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Polysialic acid (polySia) is an unusual glycan that posttranslational modifies neural cell adhesion molecule (NCAM) proteins in mammalian cells. The up-regulated expression of polySia-NCAM is associated with tumor progression in many metastatic human cancers and in neurocognitive processes. Two members of the ST8Sia family of &alpha, 2,8-polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST) both catalyze synthesis of polySia when activated cytidine monophosphate(CMP)-Sialic acid (CMP-Sia) is translocate into the lumen of the Golgi apparatus. Two key polybasic domains in the polySTs, the polybasic region (PBR) and the polysialyltransferase domain (PSTD) areessential forpolysialylation of the NCAM proteins. However, the precise molecular details to describe the interactions required for polysialylation remain unknown. In this study, we hypothesize that PSTD interacts with both CMP-Sia and polySia to catalyze polysialylation of the NCAM proteins. To test this hypothesis, we synthesized a 35-amino acid-PSTD peptide derived from the ST8Sia IV gene sequence and used it to study its interaction with CMP-Sia, and polySia. Our results showed for the PSTD-CMP-Sia interaction,the largest chemical-shift perturbations (CSP) were in amino acid residues V251 to A254 in the short H1 helix, located near the N-terminus of PSTD. However, larger CSP values for the PSTD-polySia interaction were observed in amino acid residues R259 to T270 in the long H2 helix. These differences suggest that CMP-Sia preferentially binds to the domain between the short H1 helix and the longer H2 helix. In contrast, polySia was principally bound to the long H2 helix of PSTD. For the PSTD-polySia interaction, a significant decrease in peak intensity was observed in the 20 amino acid residues located between the N-and C-termini of the long H2 helix in PSTD, suggesting a slower motion in these residues when polySia bound to PSTD. Specific features of the interactions between PSTD-CMP-Sia, and PSTD-polySia were further confirmed by comparing their 800 MHz-derived HSQC spectra with that of PSTD-Sia, PSTD-TriSia (DP 3) and PSTD-polySia. Based on the interactions between PSTD-CMP-Sia, PSTD-polySia, PBR-NCAM and PSTD-PBR, these findingsprovide a greater understanding of the molecular mechanisms underlying polySia-NCAM polysialylation, and thus provides a new perspective for translational pharmacological applications and development by targeting the two polysialyltransferases.

Polysialic acid (polySia) is a novel glycan that posttranslationally modifies neural cell adhesion molecules (NCAMs) in mammalian cells. Up-regulation of polySia-NCAM expression or NCAM polysialylation is associated with tumor cell migration and progression in many metastatic cancers and neurocognition. It has been known that two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), can catalyze polysialylation of NCAM, and two polybasic domains, polybasic region (PBR) and polysialyltransferase domain (PSTD) in polySTs play key roles in affecting polyST activity or NCAM polysialylation. However, the molecular mechanisms of NCAM polysialylation and cell migration are still not entirely clear. In this minireview, the recent research results about the intermolecular interactions between the PBR and NCAM, the PSTD and cytidine monophosphate-sialic acid (CMP-Sia), the PSTD and polySia, and as well as the intramolecular interaction between the PBR and the PSTD within the polyST, are summarized. Based on these cooperative interactions, we have built a novel model of NCAM polysialylation and cell migration mechanisms, which may be helpful to design and develop new polysialyltransferase inhibitors.

The two mammalian α2,8-polysialyltransferases (polyST's), ST8Sia II (STX) and ST8Sia IV (PST), catalyze synthesis of the α2-8-linked polysialic acid (polySia) glycans on neural cell adhesion molecules (NCAMs). The objective of this study was to clone the coding sequence of the piglet ST8Sia II and determine the mRNA expression levels of ST8Sia II, ST8Sia IV, NCAM and neuropilin-2 (NRP-2), also a carrier protein of polySia, during postnatal development. The amino acid sequence deduced from the coding sequence of ST8Sia II was compared with seven other mammalian species. Piglet ST8Sia II was highly conserved and shared 67.8% sequence identity with ST8Sia IV. Genes coding for ST8Sia II and IV were differentially expressed and distinctly different in neural and non-neural tissues at postnatal days 3 and 38. Unexpectedly, the cellular levels of mRNA coding for ST8Sia II and IV showed no correlation with the posttranslational level of polySia glycans in different tissues. In contrast, mRNA abundance coding for NCAM and neuropilin-2 correlated with expression of ST8Sia II and IV. These findings show that the cellular abundance of ST8Sia II and IV in postnatal piglets is regulated at the level of translation/posttranslation, and not at the level of transcription, a finding that has not been previously reported. These studies further highlight differences in the molecular mechanisms controlling polysialylation in adult rodents and neonatal piglets.

An elevated level of the free deaminated sialic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), was first discovered in human ovarian cancers (OCs), suggesting that KDN may be an oncodevelopmental antigen (Inoue S, Lin SL, Chang T, Wu SH, Yao CW, Chu TY, Troy FA II, Inoue Y. 1998. J Biol Chem. 273(42):27199-27204). To determine if this unexpected finding was unique to OC, we developed an LC-MS/MS glycomic approach to quantitatively determine the level of free and conjugated forms of KDN, Neu5Ac and Neu5Gc in head and neck cancers of the throat, and in a subpopulation of matched lymph nodes. These findings were correlated with tumor (T), nodal (N), metastatic (M) involvement and the differentiation status of the tumors. The following new findings are reported: (i) The level of free KDN in 49 throat cancers and a subpopulation of 10 regional lymph nodes accounted for 94.5 and 93.3%, respectively, of the total level of KDN (∼2 µg/g); (ii) in marked contrast, the level of free Neu5Ac in throat cancer and lymph nodes accounted for only 6.5 and 5.1% of the total level of Neu5Ac (85 µg/g); (3) The level of Neu5Gc (0.03 µg/g) in throat cancers was 0.30% of the level of Neu5Ac, two-thirds were conjugated and one-third was free. The central importance of these new findings is that the elevated level of free KDN relative to free Neu5Ac and Neu5GC in throat cancers showing no lymphatic metastasis, and which are poorly to moderately differentiated, suggests that free KDN may be useful as a biomarker for detecting some early-stage cancers at biopsy, and be of possible prognostic value in determining the potential degree of malignancy.

Lactoferrin (Lf) is a sialic acid (Sia)-rich, iron-binding milk glycoprotein that has multifunctional health benefits. Its potential role in neurodevelopment and cognition remains unknown. To test the hypothesis that Lf may function to improve neurodevelopment and cognition, the diet of postnatal piglets was supplemented with Lf from days 3 to 38. Expression levels of selected genes and their cognate protein profiles were quantitatively determined. The importance of our new findings is that Lf (1) upregulated several canonical signaling pathways associated with neurodevelopment and cognition; (2) influenced ~10 genes involved in the brain-derived neurotrophin factor (BDNF) signaling pathway in the hippocampus and upregulated the expression of polysialic acid, a marker of neuroplasticity, cell migration and differentiation of progenitor cells, and the growth and targeting of axons; (3) upregulated transcriptional and translational levels of BDNF and increased phosphorylation of the cyclic adenosine monophosphate (cAMP) response element-binding protein, CREB, a downstream target of the BDNF signaling pathway, and a protein of crucial importance in neurodevelopment and cognition; and (4) enhanced the cognitive function and learning of piglets when tested in an eight-arm radial maze. The finding that Lf can improve neural development and cognition in postnatal piglets has not been previously described. Electronic supplementary material The online version of this article (doi:10.1007/s12035-014-8856-9) contains supplementary material, which is available to authorized users.

