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Background:The polysialic acid (polySia) is a unique carbohydrate polymer produced on the surface Of Neuronal Cell Adhesion Molecule (NCAM) in a number of cancer cells, and strongly correlates with the migration and invasion of tumor cells and with aggressive, metastatic disease and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Selective inhibition of polySTs, therefore, presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. Heparin has been found to be effective in inhibiting the ST8Sia IV activity, but no clear molecular rationale. It has been found that polysialyltransferase domain (PSTD) in polyST plays a significant role in influencing polyST activity, and thus it is critical for NCAM polysialylation based on the previous studies.Objective:To determine whether the three different types of heparin (unfractionated hepain (UFH), low molecular heparin (LMWH) and heparin tetrasaccharide (DP4)) is bound to the PSTD; and if so, what are the critical residues of the PSTD for these binding complexes?Methods:Fluorescence quenching analysis, the Circular Dichroism (CD) spectroscopy, and NMR spectroscopy were used to determine and analyze interactions of PSTD-UFH, PSTD-LMWH, and PSTD-DP4.Results:The fluorescence quenching analysis indicates that the PSTD-UFH binding is the strongest and the PSTD-DP4 binding is the weakest among these three types of the binding; the CD spectra showed that mainly the PSTD-heparin interactions caused a reduction in signal intensity but not marked decrease in α-helix content; the NMR data of the PSTD-DP4 and the PSTDLMWH interactions showed that the different types of heparin shared 12 common binding sites at N247, V251, R252, T253, S257, R265, Y267, W268, L269, V273, I275, and K276, which were mainly distributed in the long α-helix of the PSTD and the short 3-residue loop of the C-terminal PSTD. In addition, three residues K246, K250 and A254 were bound to the LMWH, but not to DP4. This suggests that the PSTD-LMWH binding is stronger than the PSTD-DP4 binding, and the LMWH is a more effective inhibitor than DP4.Conclusion:The findings in the present study demonstrate that PSTD domain is a potential target of heparin and may provide new insights into the molecular rationale of heparin-inhibiting NCAM polysialylation.

Sialylated milk oligosaccharides (SMOs) have a multifunctional health benefit, yet the molecular details underlying their potential role in modulating intestinal maturation remains unknown. To test the hypothesis that sialyllactose (SL) may mediate intestinal maturation and function through controlling neuronal function, studies were carried out where the diet of postnatal piglets was supplemented with a mixture of 3'- and 6'-sialyllactose from postnatal day 3 to 38. Gene transcription pathways regulating enteric nervous system function, polysialic acid (polySia) synthesis, and cell proliferation were quantified. Our new findings show that SL intervention: (1) upregulated the level of gene and protein expression of the glial-derived neurotrophic factor (GDNF) in the ileum; (2) upregulated phosphorylation of the cAMP responsive element-binding protein (CREB), the downstream target of GDNF signaling pathway; (3) promoted cell proliferation based on an increase in the number and density of Ki-67 positive cells in the crypts; (4) increased the crypt width in the ileum by 10%, while gene markers for the functional cells were not affected; (5) upregulated mRNA expression level of ST8Sia IV, a key polysialyltransferase responsible for synthesis of polySia-NCAM; (6) reduced the incidence and severity of diarrhea. These results show that SL promotes intestinal maturation in neonatal piglets by upregulating GDNF, synthesis of polySia and CREB-interactive pathway.

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.

SCOPE: Lactoferrin (Lf), a sialylated milk glycoprotein, promotes early neurodevelopment and cognition. Functional concentrations of Lf, however, remain unknown. Our objective is to determine the concentration‐dependency of Lf on genes associated with neurodevelopment and cognition in neonatal piglets. METHODS AND RESULTS: Piglets are given milk replacer with Lf at concentrations of 155 (low) or 285 mg kg‐¹ day‐¹ (high) from postnatal days 3 to 38. Gene expression associated with neurodevelopment, cognition, and cognate proteins were quantitated. This study found 1) The rate of learning and long‐term memory was higher with 155 mg kg‐¹ day‐¹ assessed in an eight‐arm radial maze; 2) Global gene transcription profiling showed this lower concentration upregulated genes and functions correlated with neurodevelopment and cognition, while the higher concentration regulated cellular processes for neuroprotection; 3) Expression of BDNF genes and proteins were higher with both concentrations, while genes regulating BDNF signaling, including SLC6A3, IGF‐1 responded more to the lower concentration; 4) The lower concentration modulated genes in the five highest networks associated with cellularity and neurocognition, while the prevention of neurodevelopmental and neurological pathologies was associated with the higher concentration. CONCLUSION: The lower concentrations of Lf enhanced neurodevelopment and cognition, while higher concentrations are greater neuroprotective, findings of potential novel clinical relevance.

