane potential equal to zero. The changes in polysialic acid flux was up to 75% after 1 ms in comparison with the zero-time flux. Both a decrease of membrane conductance and an increase of polyion chain length resulted in the diminution of this effect. An increase of praseodymium ions adsorption to positively charged liposomes and an increase of the rate of segmental movement of the -CH2 and -CH3 groups, and the choline headgrup of lipid molecules, was observed in the presence of polySia. The results show that the direction of the vectorial polyion translocation depends both on the membrane electrical properties and the degree of polymerization of the polymer, and that polysialic acid can modulate the degree of ion adsorption and the dynamics of membrane lipids.     

Purification and characterization of a polysialic acid-specific sialidase from Pseudomonas fluorescens JK-0412

An enzyme with polySia degrading activity was purified from a culture filtrate of Pseudomonas fluorescens JK-0412 to apparent homogeneity using DEAE-Sepharose CL-6B column chomatography and fast performance liquid chomatography separation on a Mono-Q column. The molecular mass of the purified enzyme (tentatively named Endo-PS) was approximately 20 kDa on SDS-PAGE and 120 kDa on native-PAGE gels, suggesting that the active form is a hexamer. Although 12 residues of the Endo-PS N-terminal amino acid sequence showed 75% homology to the 21 kDa chitin binding protein (CBP21) of Serratia marcescens 2170, no significant similarity to other known proteins was observed. Apparent K _m and V _max values of Endo-PS toward the artificial substrate 4-methylumbelliferyl-sialic acid (4-MU-Neu5Ac) were 0.08 mM and 16 nmol/mg/min, respectively. The enzyme was maximally active at 37°C and pH 8.0. Interestingly, the enzyme was shown to hydrolyze the natural substrate, ??2,8-linked polySia (colominic acid), in an endo-acting manner. However, no activity toward ??2,3- or ??2,6-sialyllactose was observed. Under optimal conditions, oligoSia ranging from 2 to 30 residues long were liberated by the cleavage of polySia, as identified by HPAEC-PED. Therefore, the purified enzyme Endo-PS was found to be a polySia-specific sialidase. This is the first report to describe the properties of a bacterial polySia-specific sialidase. Therefore, this enzyme may be a useful tool for both industrial oligoSia production and research on the structure and biological functions of polySia in nature.     

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.     

Polysialic acid chains exhibit enhanced affinity for ordered regions of membranes

Polysialic acid (polySia) forms linear chains which are usually attached to the external surface of the plasma membrane mainly through the Neural Cell Adhesion Molecule (NCAM) protein. It is exposed on neural cells, several types of cancer cells, dendritic cells, and egg and sperm cells. There are several lipid raft-related phenomena in which polySia is involved; however the mechanisms of polySia action as well as determinants of its localization in lipid raft microdomains are still unknown, although the majority of NCAM molecules in the liquid-ordered raft membrane fractions of neural cells appear to be polysialylated. Here we investigate the affinity of polySia (both soluble and NCAM-dependent plasma membrane-bound) for liquid-ordered- and liquid-disordered regions of lipid vesicle and neuroblastoma cell membranes. Our studies indicate that polySia chains have a higher affinity for ordered regions of membranes as determined by the dissociation constant values for polySia-lipid bilayer complex, the fluorescence intensity of polySia bound to giant vesicles, the polySia-to-membrane FRET signal at the plasma membrane of live cells, and the decrease of the FRET signals after Endo-N treatment of the cells. These results suggest that polysialylation may be one of the determinants of protein association with liquid-ordered membrane lipid raft domains.     

Sialidase NEU4 hydrolyzes polysialic acids of neural cell adhesion molecules and negatively regulates neurite formation by hippocampal neurons

Modulation of levels of polysialic acid (polySia), a sialic acid polymer, predominantly associated with the neural cell adhesion molecule (NCAM), influences neural functions, including synaptic plasticity, neurite growth, and cell migration. Biosynthesis of polySia depends on two polysialyltransferases ST8SiaII and ST8SiaIV in vertebrate. However, the enzyme involved in degradation of polySia in its physiological turnover remains uncertain. In the present study, we identified and characterized a murine sialidase NEU4 that catalytically degrades polySia. Murine NEU4, dominantly expressed in the brain, was found to efficiently hydrolyze oligoSia and polySia chains as substrates in sialidase in vitro assays, and also NCAM-Fc chimera as well as endogenous NCAM in tissue homogenates of postnatal mouse brain as assessed by immunoblotting with anti-polySia antibodies. Degradation of polySia by NEU4 was also evident in neuroblastoma Neuro2a cells that were co-transfected with Neu4 and ST8SiaIV genes. Furthermore, in mouse embryonic hippocampal primary neurons, the endogenously expressed NEU4 was found to decrease during the neuronal differentiation. Interestingly, GFP- or FLAG-tagged NEU4 was partially co-localized with polySia in neurites and significantly suppressed their outgrowth, whereas silencing of NEU4 showed the acceleration together with an increase in polySia expression. These results suggest that NEU4 is involved in regulation of neuronal function by polySia degradation in mammals.     