As a subset of glycosyltransferases, the family of sialyltransferases catalyze transfer of sialic acid (Sia) residues to terminal non-reducing positions on oligosaccharide chains of glycoproteins and glycolipids, utilizing CMP-Neu5Ac as the activated sugar nucleotide donor. In the four known sialyltransferase families (ST3Gal, ST6Gal, ST6GalNAc and ST8Sia), the ST8Sia family catalyzes synthesis of α2, 8-linked sialic/polysialic acid (polySia) chains according to their acceptor specificity. We have determined the 3D structural models of the ST8Sia family members, designated ST8Sia I (1), II(2), IV(4), V(5), and VI(6) using the Phyre2 server. Accuracy of these predicted models are based on the ST8Sia III crystal structure as the calculated template. The common structural features of these models are: (1) Their parallel templates and disulfide bonds are buried within the enzymes and are predominately surrounded by helices; (2) The anti-parallel β-sheets are located at the N-terminal region of the enzymes; (3) The mono-sialytransferases (mono-STs), ST8Sia I and ST8Sia VI, contain only a single pair of disulfide bonds, and there are no anti-parallel β-sheets in ST8Sia VI; (4) The Nterminal region of all of the mono-STs are located some distant away from their core structure; (5) These conformational features show that the 3D structures of the mono-STs are less compact than the two polySTs, ST8Sia II and ST8Sia IV, and the oligo-ST, ST8Sia III. These structural features relate to the catalytic specificity of the monoSTs; (6) In contrast, the more compact structural features of ST8Sia II, ST8Sia IV and ST8Sia III relate to their ability to catalyze the processive synthesis of oligo- (ST8Sia III) and polySia chains (ST8Sia IIamp; ST8Sia IV); (7) Although ST8Sia II, III and IV have similar conformations in their corresponding polysialyltransferase domain (PSTD) and polybasic region (PBR) motifs, the structure of ST8Sia III is less compact than ST8Sia II and ST8Sia IV, and the amino acid components of the several three-residue-loops in the two motifs of ST8Sia III are different from that in ST8Sia II and ST8Sia IV. This is likely the structural basis for why ST8Sia III is an oligoST and not able to polysialylate and; (8) In contrast, essentially all amino acids within the threeresidue- loops in the PSTD of ST8Sia II and ST8Sia IV are highly conserved, and many amino acids in the loops and the helices of these two motifs are critical for NCAM polysialylation, as determined by mutational analysis and confirmed by our recent NMR results. In summary, these new findings provide further insights into the molecular mechanisms underlying polyST-NCAM recognition, polySTpolySia/ oligoSia interactions, and polysialylation of NCAM.

Earlier NMR studies showed that the polyisoprenols (PIs) dolichol (C95), dolichylphosphate (C95-P) and undecaprenylphosphate (C55-P) could alter membrane structure by inducing in the lamellar phospholipid (PL) bilayer a nonlamellar or hexagonal (Hex II) structure. The destabilizing effect of C95 and C95-P on host fatty acyl chains was supported by small angle X-ray diffraction and freeze-fracture electron microscopy. Our present 1H- and 31P-NMR studies show that the addition of a polyisoprenol recognition sequence (PIRS) peptide to nonlamellar membranes induced by the PIs can reverse the hexagonal structure phase back to a lamellar structure. This finding shows that the PI:PIRS docking complex can modulate the polymorphic phase transitions in PL membranes, a finding that may help us better understand how glycosyl carrier-linked sugar chains may traverse membranes. Using an energy-minimized molecular modeling approach, we also determined that the long axis of C95 in phosphatidylcholine (PC) membranes is oriented approximately parallel to the interface of the lipid bilayer, and that the head and tail groups are positioned near the bilayer interior. In contrast, the phosphate head group of C95-P is anchored at the PC bilayer, and the angle between the long axis of C95-P and the bilayer interface is about 758, giving rise to a preferred conformation more perpendicular to the plane of the bilayer. Molecular modeling calculations further revealed that up to five PIRS peptides can bind cooperatively to a single PI molecule, and this tethered structure has the potential to form a membrane channel. If such a channel were to exist in biological membranes, it could be of functional importance in glycoconjugate translocation, a finding that has not been previously reported.

The pre-existence of α2→8-linked disialic acid (di-Sia) and oligosialic acid (oligo-Sia) structures with up to 7 Sia residues was shown to occur on a large number of brain glycoproteins, including neural cell adhesion molecules (N-CAMs), by two highly sensitive chemical methods (Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1998) Anal. Biochem. 261, 191–197; Sato, C., Inoue, S., Matsuda, T., and Kitajima, K. (1999) Anal. Biochem. 266, 102–109). This unexpected finding was also confirmed using a newly developed antibody prepared using a copolymer of α2→8-linked N-acetylneuraminylp-vinylbenzylamide and acrylamide as an immunogen and known antibodies whose immunospecificities were determined to be di- and oligo-Sia residues with defined chain lengths. The major significance of the new finding that di- and oligo-Sia chains exist on a large number of brain glycoproteins is 2-fold. First, it reveals a surprising diversity in the number and M r of proteins distinct from N-CAM that are covalently modified by these short sialyl glycotopes. Second, it suggests that synthesis of di- and/or oligo-Sia units may be catalyzed by α2→8-sialyltransferase(s) that are distinct from the known polysialyltransferases, STX and PST, which are partially responsible for polysialylation of N-CAM.