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.

Polysialic acid plays a key role in cancer metastasis and neurodevelopment. Our aim was to determine the developmental gene-expression profiles for the two polysialyltransferases ST8Sia II and ST8Sia IV, neural cell-adhesion molecules (NCAMs), SynCAM 1, neuropilin-2 (NRP2) and their polysialylated cognate glycans in different regions of the piglet brain during postnatal development. Our findings show that: 1) the cellular levels of mRNA coding for ST8Sia II and ST8Sia IV, NCAMs, SynCAM 1, NRP2 and polySia are age-dependent and cell-type-specific during neonatal brain development, 2) there was a lack of correlation between abundance level of mRNA coding for ST8Sia II and ST8Sia IV and the abundance level of the post-translation expression of polySia in all nine brain regions, 3) expression levels of polySia did not correlate with the levels of the carrier proteins NCAM-140, SynCAM 1 and NRP2 in nine brain regions, and 4) the cellular abundance of ST8Sia II and ST8Sia IV in nine subregions of piglet brain is regulated at the level of translation/post-translation, and not at the level of transcription. Collectively, our findings suggest that neuronal and glial cells within different regions of the brain have different transcriptional programs that can direct cell division at different rates based on the activity levels of ST8Sia II and ST8Sia IV and the level of their carrier proteins during neurodevelopment.

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.

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.

α2,8-Linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal α2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-β-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs ∼50–60 or less, a subpopulation of chains with DPs ∼150 to ∼180 and extending to DP ∼400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-β-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.

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.

Synthesis of α2,8-polysialic acid (polySia) glycans are catalyzed by two highly homologous mammalian polysialyltransferases (polySTs), ST8Sia II (STX) and ST8Sia IV (PST), which are two members of the ST8Sia gene family of sialytransferases. During polysialylation, both STX and PST catalyze the transfer of multiple Sia residues from the activated sugar nucleotide precursor, CMP-Neu5Ac (Sia), to terminal Sia residues on N- and Olinked oligosaccharide chains on acceptor glycoproteins, including the neural cell adhesion molecule (NCAM), which is the major carrier protein of polySia. Based on our new findings and previously published studies, this review summarizes the present concepts regarding the molecular mechanism underlying regulation of protein-specific polysialylation of NCAM that includes the following: (1) Determination of the catalytic domains and specific regions within ST8Sia IV for recognizing and catalyzing the efficient polysialylation of NCAM; (2) Identification of key amino acid residues within the PSTD motif of ST8Sia IV that are essential for polysialylation; (3) Verification of key amino acids in the PBR domain of ST8Sia IV required for NCAM-specific polysialylation; and (4) a 3D conformational study of ST8Sia IV based on the Phyre2 server to discover the relationship between the structure and its functional domains of the polyST. Based on these results, our 3D model of ST8Sia IV was used to identify and characterize the catalytic domains and amino acid residues critical for catalyzing polysialylation, and have provided new structural information for supporting a detailed mechanism of polyST-NCAM interaction required for polysialylation of NCAM, findings that have not been previously reported.