The effect of long-chain bases on polysialic acid-mediated membrane interactions

Negatively-charged polysialic acid (polySia) chains are usually membrane-bound and are often expressed on the surface of neuroinvasive bacterial cells, neural cells, and tumor cells. PolySia can mediate both repulsive and attractive cis interactions between membrane components, and trans interactions between membranes. Positively-charged long-chain bases are widely present in cells, are often localized in membranes and can function as bioactive lipids. Here we use Langmuir monolayer technique, fluorescence spectroscopy and electron microscopy of lipid vesicles to study the role of a simple long-chain base, octadecylamine (ODA), in both cis and trans interactions mediated by polySia in model membranes composed of ODA and dioleoylphospatidycholine (DOPC). When added free to an aqueous solution, polySia increases the collapse pressure of ODA/DOPC monolayers, reduces the effect of ODA on the limiting molecular area, inverses the values of excess area per molecule and of excess free energy of mixing from positive to negative, and induces fusion of ODA/DOPC vesicles. These results suggest that a polySia chain can act as a multi-bridge that mediates cis interactions between different components of a lipid membrane, disrupts membrane aggregates, and mediates trans interactions between lipids in apposing membranes. These observations imply that polySia in cellular systems can act in a similar way.     

Studies on the linkage between teichoic acid and peptidoglycan in a bacteriophage-resistant mutant of Staphylococcus aureus H.

1. In addition to poly(ribitol phosphate) the walls of a bacteriophage-resistant mutant of Staphylococcus aureus H contain glycerol phosphate residues that are not removed on digestion with trypsin or extraction with phenol. 2. The glycerol phosphate is present in a chain, containing three or four glycerol phosphate residues, which is covalently attached to the peptidoglycan through a phosphodiester linkage to muramic acid; this linkage is readily hydrolysed by dilute alkali. 3. The degradative studies described suggest that the poly(ribitol phosphate) chains of the wall teichoic acid may be attached to the wall by linkage to this glycerol phosphate oligomer.     

A new sialic acid analogue, 9-O-acetyl-deaminated neuraminic acid, and alpha -2,8-linked O-acetylated poly(N-glycolylneuraminyl) chains in a novel polysialoglycoprotein from salmon eggs

A new polysialoglycoprotein, designated PSGP(On), was isolated from the unfertilized eggs of the kokanee salmon, Oncorhynchus nerka adonis. 400-MHz 1H NMR analyses showed the O. nerka adonis PSGP contained alpha -2,8-linked oligo- and polysialic acid (polySia) chains that were made up of 4-O-Ac-, 7-O-Ac-, and 9-O-Ac esters of N-glycolylneuraminic acid (Neu5Gc) residues. The presence of a new sialic acid derivative, identified by 1H NMR as 9-O-acetyl-2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (trivial name, 9-O-acetyldeaminated neuraminic acid; 9-O-Ac-KDN), was also shown to be present as a minor component. The O-acetylated KDN residues appear to cap the nonreducing termini of the O-acetylated poly(Neu5Gc) chains. The O-acetylated polySia chains were resistant to depolymerization by bacterial exosialidases and a bacteriophage-derived endo-N-acylneuraminidase that is specific for catalyzing the hydrolysis of alpha -2,8-linkages in polySia containing either N-acetylneuraminic acid or Neu5Gc residues. After de-O-acetylation by mild alkali, the polySia chains were sensitive to digestion by endo-N-acylneuraminidase, yet partially resistant to exosialidase. These data confirm the alpha -2,8-ketosidic linkage in these chains and the nonreducing terminal location of the KDN residues. These results extend further the range of structural diversity in polySia-containing glycoconjugates, and in the family of naturally occurring sialic acids. They also suggest that the O-acetylated Neu5Gc and 9-O-Ac-KDN residues may have an important role during oogenesis.     