Although the deaminoneuraminic acid or KDN glycotope (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) is expressed in glycoconjugates that range in evolutionary diversity from bacteria to man, there is little information as to how this novel sugar is synthesized. Accordingly, biosynthetic studies were initiated in trout testis, an organ rich in KDN, to determine how this sialic acid is formed. These studies have shown that the pathway consists of the following three sequential reactions: 1) Man + ATP --> Man-6-P + ADP; 2) Man-6-P + PEP --> KDN-9-P + P(i); 3) KDN-9-P --> KDN + P(i). Reaction 1, catalyzed by a hexokinase, is the 6-O-phosphorylation of mannose to form D-mannose 6-phosphate (Man-6-P). Reaction 2, catalyzed by KDN-9-phosphate (KDN-9-P) synthetase, condenses Man-6-P and phosphoenolpyruvate (PEP) to form KDN-9-P. Reaction 3, catalyzed by a phosphatase, is the dephosphorylation of KDN-9-P to yield free KDN. It is not known if a kinase specific for Man (Reaction 1) and a phosphatase specific for KDN-9-P (Reaction 3) may exist in tissues actively synthesizing KDN. In this study, the KDN-9-P synthetase, an enzyme that has not been previously described, was identified as at least one key enzyme that is specific for the KDN biosynthetic pathway. This enzyme was purified 50-fold from rainbow trout testis and characterized. The molecular weight of the enzyme was estimated to be about 80,000, and activity was maximum at neutral pH in the presence of Mn(2+). N-Acetylneuraminic acid 9-phosphate (Neu5Ac-9-P) synthetase, which catalyzes the condensation of N-acetyl-D-mannosamine 6-phosphate and phosphoenol-pyruvate to produce Neu5Ac-9-P, was co-purified with the KDN-9-P synthetase. Substrate competition experiments revealed, however, that syntheses of KDN-9-P and Neu5Ac-9-P were catalyzed by two separate synthetase activities. The significance of these studies takes on added importance with the recent discovery that the level of free KDN is elevated in human fetal cord but not matched adult red blood cells and in ovarian cancer cells (Inoue, S., Lin, S-L., Chang, T., Wu, S-H., Yao, C-W., Chu, T-Y., Troy, F. A., II, and Inoue, Y. (1998) J. Biol. Chem. 273, 27199-27204). This unexpected finding emphasizes the need to understand more fully the role that free KDN and KDN-glycoconjugates may play in normal hematopoiesis and malignancy.

Red meat and dairy products contain high sialic acid (Sia) levels, but the metabolic fate and health impact in children remain unknown. The aims of the present study were to quantify the levels of urinary Sia N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc) and ketodeoxynonulosonic acid (KDN) and to determine their relationship with dietary Sia intake. Spot urine samples were collected from 386 healthy children aged 3 (n 108), 4 (n 144) and 5 (n 134) years at 06.30–07.00, 11.30–12.00 and 16.30–17.00 hours. Food intake levels were recorded on the day of urine sample collection. Sia levels were quantified using LC–MS/MS with [13C3]Sia as an internal standard. We found that (1) total urinary Sia levels in healthy pre-school children ranged from 40 to 79 mmol Sia/mol creatinine; (2) urinary Sia levels were independent of age and consisted of conjugated Neu5Ac (approximately 70·8 %), free Neu5Ac (approximately 21·3 %), conjugated KDN (approximately 4·2 %) and free KDN (approximately 3·7 %); Neu5Gc was detected in the urine of only one 4-year-old girl; (3) total urinary Sia levels were highest in the morning and declined over time in 4- and 5-year-old children (P

The CMP-sialic acid:poly alpha 2,8sialosyl sialyltransferase (polyST) in neurotropic Escherichia coli K1 inner membranes catalyzes synthesis of the alpha 2,8-linked polysialic acid capsule. The capsule is a neurovirulent determinant associated with neonatal meningitis in humans. A functionally similar polyST in human neuroblastomas polysialylates neural cell adhesion molecules. While bacteria do not synthesize glycosphingolipids (GSLs), we report here that the E. coli K1 polyST can selectively polysialylate several structurally related GSLs, when added as exogenous sialyl acceptors. A structural feature common to the preferred sialyl acceptors (GD3 > GT1a > GQ1b = GT1b > GD2 = GD1b = GD1a > GM1) was the disialyl glycotope, Sia alpha 2,8Sia, alpha 2,3-linked to galactose (Sia is sialic acid). A linear tetrasaccharide with a terminal Sia residue (e.g., GD3) was the minimum length oligosaccharide recognized by the polyST. Endo-N-acylneuraminidase was used to confirm the alpha 2,8-specific polysialylation of GSL. Ceramide glycanase was used to release the polysialyllactose chains from the ceramide moiety. Size analysis of these chains showed that 60-80 Sia residues were transferred to the disialyllactose moiety of GD3. The significance of these findings is two-fold. (i) The E. coli K1 polyST can be used as a synthetic reagent to enzymatically engineer the glycosyl moiety of GSL, thus creating oligo- or polysialylated GSLs. Such "designer" GSLs may have potentially important biological and pharmacological properties. (ii) The use of GSLs as exogenous sialyl acceptors increases the sensitivity of detecting polyST activity. The practical advantage of this finding is that polyST activity can be identified and studied in those eukaryotic cells that express low levels of this developmentally regulated enzyme and/or its acceptor.

The kinetics of acid- and sialidase-catalyzed hydrolysis of the interketosidic linkages of two different disialic acids, Neu5Gc alpha 2--5-OglycolylNeu5Gc and Neu5Gc alpha 2--8Neu5Gc, were studied. The former sequence was recently identified in the polysialic acid chains of a sialic acid-rich glycoprotein isolated from the egg jelly coat of two different species of sea urchins, and the latter was previously found in the cortical alveolar-derived polysialoglycoprotein from rainbow trout eggs. At pH values3.8, the rate of hydrolysis of Neu5Gc alpha 2--5-OglycolylNeu5Gc was greater than that of Neu5Gc alpha 2--8Neu5Gc. Paradoxically, however, Neu5Gc alpha 2--5-OglycolylNeu5Gc was more stable than Neu5Gc alpha 2--8Neu5Gc at pH values3.8. These findings indicate a greater contribution of intramolecular general acid catalysis to the lability of the alpha 2--5-ketosidic linkage. Neu5Gc alpha 2--5-OglycolylNeu5Gc was a poor substrate for Arthrobacter ureafaciens, Clostridium perfringens, and Vibrio cholerae sialidases, in contrast to Neu5Gc alpha 2--8Neu5Gc. Neu5Gc alpha 2--5-OglycolylNeu5Gc was essentially resistant to hydrolysis by A. ureafaciens sialidase.

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.

Chemical studies have shown the occurrence of the deaminated sialic acid 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) in paired samples of blood obtained from mothers and newborns of healthy human individuals. Most of the KDN was found in red blood cells, although low levels were detected in mononuclear cells. No N-glycolylneuraminic acid was detected. Unexpectedly, nearly all of the KDN in fetal cord and matched maternal red blood cells was present as the free sugar and comparatively little occurred conjugated or as cytidine 5'-KDN phosphate. The amount of free KDN in fetal newborn red blood cells was 2.4-fold higher than in red blood cells from the mothers or from healthy nonpregnant women. Free KDN was also identified in normal human ovaries, in ovarian tumors, and in ascites cells obtained from ovarian cancer patients. Importantly, as in fetal cord red blood cells, a distinguishing feature of KDN expression in ovarian tumor cells was an elevated level of free KDN compared with normal controls. A positive correlation was found between an increase in the ratio of free KDN/N-acetylneuraminic acid in ovarian adenocarcinomas and the stage of malignancy. This was particularly evident in tumor cells isolated from the ascites fluid. The central importance of these new findings is 2-fold. First, they show that free KDN is a minor but ubiquitous sialic acid in human red blood cells and that its elevated expression in red blood cells from fetal cord blood compared with maternal red blood cells may be developmentally related to blood cell formation during embryogenesis. Second, the enhanced expression of KDN in ovarian cancer cells suggests that this sialic acid, like the alpha2,8-linked polysialic acid glycotope, may be an oncofetal antigen in these tumors and thus could be an "early warning" signal for onset of disease and/or a marker for detection of recurrence of disease. These new findings highlight the importance of elucidating the role that KDN and KDN-containing glycoconjugates may play in normal development and malignancy.