During brain development, polysialylated (polySia) neural cell adhesion molecules (polySia–NCAMs) modulate cell–cell adhesive interactions involved in synaptogenesis, neural plasticity, myelination, and neural stem cell (NSC) proliferation and differentiation. Our findings show that polySia–NCAM is expressed on NSC isolated from adult guinea pig spiral ganglion (GPSG), and in neurons and Schwann cells after differentiation of the NSC with epidermal, glia, fibroblast growth factors (GFs) and neurotrophins. These differentiated cells were immunoreactive with mAb’s to polySia, NCAM, β-III tubulin, nestin, S-100 and stained with BrdU. NSC could regenerate and be differentiated into neurons and Schwann cells. We conclude: (1) polySia is expressed on NSC isolated from adult GPSG and on neurons and Schwann cells differentiated from these NSC; (2) polySia is expressed on neurons primarily during the early stage of neuronal development and is expressed on Schwann cells at points of cell–cell contact; (3) polySia is a functional biomarker that modulates neuronal differentiation in inner ear stem cells. These new findings suggest that replacement of defective cells in the inner ear of hearing impaired patients using adult spiral ganglion neurons may offer potential hope to improve the quality of life for patients with auditory dysfunction and impaired hearing disorders.

Propose To design a set of brain templates for postnatal piglet brains based on high-resolution T1-weighted imaging for voxel-based morphometric analysis. Materials and methods Using a 3.0 T magnetic resonance (MR) scanner, a population-based whole brain template was developed by averaging forty T1 images in the brains of postnatal piglets at 38 days of age. The templates for gray and white matter, and cerebrospinal fluid were designed based on the corresponding probability maps by adapting individual data sets using statistical parametric mapping. Anatomical labeling maps were generated from labeling propagation derived from the established Pig Brain Atlas. Differences in the coordinates from four significant structural landmarks in the template, plus an additional 12 normalized images and anatomical labeling maps were measured to validate the accuracy of the registration of the template. Results A whole brain template, a set of tissue-specific probability and anatomical labeling maps were developed. The location deviation of the four significant structural landmarks, including the anterior and posterior regions in the corpus callosum, and the left and right caudate nucleus, was found to be Conclusion A whole brain template map and a set of tissue-specific probability and anatomical labeling maps were developed to analyze the morphometric imaging of the postnatal piglet brain, an animal model of the human infant.

Background: Lactoferrin (Lf) is an iron-binding, sialylated glycoprotein prevalent in milk, and known to have multifunctional health benefits. Its potential role in neurodevelopment and cognition r...

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.

The molecular mechanisms underlying how dietary lactoferrin (Lf) impacts gut development and maturation and protects against early weaning diarrhea are not well understood. In this study, we supplemented postnatal piglets with an Lf at a dose level of 155 and 285 mg/kg/day from 3 to 38 days following birth. Our findings show that the high dose of Lf up-regulated messenger RNA expression levels of genes encoding brain-derived neurotrophic factor (BDNF) and ubiquitin carboxy-terminal hydrolase L1 (ubiquitin thiolesterase (UCHL1) and, to a lesser extent, glial cell line-derived neurotrophic factor, in the duodenum (P

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.

The glycosyl carrier lipids, dolichylphosphate (C(95)-P) and undecapreylphosphate (C(55)-P) are key molecular players in the synthesis and translocation of complex glycoconjugates across cell membranes. The molecular mechanism of how these processes occur remains a mystery. Failure to completely catalyze C(95)-P-mediated N-linked protein glycosylation is lethal, as are defects in the C(55)-P-mediated synthesis of bacterial cell surface polymers. Our recent NMR studies have sought to understand the role these "super-lipids" play in biosynthetic and translocation pathways, which are of critical importance to problems in human biology and molecular medicine. The PIs can alter membrane structure by inducing in the lamellar phospholipids (PL) bilayer a non-lamellar or hexagonal (Hex(II)) structure. Membrane proteins that bind PIs contain a transmembrane binding motif, designated a PI recognition sequence (PIRS). Herein we review our recent combination of (1)H- and (31)P NMR spectroscopy and energy minimized molecular modeling studies that have determined the preferred orientation of PIs in model phospholipids membranes. They also show that the addition of a PIRS peptide to nonlamellar membranes induced by the PIs can reverse the Hex(II) phase back to a lamellar structure. Our molecular modeling calculations have also shown that as many as five PIRS peptides can bind to a single PI molecule. These findings lead to the hypothesis that the PI-induced Hex(II) structure may have the potential of forming a membrane channel that could facilitate glycoconjugate translocation processes. This is an alternate hypothesis to the possible existence of hypothetical "flippases" to accomplish movement of hydrophilic sugar chains across hydrophobic membranes.