A challenge to the ultrasensitive chemical method for the analysis of oligo- and polysialic acids at a nanogram level of colominic acid and a milligram level of brain tissues

Polysialic acid (polySia) is a functional epitope and is known: 1) to regulate normal fertilization of lower vertebrates and invertebrates; 2) to be expressed on neural cell adhesion molecule (NCAM) when the formation or re-arrangement of nervous tissues takes place during embryonic stages as well as in adults of higher vertebrates; and 3) to be re-expressed in several human tumors. Thus, polySia serves as oncodevelopmental antigen. To date sensitive biochemical diagnostic probes (antibodies and endo-N-acylneuraminidase) to detect polySia are known. However, these reagents are not commercially available yet and they are only reactive to specific types of polySia structure. Moreover, precise information not only on diversity but also on the length or degree of polymerization (DP) of extended polySia chains is considered important in understanding the molecular mechanism of biosynthesis of polySia chains and fine-tuning of NCAM-NCAM adhesive interaction by polySia chain but cannot be obtained with these biochemical probes. We have been continuously making efforts to develop and improve the sensitivity of chemical methods for polySia analysis toward these challenging problems. This article presents our most recently developed chemical method for polySia analysis and its use in obtaining new information on DP of colominic acid samples and polySia chains present in rat brain tissues with the highest sensitivity that has ever been attained.     

Polysialic acid, a glycan with highly restricted expression, is found on human and murine leukocytes and modulates immune responses

Polysialic acid (polySia) is a large glycan with restricted expression, typically found attached to the protein scaffold neural cell adhesion molecule (NCAM). PolySia is best known for its proposed role in modulating neuronal development. Its presence and potential functions outside the nervous systems are essentially unexplored. Herein we show the expression of polySia on hematopoietic progenitor cells, and demonstrate a role for this glycan in immune response using both acute inflammatory and tumor models. Specifically, we found that human NK cells modulate expression of NCAM and the degree of polymerization of its polySia glycans according to activation state. This contrasts with the mouse, where polySia and NCAM expression are restricted to multipotent hematopoietic progenitors and cells developing along a myeloid lineage. Sialyltransferase 8Sia IV(-/-) mice, which lacked polySia expression in the immune compartment, demonstrated an increased contact hypersensitivity response and decreased control of tumor growth as compared with wild-type animals. This is the first demonstration of polySia expression and regulation on myeloid cells, and the results in animal models suggest a role for polySia in immune regulation.     

Large-scale production and homogenous purification of long chain polysialic acids from E. coli K1

The study of new biomaterials is the objective of many current research projects in biotechnological medicine. A promising scaffold material for the application in tissue engineering or other biomedical applications is polysialic acid (polySia), a homopolymer of alpha2,8-linked sialic acid residues, which represents a posttranslational modification of the neural cell adhesion molecule and occurs in all vertebrate species. Some neuroinvasive bacteria like, e.g. Escherichia coli K1 (E. coli K1) use polySia as capsular polysaccharide. In this latter case long polySia chains with a degree of polymerization of >200 are linked to lipid anchors. Since in vertebrates no polySia degrading enzymes exist, the molecule has a long half-life in the organism, but degradation can be induced by the use of endosialidases, bacteriophage-derived enzymes with pronounced specificity for polySia. In this work a biotechnological process for the production of bacterial polysialic acid is presented. The process includes the development of a multiple fed-batch cultivation of the E. coli K1 strain and a complete downstream strategy of polySia. A controlled feed of substrate at low concentrations resulted in an increase of the carbon yield (C(product)/C(substrate)) from 2.2 to 6.6%. The downstream process was optimized towards purification of long polySia chains. Using a series of adjusted precipitation steps an almost complete depletion of contaminating proteins was achieved. The whole process yielded 1-2g polySia from a 10-l bacterial culture with a purity of 95-99%. Further product analysis demonstrated maximum chain length of >130 for the final product.     