The alpha2,8-polysialyltransferases (polySTs) from embryonic chick brain catalyze the alpha2,8-specific polysialylation of endogenous neural cell adhesion molecules (N-CAMs). This posttranslation glycosylation decreases N-CAM-dependent cell adhesion and migration. The enzymatic properties of the membrane-bound form of the polyST activity was investigated in vitro. Our results show that the polyST activity was developmentally expressed with maximum specific activity appearing about 12 days after fertilization. This time shortly precedes maximal expression of the cognate polysialylated N-CAMs. Kinetic studies showed the KMand Vmaxfor CMP-Neu5Ac were 133 microM and 0.13 microM/h, respectively, at pH 6.1, 33 degrees C. CMP-Neu5Gc was not a donor substrate. PolyST activity was increased 5- to 6-fold in the presence of 10 mM MnCl2,the preferred divalent cation, and 1 mM dithiothreitol (DTT). Heparin (3 kDa) was a noncompetitive inhibitor of polysialylation with a Kiof 9 microM. Based on the affinity of the enzyme for heparin, the polyST activity was partially purified ( approximately 30-fold) by heparin-Sepharose affinity chromatography, after differential solubilization with the zwitterionic detergent, CHAPS. DTT and chemical modification studies using the thiol-directed alkylating reagents, N-ethylmaleimide (NEM) and iodoacetamide (IAA), were used to show that at least one cysteinyl residue in the polyST was of critical importance for polysialylation, but of lesser importance for monosialylation, catalyzed by the alpha2,3-, alpha2,6-, and alpha2,8-monosialyltransferases (monoSTs). A sulfhydryl residue is implicated in chain initiation. Two important structural differences between the mono- and polySTs were revealed by sequence analyses. First, the polySTs contain heparin-like, positively charged amino acid clusters upstream of both sialylmotif L and S. Second, the polySTs contain a uniquely extended basic amino acid region (pI 11. 6-12.0) of 31 residues immediately upstream of sialylmotif S. This extended, positively charged region may function in the processive mechanism of polymerization by allowing nascent polySia chains to remain bound to the polyST during the repetitive addition of each new Sia residue to the nonreducing termini of the growing chain. The importance of these studies is that they provide new information on the enzymatic basis of polysialylation. They also reveal that sulfhydryl residues and extended basic amino acid domains are two structural features unique to polysialylation, in contrast to monosialylation. Both may be important distinguishing features between the classes of distributive (monoSTs) and processive polysialyltransferases, which have not been previously described.

A rapid, sensitive, and facile method for screening and characterizing anti-polysialic acid (polySia) antibodies using lipid-conjugated oligo/polysialic acids (oligo/polySia) was developed, which is based on an enzyme-linked immunosorbent assay. Homooligo/polymers of α2→8-linked N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid, and 2-keto-3-deoxy-D-galacto-nononic acid (KDN) were conjugated with phosphatidylethanolamine dipalmitoyl (PE) by reductive amination to prepare neo-oligo/polysialoglycolipids (oligo/polySia-PE). Using this method, the anti-polySia equine antibody, H.46, bound to (→8Neu5Acα2→)n-PE, where n = 9 or more residues, a result in confirmation of previous binding studies using radiolabeled oligo/polyNeu5Ac. The antigenic specificity and sensitivity of two monoclonal anti-poly/oligoNeu5Ac antibodies (mAb.12E3 and mAb.5A5) and one anti-oligoKDN antibody (mAb.kdn8kdn), were also determined. mAb.12E3 could detect as little as 25 pg/well of oligo/polyNeu5Ac-PE, while mAb.5A5 and polyclonal antibody H.46 required at least 0.4 ng/well of oligo/polyNeu5Ac-PE to be detected. mAb.kdn8kdn detected as little as 12 ng/well of oligoKDN-PE. Using a series of oligo/polySia-PE with defined degrees of polymerization (DP), the minimum chain length for immunoreactivity of the anti-polySia antibodies was determined to be: DP 5 for mAb.12E3; DP 3 for mAb.5A5; DP 2 for mAb.kdn8kdn; and DP 8 for H.46. Thus, mAb.12E3 and mAb.5A5 recognize shorter oligomers of Neu5Ac than H.46, a finding that is of practical value for identifying shorter oligoSia chains in glycoconjugates. Because mAb.12E3 and mAb.5A5 also recognize extended polySia chains, these antibodies cannot be used, however, to differentiate between short and long chains of polySia when both are expressed on the same molecule.

Polysialic acids (polySia)* are a structurally diverse family of linear carbohydrate chains that consist of N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc) residues, usually joined internally by α2,8-, α2,9-, or alternating α2,8-/α2,9-ketosidic linkages. 3-Deoxy-D-glycero-D-galacto-2- nonulosonic acid (KDN) is a unique deaminated form of Sia, and polyKDN chains share many properties in common with polySia (Table I). The finding of poly(Neu5Ac), poly(Neu5Gc), poly(Neu5Ac,Neu5Gc), poly(KDN) chains and their partially O-acetylated and O-lactylated forms in salmonid fish egg glycoproteins demonstrates the natural occurrence of multiple forms of these unique sugar chains.

A novel glycosyltransferase which catalyses transfer of deaminated neuraminic acid, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid) from CMP-KDN to the non-reducing termini of oligo-polysialyl chains of polysialoglycoprotein (PSGP), was discovered in the ovary of rainbow trout (Oncorhynchus mykiss). The KDN-transferase activity was optimal at neutral pH, and stimulated 2 to 2.5-fold by 2-5 mM Mg2+ or Mn2+. Expression of KDN-transferase was developmentally regulated in parallel with expression of the alpha 2--8-polysialyltransferase, which catalyses synthesis of the oligo-polysialyl chains in PSGP. Incorporation of the KDN residues into the oligo-polysialyl chains prevented their further elongation, resulting in 'capping' of the oligo-polysialyl chains. This is the first example of a glycosyltransferase that catalyses termination of alpha 2--8-polysialylation in glycoproteins.

Publisher Summary This chapter provides a description of the strategies and methods used for detecting, analyzing, and modifying polysialic acids (polySia) and their derivatives. Several highly specific and sensitive anti-polySia and anti-2-keto-3-deoxy- D -glycero- D -galacto-nononic acid (KDN) antibodies have been developed to identify α -2,8-linked polySia, (KDN)G M3 , or oligoKDN expression in prokaryotic and eukaryotic cells. Endo N is the key enzyme for confirming the presence of polySia. It is specific for hydrolyzing oligo- or polySia residues in sources as distinct as bacteria and human brain. The K1F Endo N has been widely used in both immunodetection procedures and polysialyltransferase reactions to prove the existence of α -2,8-polySia by catalyzing its depolymerization. The chapter presents immunochemical methods for rapidly detecting α -2,8-linked oligo/polySia chains in cell or tissue homogenates that allow this epitope to be readily detected by Western blot or rocket immunoelectrophoresis, using the H.46 antibody. Similar procedures using monoclonal antibodies, enzyme-linked immunnosorbent assays (ELISAs), and immunocytochemistry are also described.