To determine the molecular basis of eukaryotic polysialylation, the function of a structurally unique polybasic motif of 32 amino acids (pI approximately 12) in the polysialyltransferases (polySTs), ST8Sia II (STX and ST8Sia IV (PST) was investigated. This motif, designated the "polysialyltransferase domain" (PSTD), is immediately upstream of the sialylmotif S (SM-S). PolyST activity was lost in COS-1 mutants in which the entire PSTD in ST8Sia IV was deleted, or in mutants in which 10 and 15 amino acids in either the N- or C- terminus of PSTD were deleted. Site-directed mutagenesis showed that Ile(275), Lys(276) and Arg(277) in the C-terminus of PSTD in ST8Sia IV, which is contiguous with the N-terminus of sialylmotif-S, were essential for polysialylation. Arg(252) in the N-terminus segment of the PSTD was also required, as was the overall positive charge. Thus, multiple domains in the polySTs can influence their activity. Immunofluorescent microscopy showed that the mutated proteins were folded correctly, based on their Golgi localization. The structural distinctness of the conserved PSTD in the polySTs, and its absence in the mono- oligoSTs, suggests that it is a "polymerization domain" that distinguishes a polyST from a monosialyltransferases. We postulate that the electrostatic interaction between the polybasic PSTD and the polyanionic polySia chains may function to tether nascent polySia chains to the enzyme, thus facilitating the processive addition of new Sia residues to the non-reducing end of the growing chain. In accord with this hypothesis, the polyanion heparin was shown to inhibit recombinant human ST8Sia II and ST8Sia IV at 10 microM.

Alpha2,8-linked polysialic acid (polySia) is a structurally unique antiadhesive glycotope that covalently modifies N-linked glycans on neural cell adhesion molecules (N-CAMs). These sugar chains play a key role in modulating cell-cell interactions, principally during embryonic development, neural plasticity, and tumor metastasis. The degree of polymerization (DP) of polySia chains on N-CAM is postulated to be of critical importance in regulating N-CAM function. There are limitations, however, in the conventional methods to accurately determine the DP of polySia on N-CAM, the most serious being partial acid hydrolysis of internal alpha2,8-ketosidic linkages that occur during fluorescent derivatization, a step necessary to enhance chromatographic detection. To circumvent this problem, we have developed a facile method that combines the use of Endo-beta-galactosidase to first release linear polySia chains from N-CAM, with high resolution high pressure liquid chromatography profiling. This strategy avoids acid hydrolysis prior to chromatographic profiling and thus provides an accurate determination of the DP and distribution of polySia on N-CAM. The potential of this new method was evaluated using a nonpolysialylated construct of N-CAM that was polysialylated in vitro using a soluble construct of ST8Sia II or ST8Sia IV. Whereas most of the oligosialic acid/polySia chains consisted of DPs approximately 50-60 or less, a subpopulation of chains with DPs approximately 150 to approximately 180 and extending to DP approximately 400 were detected. The DP of this subpopulation is considerably greater than reported previously for N-CAM. Endo-beta-galactosidase can also release polySia chains from polysialylated membranes expressed in the neuroblastoma cell line, Neuro2A, and native N-CAM from embryonic chick brains.