Specificity of fatty acid acylation of cellular proteins

Labeling of the BC3H1 muscle cell line with [3H] palmitate and [3H]myristate results in the incorporation of these fatty acids into a broad spectrum of different proteins. The patterns of proteins which are labeled with palmitate and myristate are distinct, indicating a high degree of specificity of fatty acylation with respect to acyl chain length. The protein-linked [3H]palmitate is released by treatment with neutral hydroxylamine or by alkaline methanolysis consistent with a thioester linkage or a very reactive ester linkage. In contrast, only a small fraction of the [3H]myristate which is attached to proteins is released by treatment with hydroxylamine or alkaline methanolysis, suggesting that myristate is linked to proteins primarily through amide bonds. The specificity of fatty acid acylation has also been examined in 3T3 mouse fibroblasts and in PC12 cells, a rat pheochromacytoma cell line. In both cells, palmitate is primarily linked to proteins by a hydroxylamine-labile linkage while the major fraction of the myristic acid (60-70%) is linked to protein via amide linkage and the remainder via an ester linkage. Major differences were noted in the rate of fatty acid metabolism in these cells; in particular in 3T3 cells only 33% of the radioactivity incorporated from myristic acid into proteins is in the form of fatty acids. The remainder is presumably the result of conversion of label to amino acids. In BC3H1 cells, palmitate- and myristate-containing proteins also exhibit differences in subcellular localization. [3H]Palmitate-labeled proteins are found almost exclusively in membranes, whereas [3H]myristate-labeled proteins are distributed in both the soluble and membrane fractions. These results demonstrate that fatty acid acylation is a covalent modification common to a wide range of cellular proteins and is not restricted solely to membrane-associated proteins. The major acylated proteins in the various cell lines examined appear to be different, suggesting that the acylated proteins are concerned with specialized cell functions. The linkages through which fatty acids are attached to proteins also appear to be highly specific with respect to the fatty acid chain length.     

Direct binding of polysialic acid to a brain-derived neurotrophic factor depends on the degree of polymerization

Polysialic acid (polySia) is the homopolymer of sialic acid and negatively regulates neuronal cell-cell and cell-extracellular matrix interactions through steric and repulsive hindrance due to its bulky polyanionic structure. Whether polySia also functions as a positive regulator in the nervous system through binding to specific ligands is not known. In the present study, we demonstrated that a brain-derived neurotrophic factor (BDNF) dimer binds directly to polySia to form a large complex with an M(r) greater than 2000 kDa under physiologic conditions. Although somewhat affected by the linkage and type of sialic acid components in the polySia, the complex formation is highly dependent on the polySia chain length. The minimum degree of polymerization required for the complex formation is 12. This is the first study to demonstrate the biologic significance of the degree of polySia polymerization in eukaryotes. Similar large polySia complexes form with other neurotrophic factors such as nerve growth factor, neurotrophin-3, and neurotrophin-4. Furthermore, the BDNF, after making a complex with polySia, can bind to the BDNF receptors, TrkB and p75NTR. The complex formation of BDNF with polySia upregulates growth or/and survival of neuroblastoma cells. These findings suggest that polySia functions as a reservoir of BDNF and other neurotrophic factors and may serve to regulate their local concentrations on the cell surface.     

Thiopalmitoylation of myelin proteolipid protein epitopes enhances immunogenicity and encephalitogenicity

Proteolipid protein (PLP) is the most abundant protein of CNS myelin, and is posttranslationally acylated by covalent attachment of long chain fatty acids to cysteine residues via a thioester linkage. Two of the acylation sites are within epitopes of PLP that are encephalitogenic in SJL/J mice (PLP(104-117) and PLP(139-151)) and against which increased immune responses have been detected in some multiple sclerosis patients. It is known that attachment of certain types of lipid side chains to peptides can result in their enhanced immunogenicity. The aim of this study was to determine whether thioacylated PLP peptides, as occur in the native protein, are more immunogenic than their nonacylated counterparts, and whether thioacylation influences the development of autoreactivity and experimental autoimmune encephalomyelitis. The results show that in comparison with nonacylated peptides, thioacylated PLP lipopeptides can induce greater T cell and Ab responses to both the acylated and nonacylated peptides. They also enhanced the development and chronicity of experimental autoimmune encephalomyelitis. Synthetic peptides in which the fatty acid was attached via an amide linkage at the N terminus were not encephalitogenic, and they induced greater proportions of CD8+ cells in initial in vitro stimulation. Therefore, the lability and the site of the linkage between the peptide and fatty acid may be important for induction of encephalitogenic CD4+ T cells. These results suggest that immune responses induced by endogenous thioacylated lipopeptides may contribute to the immunopathogenesis of chronic experimental demyelinating diseases and multiple sclerosis.     