When neuroinvasive Escherichia coli K1 cells are grown at temperatures below 20 degrees C, they fail to synthesize the alpha-2,8-linked polysialic acid (polySia) capsule. The objective of this study was to use a genetic and biochemical approach to analyse why capsule expression was defective at cold temperatures. The strategy was to construct E.coli K1-derived mutants with defects in activation and degradation of Sia. The inability to degrade Sia because of a defect in the Sia-specific aldolase permitted accurate quantitation of Sia and CMP-Sia. Strains EV5 and EV90 possessed a defective CMP-Sia synthetase and were unable to activate Sia. These mutants were then used to study how synthesis of Sia, CMP-Sia, and the polySia capsule was affected by growth at 15 degrees C. In contrast to wild type strains, the mutants accumulated Sia in considerable quantities (up to 100 nmol mg protein-1) at 37 degrees C. However, no Sia was detected after growth at 15 degrees C. A temperature upshift experiment showed that the intracellular concentration of Sia increased ca. 3-fold within 5-10 min after shift from 15 to 37 degrees C, even in the presence of inhibitors of protein synthesis or transcription initiation. An in vitro assay for Sia synthase showed that Sia was synthesized at 37 degrees C in cell-free extracts from both 37 and 15 degrees C grown cells, but that no synthesis occurred when the same extracts were assayed at 15 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

An experimental procedure for detecting and characterizing tumor-associated, virion, and histocompatibility antigens has been developed. The method takes advantage of the high resolution that proteins, solubilized by Triton X-100 and reduced, display after sodium dodecyl sulfate gel electrophoresis. The antigens can be detected as distinct molecular weight species by a highly sensitive inhibition of cytotoxic reaction. When coupled to the lactoperoxidase-catalyzed iodination of intact cells, the procedure permits the determination of externally exposed antigens. In the present study, the method has been applied to the Moloney leukemia virus-induced YAC lymphoma cells of strain A mice, which express a Moloney leukemia virus-determined cell surface antigen (MCSA) in addition to the type C viral proteins gp71, p30, p15, p15(E), p12, and p10. MCSA was identified as an exposed surface protein distinct in size and antigenic determinants from the major envelope and core protein of Moloney leukemia virus and the histocompatibility antigens. Multiple molecular weight species possessing antigenic determinants for MCSA, gp71, and H-2 a have been detected. These results provide direct confirmation that MCSA is unrelated to the known virion structural proteins or to the H-2 a antigen. This method should permit the direct identification and molecular weight characterization of any antigen whose determinants are not solely dependent on a complex quaternary structure and for which serological reagents are available.

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.

A rapid procedure utilizing high-performance liquid chromatography was developed for the separation of homooligomers of sialic acid (N-acetylneuraminic acid). The method utilizes the anion exchanger Mono-Q HR 5 5 and can resolve sialyl oligomers with degrees of polymerization (DP) from 2 to 20 in 25 min. Previous methods required 1 to 9 days. Recoveries are quantitative and the method can be used either analytically to analyze the enzymatic digestion products of polysialic acid or semipreparatively to prepare sialyl oligomers of defined length. The method is potentially useful for analyzing other anionic oligosaccharides.

Membrane-associated sialytransferase complexes from Escherichia coli K-235 catalyze the incorporation of N-acetylneuraminic acid (NeuNAc) from cytidine 5-monophospho-N-acetylneuraminic acid (CMP-NeuNAc) into polymeric products and a lipid fraction. Reconstitution of enzyme activity in lipid-depleted membrane complexes had an absolute dependence on a purified phospholipid which was characterized by high resolution mass spectrometry following dephosphorylation as undecaprenol. An identical mass spectrum was obtained on the phosphorylated lipid confirming that the active derivative was undecaprenyl phosphate. This C55-isoprenoid alcohol accounted for 95% of the polyisoprenol and contained 11 isoprene units, each one unsaturated. The remaining 5% was composed of the C60 homologue, dodecaprenol. Ficarprenyl phosphate, an isomer of undecaprenyl phosphate, also restored enzymatic activity although on a molar basis, it was less active than undecaprenyl phosphate. These results provide direct evidence that sialyl polymer synthesis has an obligatory requirement for undecaprenyl phosphate, a membrane-bound lipid coenzyme which functions as an intermediate carrier of glycosyl residues in the biosynthesis of a variety of microbial cell surface polysaccharides. These data extend further the general class of polysaccharides whose synthesis involves undecaprenyl phosphate and are in accord with the hypothesis that this lipid acts as an intermediate carrier of sialyl residues in sialyl polymer synthesis according to the following reaction: CMP-NeuNAc plus P-undecaprenol in equilibrium NeuNAc-P-undecaprenol plus CMP Although the sialylated lipid remains to be characterized, evidence in support of this conclusion was obtained by kinetic analysis of N-acetylneuraminic acid transfer into the lipid soluble fraction and sialyl polymers. These studies showed a rapid incorporation of N-acetylneuraminic acid into the lipid-soluble fraction prior to maximal sialyl polymer formation, an observation consistent with a possible precursortion, an observation consistent with a possible precursor-product relationship. Confirmation that the radioactivity in the lipid-soluble fraction was lipid-linked N-acetylneuraminic acid was provided by the demonstration of the formation of a transitory sialyl-lipid with chromatographic properties expected of sialyl-undecaprenyl phosphate. Three additional lines of investigation implicated a functional role for undecaprenyl phosphate in sialyl polymer synthesis...

Prokaryotic derived probes that specifically recognize alpha-2,8-ketosidically linked polysialosyl units were developed to identify and study the temporal expression of these unique carbohydrate moieties in developing neural tissue (Vimr, E. R., McCoy, R. D., Vollger, H. F., Wilkison, N. C., and Troy, F. A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1971-1975). These polysialosyl units cap N-linked oligosaccharides of the complex-type on neural cell adhesion molecules (N-CAM). A Golgi-enriched fraction from 20-day-old fetal rat brain contains a membrane-associated sialyltransferase that catalyzes the incorporation of [14C]N-acetylneuraminic acid [( 14C]NeuNAc) from CMP-[14C] NeuNAc into polymeric products. At pH 6.0, 84 pmol of NeuNAc mg of protein-1 h-1 were incorporated. In sodium dodecyl sulfate-polyacrylamide gels, the major radiolabeled species migrated with a mobility expected for N-CAM. A bacteriophage-derived endoneuraminidase specific for polysialic acid was used to demonstrate that at least 20-30% of the [14C]NeuNAc was incorporated into alpha-2,8-linked polysialosyl units. This was confirmed by structural studies which showed that the endoneuraminidase-sensitive brain material consisted of multimers of sialic acid. The addition of a partially purified preparation of chick N-CAM to the membranous sialyltransferase stimulated sialic acid incorporation 3-fold. The product of this reaction was also sensitive to endoneuraminidase and contained alpha-2,8-linked polysialosyl chains, thus showing that N-CAM can serve as an exogenous acceptor for sialylation in vitro. Sialic acid incorporated into adult rat brain membranes was resistant to endoneuraminidase, indicating that the poly-alpha-2,8-sialosyl sialyltransferase activity is restricted to an early developmental epoch. It is recommended that the enzyme described here be designated CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase and the trivial name poly-alpha-2,8-sialosyl sialyltransferase be adopted.

Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP-134. The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46). These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F. A., Hallenbeck, P. C., McCoy, R. D., and Vimr, E. R. (1987) Methods Enzymol. 138, 169-185). Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D-[3H]GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of [14C] NeuNAc residues in an Escherichia coli K1 sialyltransferase assay. In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of [14C]NeuNAc from CMP-[14C]NeuNAc to exogenous acceptors containing at least 3 sialyl residues. In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used. Limit Endo-N digestion of the 3H-glycopeptides from the [3H] GlcN-labeled cells released short [3H]sialyl oligomers [( 3H]DP1-6) which were degraded to [3H]NeuNAc by exosialidase. Partial Endo-N digestion released a series of [3H]sialyl oligomers extending up to DP55. The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H]DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked [3H]NeuNAc residues. In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with [14C]NeuNAc by E. coli K1 sialyltransferase. The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels. Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)

A cell envelope particulate fraction from an encapsulated strain of Bacillus licheniformis ATCC 9945A contains a polyglutamyl synthetase which catalyzes the polymerization of l-glutamic acid to form a high molecular weight polymer of γ-d-glutamic acid. The reaction, which is specific for l-glutamic acid, requires ATP and Mg2+ ion and is stimulated by K+ ion and dithiothreitol. A nearly 2-fold increase in activity is observed in the presence of optimal levels of glycerol (0.1 m) and dimethylsulfoxide (0.1 m). The Km for l-glutamic acid is 0.5 mm. d-Glutamic acid is not incorporated nor does it influence the polymerization of the l isomer. Glutamyl dipeptides inhibited polyglutamyl synthetase activity. The cell envelope-associated polyglutamyl synthetase does not require an RNA template since it is refractory to treatment with RNase. It is also not inhibited by chloramphenicol, actinomycin D, puromycin, or rifampicin. Thus, the mechanism of capsular polymer formation is uniquely different from that involved in normal protein synthesis. Pre-treatment of the particulate fraction with Pronase destroyed its ability to synthesize the capsular polymer. Since a molar excess of [12C]α-ketoglutarate had no effect on the incorporation of l-[14C]glutamic acid, the subsequent interconversion of the glutamyl optical antipodes does not involve a series of glutamyl transamination and alanine racemization reactions in which α-ketoglutarate is a key intermediate. These reactions have been demonstrated in soluble extracts from B. licheniformis and have been shown to be one mechanism to account for the formation of d-glutamic acid from the l isomer. The observation that l-glutamine also has no effect on l-glutamic acid incorporation rules out the possible involvement of a transamidation reaction between glutamine and l- or d-glutamic acid. Polymer formation does not involve a series of transpeptidation reactions between glutamyl dipeptides since dipeptides inhibit polymer synthesis. Although the precise mechanism of activation, racemization, and polymerization is not fully understood, the results of these studies establish that the poly(γ-d-glutamyl) capsule is synthesized by a sequence of membrane-associated enzymatic reactions. No evidence for the involvement of small molecular weight oligopeptides as free intermediates has been obtained. The enzymatically synthesized α-d-glutamyl polymers were readily isolated by virtue of their complete resistance to hydrolysis by Pronase. After treatment with this protease, molecular sieve chromatography resulted in the quantitative isolation of the capsular polymers. Glutamic acid was the only labeled amino acid recovered following acid-catalyzed hydrolysis of the purified product. The specific activity of the glutamic acid was 90% that of the initial specific activity which indicates a considerable amount of de novo synthesis. Subsequent chemical, physical, and enzymatic analyses have confirmed that the enzymatically synthesized polymers are structurally identical with the capsular γ-d-glutamyl polymers synthesized in vivo.

Treatment of 14C-uniformly labeled cell walls of Penicillium chrysogenum with chitobiase-containing chitinase resulted in 73% solubilization. The solubilized components were isolated, identified, and determined quantitatively. N-Acetylglucosamine (GlcNAc) and N,N'-diacetylchitobiose ((GlcNAc)2) accounted for 42% of the cell wall. Since kinetic analysis showed that GlcNAc arose from (GlcNAc)2, at least 42% of the cell wall contains GlcNAc in β-d-(1 → 4)-glycosidic linkage. When the same experiment was performed with a chitobiase-free chitinase preparation nearly identical results were obtained, except, as expected, essentially all of the GlcNAc was present as (GlcNAc)2. Small amounts of glucose and laminaritriose were also released. In addition, a nondialyzable component, Fraction 5A, accounting for 14% of the wall, was isolated. This fraction, devoid of GlcNAc, contains 20 times the amount of galactose and 2 times the amount of mannose with respect to glucose found in cell wall hydrolysates. An additional 13% of the wall was solubilized when the cell wall residue, following treatment with chitinase, was treated with chitinase-free β-d-(1 → 3)-glucanase. Therefore, 86% of the wall was solubilized when treated first with chitinase, then with β-d-(1 → 3)-glucanase. Initial treatment of cell walls with β-d-(1 → 3)-glucanase resulted in 48% solubilization. From an analysis of the solubilized components the conclusion was reached that at least 40% of the wall contains glucose in β-d-(1 → 3)-glycosidic linkage. An additional 44% of the wall was solubilized when the residue, following treatment with β-d-(1 → 3)-glucanase, was treated with chitinase. Thus, 92% of the wall was solubilized when treated first with β-d-(1 → 3)-glucanase, then with chitinase. The observations that β-d-(1 → 3)-glucanase-free chitinase released in addition to GlcNAc and (GlcNAc)2 also glucose, laminaritriose, and a fraction containing mannose, galactose, and glucose (Fraction 5A) and that treatment of this fraction with β-d-(1 → 3)-glucanase released glucose and laminaribiose suggest that glucose may exist in cell walls in one or more forms other than the major glucan. From the available data it is not clear whether Fraction 5A contains one or several polymers. An analysis of the monomeric cell wall constituents released by acid hydrolysis is in excellent agreement with the results obtained by enzymatic digestion. These results showed the cell wall to contain about 48% glucosamine and about 52% neutral hexoses, which consisted of glucose, galactose, and mannose in the molar proportion of 10:1:2. Thus, if 40% of the wall is glucose, then galactose and mannose account for 4% and 8% of the wall, respectively.