The objective of these studies was to test the hypothesis that proteins that contain potential polyisoprenyl recognition sequences (PIRSs) in their transmembrane-spanning domain can bind to the polyisoprenyl (PI) glycosyl carrier lipids undecaprenyl phosphate (C55-P) and dolichyl phosphate (C95-P). A number of prokaryotic and eukaryotic glycosyltransferases that utilize PI coenzymes contain a conserved PIRS postulated to be the active PI binding domain. To study this problem, we first determined the 3D structure of a PIRS peptide, NeuE, by homonuclear 2D 1 H‐nuclear magnetic resonance (NMR) spectroscopy. Experimentally generated distance constraints derived from nuclear Overhauser enhancement and torsion angle constraints derived from coupling constants were used for restrained molecular dynamics and energy minimization calculations. Molecular models of the NeuE peptide were built based on calculations of energy minimization using the DGII program NMRchitect. 3D models of dolichol (C95) and C95-P were built based on our 2D 1 H-NMR nuclear Overhauser enhancement spectroscopy (NOESY) results and refined by energy minimization with respect to all atoms using the AMBER (assisted modeling with energy refinements) force field. Our energy minimization studies were carried out on a conformational model of dolichol that was originally derived from small-angle X-ray scattering and molecular mechanics methods. These results revealed that the PIs are conformationally nearly identical tripartite molecules, with their three domains arranged in a coiled, helical structure. Analyses of the intermolecular cross-peaks in the 2D NOESY spectra of PIRS peptides in the presence of PIs confirmed a highly specific interaction and identified key contact amino acids in the NeuE peptide that constituted a binding motif for interacting with the PIs. These studies also showed that subtle conformational changes occurred within both the PIs and the NeuE peptide after binding. 3D structures of the resulting molecular complexes revealed that each PI could bind more than one PIRS peptide. These studies thus represent the first evidence for a direct physical interaction between specific contact amino acids in the PIRS peptides and the PIs and supports the hypothesis of a bifunctional role for the PIs. The central idea is that these superlipids may serve as a structural scaffold to organize and stabilize in functional domains PIRS-containing proteins within multiglycosyltransferase complexes that participate in biosynthetic and translocation processes.

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.

Recently, we reported the discovery of a new type of sialidase, KDNase, which specifically hydrolyzes the ketosidic linkages of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN), but not N-acylneuraminyl linkages. We now report that this enzyme, designated KDNase SM, is an inducible enzyme that is localized in the periplasm of Sphingobacterium multivorum. Growth of S. multivorum in the presence of KDN-containing oligosaccharide alditols, KDNalpha2--3Galbeta1--3GalNAc alpha1--3[KDNalpha2--(8KDN alpha2--)n--6]GalNAcol, as a sole carbon source induced KDNase SM activity 15 40-fold, compared with growth in the absence of inducer. KDN, Neu5Ac, or Neu5Ac oligomers were ineffective as inducers. The enzyme was released from the periplasm of induced cells by cold osmotic shock and purified 700-fold to homogeneity. The specific activity of the pure enzyme was 82,100 units/mg of protein. KDNase SM activity resided in a single polypeptide chain with an estimated molecular weight of approximately 47,500. Enzyme activity was maximal at near neutral pH. The availability of pure KDNase will now make it possible to study the structure and functional role of KDN-glycoconjugates and to determine the molecular mechanism whereby the enzyme can discriminate between KDN and N-acylneuraminic acid.

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.

Immune complexes (IC) were isolated from sera of patients with breast cancer (BC), from effusions from patients with breast and ovarian cancer (OC) and from effusions from patients with non-malignant diseases. Isolation was carried out with the aid of tubes coated with rheumatoid factor (RF) as immunoadsorbents. The effusions were first filtered through Sephacryl S-300 columns and the large molecular weight material eluted in front of the IgG peak was used as the starting material, while most of the sera were processed without prior filtration. The IC eluted from the RF-coated tubes were radioiodinated and analyzed by electrophoresis on polyacrylamide gels, followed by autoradiography. Four putative IC-derived proteins were identified in the RF eluates and designated according to their molecular weights (in Kdalton): p94, p46, p 17 and p 13. p94 was found in 1/8 BC effusions, but in none of the other samples tested; “p46”, which was actually a complex of several closely spaced proteins, was identified in at least 3/16 BC sera, 6/8 BC effusions, 3/4 OC effusions and 1/5 non-malignant effusions, but in none of 17 normal sera. The upper band of the complex seemed to be present mainly, if not exclusively, in effusions from patients with neoplasia. p17 and p13 were found more often and in higher concentrations in the malignant effusions. All of these proteins could be precipitated by treating the RF eluates with anti human Ig serum, suggesting that they were associated with Ig, presumably as immune complexes.