Attachment of the main chain to the linkage unit in biosynthesis of teichoic acids

The main chain of teichoic acids can be assembled in cell-free membrane preparations by the transfer of residues from the appropriate nucleotide precursors to an incompletely characterized amphiphilic molecule, lipoteichoic acid carrier (LTC). However, in the cell wall, the main chain is attached to peptidoglycan through a linkage unit which is synthesized independently. It is believed that, in these cell-free systems, lipid intermediates carrying linkage units are also able to accept residues directly from nucleotide precursors to build up the main chain. In this paper, we have shown that the main chain attached to LTC was transferred from LTC to lipids containing the linkage unit. Thus, in these systems, there appear to be two routes to the biosynthesis of teichoic acid-linkage unit complexes, one by direct assembly of the main chain on linkage unit lipids and the other by transfer of the preassembled main chain from LTC to the linkage unit. It was also shown that linkage unit lipids from different organisms were interchangeable and that these were used for polymer synthesis by Bacillus subtilis 3610, in which the teichoic acid is a poly(glycerol phosphate).     

Identifying regions of membrane proteins in contact with phospholipid head groups: covalent attachment of a new class of aldehyde lipid labels to cytochrome c oxidase

A series of amine-specific reagents based on the benzaldehyde reactive group have been synthesized, characterized, and used to study beef heart cytochrome c oxidase reconstituted in phospholipid bilayers. The series contained three classes of reagents: lipid-soluble phosphodiesters having a single hydrocarbon chain, phospholipid analogues, and a water-soluble benzaldehyde. All reagents were either radiolabeled or spin-labeled or both. The Schiff bases formed by these benzaldehydes with amines were found to be reversible until the addition of the reducing agent sodium cyanoborohydride, whereas attachment of lipid-derived aliphatic aldehydes was not readily reversible in the absence of the reducing agent. The benzaldehyde group provides a convenient method of controlling and delaying permanent attachment to integral membrane proteins until after the reconstitution steps. This ensures that the lipid analogues are located properly to identify amine groups at the lipid-protein interface rather than reacting indiscriminately with amines of the hydrophilic domains of the protein. The benzaldehyde lipid labels attach to cytochrome c oxidase with high efficiency. Typically, 20% of the amount of lipid label present was covalently attached to the protein, and the number of moles of label incorporated per mole of protein ranged from 1 to 6, depending on the molar ratios of label, lipid, and protein. The efficiency of labeling by the water-soluble benzaldehyde was much less than that observed for any of the lipid labels because of dilution effects, but equivalent levels of incorporation were achieved by increasing the label concentration. Electron spin resonance spectra of a nitroxide-containing phospholipid analogue covalently attached to reconstituted cytochrome c oxidase exhibited a large motion-restricted component, which is characteristic of spin-labeled lipids in contact with the hydrophobic surfaces of membrane proteins. The line shape and splittings were similar for covalently attached label and label free to diffuse and contact the protein molecules in the bilayer, providing independent evidence that the coupling occurs at the protein-lipid interface. The distribution of the benzaldehyde reagents attached to the polypeptide components of cytochrome c oxidase was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The labeling pattern observed for the lipid analogues was not affected by the presence of the nitroxide moiety on the acyl chains but was dependent on the molar ratio of labeling reagent to protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemical analysis of the developmental pattern of polysialylation in chicken brain. Expression of only an extended form of polysialyl chains during embryogenesis and the presence of disialyl residues in both embryonic and adult chicken brains

Recent studies have demonstrated the involvement of two polysialyltransferases in neural cell adhesion molecule (N-CAM) polysialylation. The availability of cDNAs encoding these enzymes facilitated studies on polysialylation of N-CAM. However, there is a dearth of detailed structural information on the degree of polymerization (DP), DP ranges, and the influence of embryogenesis on the DP. It is also unclear how many polysialic acid (polySia) chains are attached to a single core N-glycan. In this paper we applied new, efficient, and sensitive high pressure liquid chromatography methods to qualitatively and quantitatively analyze the polySia structures expressed on embryonic and adult chicken brain N-CAM. Our studies resulted in the following new findings. 1) The DP of the polySia chains was invariably 40-50 throughout developmental stages from embryonic day 5 to 21 after fertilization. In contrast, glycopeptides containing polySia with shorter DPs, ranging from 15 to 35, were isolated from adult brain. 2) Chemical evidence showed glycan chains abundant in Neu5Acalpha2,8Neu5Ac were expressed during all developmental stages including adult. 3) Levels of both di- and polySia were found to show distinctive changes during embryonic development.