Aerobacter aerogenes produces a capsular polysaccharide composed of tetrasaccharide repeating units containing galactose, mannose, and glucuronic acid in a molar ratio of 2:1:1. Particulate cell envelope fractions of the organism catalyze the incorporation of galactose, mannose, and glucuronic acid from their respective nucleotide derivatives into capsular polysaccharide and into a lipid fraction. Kinetic studies of galactose-14C transfer from UDP-galactose-14C into the lipid fraction and into capsular polysaccharide suggested that a lipid-bound oligosaccharide intermediate is a precursor of the capsular polysaccharide. The biosynthesis of the intermediate oligosaccharide was shown to be initiated by the incorporation of galactose into an endogenous phospholipid. This reaction was characterized as a phosphogalactosyltransferase on the basis of its reversibility by UMP, but not UDP, and the alkali-catalyzed release of cyclic galactose 1,2-phosphate from the intermediate, indicating that galactose is bound to the lipid by a pyrophosphate linkage. The carrier phospholipid was isolated and purified by column chromatography on silicic acid and DEAE-cellulose, and finally, by preparative thin layer chromatography. The pure phospholipid, identified as undecaprenyl phosphate by mass spectrometry, restored the polysaccharide-synthesizing capacity of enzyme preparations which had been depleted of endogenous lipid by treatment with organic solvents. The particulate fraction catalyzes the sequential transfer of mannose, glucuronic acid, and a second galactose moiety to galactosyl-P-P-undecaprenol to form the di-, tri-, and tetrasaccharide derivatives, each of which was isolated and characterized, establishing the following sequence: UDP-Gal + P-lipid ⇌ Gal-P-P-lipid + UMP Gal-P-P-lipid + GDP-Man → Man-Gal-P-P-lipid + (GDP) Man-Gal-P-P-lipid + UDP-GlcUA → GlcUA-Man-Gal-P-P-lipid + (UDP) GlcUA-Man-Gal-P-P-lipid + UDP-Gal → Gal-(GlcUA)-Man-Gal-P-P-lipid + (UDP) The lipid-linked tetrasaccharide is polymerized to form a macromolecular product which is identical with native capsular polysaccharide, based on its composition and its susceptibility to degradation by a specific, phage-induced depolymerase.

Gas-phase zwitterionic amino acids were formed in complexes of underivatized beta-cyclodextrin through reactions with a neutral base, n-propylamine. The reaction was performed in the analyzer cell of an electrospray ionization-Fourier transform mass spectrometer. Most of the natural amino acids were studied with three cyclodextrin hosts including alpha-, beta-, and gamma-cyclodextrin to understand better the structural features that lead to the stabilization of the zwitterionic complexes. Molecular dynamics calculations were performed to provide insight into the structural features of the complexes. The rate constants of the reactions were obtained through kinetic plots. Examination of both L- and D-enantiomers of the amino acid showed that the reaction was enantioselective. The reaction was then employed to analyze mixtures of Glu enantiomers naturally occurring in the bacteria Bacillus licheniformis.

The transbilayer diffusion rates and self-association of spin-labeled glycosyl carrier lipids and other isoprenoids have been studied in model phospholipid membranes. Transposition rates of phosphorylated species in small (300 A) or large (greater than or equal to 1000 A) diameter unilamellar phosphatidylcholine (PC) vesicles are slow (t1/2 greater than 5 h at 25 degrees C); this argues against their proposed role in the transbilayer portage of sugar units during polysaccharide and glycoprotein assembly. The same probes mix well with several host lipids, remaining monomolecularly dispersed even at high ionic strength (0.5 M NaCl) or in the presence of various polyvalent cations. This behavior persists for several degrees beneath the transition temperature (Tc) of saturated lecithins. In contrast, neutral carboxylate analogues undergo pronounced reversible self-association. Aggregation is quite dependent on the probe concentration and nature of the host but only weakly temperature dependent above the Tc. Even at low probe concentrations (less than 0.5 mol %) in fluid membranes, aggregates persist above 75 degrees C. Unsaturation in the lecithin fatty acyl chains dramatically increases isoprenoid monomer solubility. Segregation appears to involve relocation of the entire molecule in the membrane interior.

In Escherichia coli, synthesis of sialic acid is not regulated by allosteric inhibition mediated by cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuNAc). Evidence for the lack of metabolic control by feedback inhibition was demonstrated by measuring the intracellular level of sialic acid and CMP-NeuNAc in mutants defective in sialic acid polymerization and in CMP-NeuNAc synthesis. Polymerization-defective mutants could not synthesize the polysialic acid capsule and accumulated ca. 25-fold more CMP-NeuNAc than the wild type. Mutants unable to activate sialic acid because of a defect in CMP-NeuNAc synthetase accumulated ca. sevenfold more sialic acid than the wild type. An additional threefold increase in sialic acid levels occurred when a mutation resulting in loss of N-acylneuraminate pyruvate-lysase (sialic acid aldolase) was introduced into the CMP-NeuNAc synthetase-deficient mutant. The aldolase mutation could not be introduced into the polymerization-defective mutant, suggesting that any further increase in the intracellular CMP-NeuNAc concentration was toxic. These results show that sialic acid aldolase can regulate the intracellular concentration of sialic acid and therefore the concentration of CMP-NeuNAc. We conclude that regulation of aldolase, mediated by sialic acid induction, is necessary not only for dissimilating sialic acid (E.R. Vimr and F. A. Troy, J. Bacteriol. 164:845-853, 1985) but also for modulating the level of metabolic intermediates in the sialic acid pathway. In agreement with this conclusion, an increase in the intracellular sialic acid concentration was correlated with an increase in aldolase activity. Direct evidence for the central role of aldolase in regulating the metabolic flux of sialic adid in E. coli was provided by the finding that exogenous radiolabeled sialic acid was specifically incorporated into sialyl polymer in aldolase-negative strain but not in the wild type.

2H-NMR investigation of polyisoprenols (PIs) in model membranes has revealed information about their motions, relative order, and locale within the membrane. Initial 2H-NMR studies of the organization of the shorter chain homologues geraniol (C10), farnesol (C15), and solanesol (C45) were carried out by incorporating 2H-acetyl esters of the alcohol or the di-perdeuterome-thylated derivatives of the omega-labeled prenols into multilamellar phosphatidylcholine (PC) vesicles. 2H-NMR powder patterns interpretable in terms of quadrupole splittings and spin-lattice relaxation times were obtained. Similar experiments have now been carried out with the labeled free alcohol, acetyl ester, and phosphate ester of dolichol (C95) and undecaprenol (C55). 2H-NMR results show that the head and tail 2H-labeled sites of C55 and C95 exhibit a fast motion isotropic signal only; no slower motion anisotropy, as exhibited by the short chain PIs, was observed. These data suggest that C55 and C95 either have substantially different (faster) motions and/or conformations relative to the shorter chain PIs within the membrane, and that the longer PIs alter the membrane host packing matrix. This conclusion was supported by 31P-NMR studies of C55 and C95 derivatives in PC and PE/PC membranes, which showed new pronounced spectral changes relative to the results obtained with the shorter chain PIs. These spectral changes indicate that undecaprenol and dolichol derivatives appear to induce a non-bilayer (isotropic) organization of phospholipid molecules in PE/PC (2:1) vesicles. The possible physiological consequences of this perturbation remains to be determined.

Three prokaryotic-derived probes to identify and study the temporal expression of polysialosyl units in neuronal tissue have been developed. A polyclonal antibody, a bacteriophage-derived endo-neuraminidase, and an Escherichia coli K1 sialyltransferase are all specific for either recognizing or synthesizing poly(sialic acid) containing alpha-2,8-ketosidic linkages. Polysialosyl immunoreactivity with apparent Mr values of 180,000-240,000 was specific for developing neuronal tissue; it was not detected in neonatal liver or kidney or in adult brain tissue. The developmentally regulated disappearance in poly(sialic acid) is consistent with the probes described here recognizing the polysialosyl carbohydrate units of a neuronal cell adhesion molecule (N-CAM). Treatment of brain extracts with a bacteriophage-derived endo-neuraminidase specific for alpha-2,8-linked polysialosyl units abolished the immunoreactivity. The material solubilized by endo-neuraminidase was isolated, reduced with borotritide, and shown to contain oligomers of sialic acid with three to six sialyl units. Treatment of the 3H-labeled oligosialic acid with exo-neuraminidase quantitatively converted the radioactivity to sialitol, establishing that the brain-derived oligomers were composed solely of sialic acid. A membranous sialytransferase from E. coli K1 that can transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid) also recognized rat brain membranes, further substantiating the presence of poly(sialic acid) in rat brain. This conclusion was confirmed by using a mutant of E. coli K1 that was defective in the synthesis of poly(sialic acid) and could only transfer sialic acid to exogenous acceptors of oligo- or poly(sialic acid). Sialyl polymer synthesis was restored in the mutant when brain membranes were added as exogenous acceptor.

A phage endoneuraminidase that specifically cleaves alpha-2, 8-linked polysialic acid has been found to be a useful probe for examining the biological role of this sugar moiety on the neural cell adhesion molecule (NCAM). The enzyme caused a 3.3-fold increase in the rate of NCAM-dependent aggregation of membrane vesicles from chicken embryonic brain, without the nonspecific effects previously encountered with the use of exoneuraminidases. The enhancement of aggregation was closely correlated with removal of sialic acid as assessed by electrophoretic mobility. Extension of this analysis to cultures of spinal ganglia indicated that removal of sialic acid by the endoneuraminidase results in an increase in the thickness of neurite bundles. This enhancement of fasciculation was reversed by addition of anti-NCAM Fab, suggesting that the enzyme treatment was not toxic and did not produce nonspecific effects on adhesion. Injection of the enzyme into the eyes of 3.5-d chicken embryos consistently produced a striking array of abnormalities in those parts of the neural retina that contained the highest concentrations of NCAM at the time of injection. These perturbations included a dramatic thickening of the neural epithelium in the posterior eye, a failure of cells in this region to elongate radially, formation of an ectopic optic fiber layer, and an incomplete association of the presumptive pigmented epithelium with the neural retina. These results provide the first direct evidence that the polysialic acid on NCAM has a regulatory effect on adhesion between living cells, and that the amount of this carbohydrate is critical for the normal morphogenesis of nerve tissue.

The synthesis, purification, and characterization of two types of spin-labeled glycosyl carrier lipids and shorter chain isoprenols are described. As models for phosphorylated lipids intermediates, phosphodiesters of tempol and the prenols dolichol, ficaprenol, solanesol, phytol, and farnesol were prepared. For analogues of neutral species each prenol was esterified with a pyrrolidinecarboxylic acid based label. Tripropylbenzenesulfonyl chloride was used as the condensing agent in both cases. Phosphodiester yields ranged from 36% for the 55-carbon compound to greater than 66% for the 95-carbon prenol. Both types of probes were incorporated into phospholipid bilayers, where each became oriented with the artificial head group at, or very close to, the water--hydrocarbon interface. Electron spin resonance spectra of the phosphodiesters are matrix dependent, indicating rapid isotropic tumbling in chloroform but highly anisotropic reorientation in unsaturated phosphatidylcholine (PC) hosts. Rotation or large amplitude oscillation about either or both the tempo C4--O linkage and the P--O (chain) bond as well as whole molecule rotation within the bilayer could account for the observed x-axis anisotropy. Segmental motion within the polyprene chain does not appear to be a determinant.

Publisher Summary This chapter describes the in vitro conditions that permit detection of the sialyltransferase and procedures to quantitate the amount of N -Acetylneuraminic acid (NeuNAc) incorporated specifically into α-2,8-1inked polysialosyl units in neural cell adhesion molecule (N-CAM). Determination of the reaction catalyzed by the membranous poly-α-2,8-sialosyl sialyltransferase is based on the transfer of [ 14 C]NeuNAc from CMP-[ I4 C]NeuNAc to high molecular weight endogenous acceptors believed to be N-CAM molecules. The chapter discusses the procedures for preparing the membranes and for measuring CMP-NewNAc, that is Poly-α-2,8-Sialosyl Sialystransferase activity. There is a discussion about the effect of endo-N on the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic profile of 14 C-labeled polysialic acid synthesized endogenously by fetal rat brain. The chapter discusses an experiment in which [ I4 C]NeuNAc from CMP-[ I4 C]NeuNAc is incorporated into fetal rat brain membranes in the presence of exogenous N-CAM. Data from the experiment support that, N-CAM can serve as an exogenous acceptor for sialylation in vitro . Treatment of the sialyl trimer with exoneuraminidase quantitatively converts the label to [ 14 C]NeuNAc, thus, proving that the oligomers are composed of sialic acid.

Recent studies have shown a relationship between the level of the sialic acid (Sia), N-glycolylneuraminic acid (Neu5Gc) in red meat and its risk in cancer, cardiovascular and inflammatory diseases. Unresolved is the Sia concentration in different organs of piglets during development. Our aim was to determine the level of free and conjugated forms of Neu5Gc, N-acetylneuraminic acid (Neu5Ac) and ketodeoxynonulsonic acid (Kdn) in fresh and cooked spleen, kidney, lung, heart, liver, and skeletal muscle from 3-days-old (n = 4-8), 38-days-old (n = 10) and adult piglets (n = 4) by LC-MS/MS. Our findings show: (1) Lung tissue from 3 days-old piglets contained the highest level of total Sia (14.6 μmol/g protein) compared with other organs or age groups; (2) Unexpectedly, Neu5Gc was the major Sia in spleen (67-79 %) and adult lung (36-49 %) while free Kdn was the major Sia in skeletal muscle. Conjugated Neu5Ac was the highest Sia in other organs (61-84 %); (3) Skeletal muscle contained the lowest concentration of Neu5Gc in fresh and cooked meat; (4) Kdn accounted for