Functional Correlates and Impact of Dietary Lactoferrin Intervention and its Concentration-dependence on Neurodevelopment and Cognition in Neonatal Piglets.

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.

Molecular Mechanisms Underlying How Sialyllactose Intervention Promotes Intestinal Maturity by Upregulating GDNF Through a CREB-Dependent Pathway in Neonatal Piglets.

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.

The Inhibition of Polysialyltranseferase ST8SiaIV Through Heparin Binding to Polysialyltransferase Domain (PSTD).

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.

Biochemical Characterization and Analyses of Polysialic-Acid-Associated Carrier Proteins and Genes in Piglets during Neonatal Development.

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.

Developmental changes in the level of free and conjugated sialic acids, Neu5Ac, Neu5Gc and KDN in different organs of pig: a LC-MS/MS quantitative analyses.

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 <5 % of the total Sia in most organs; (5) During development, the total Sia concentration showed a 44-79 % decrease in all organs; (6) In adult piglets, the high to low rank order of total Sia was lung, heart, spleen, kidney, liver and skeletal muscle. In conclusion, the high level of Neu5Gc in all organs compared to skeletal muscle is a potential risk factor suggesting that dietary consumption of organ meats should be discouraged in favor of muscle to protect against cancer, cardiovascular and other inflammatory diseases.

Molecular characterization and expression analyses of ST8Sia II and IV in piglets during postnatal development: lack of correlation between transcription and posttranslational levels.

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.

LC-MS/MS glycomic analyses of free and conjugated forms of the sialic acids, Neu5Ac, Neu5Gc and KDN in human throat cancers.

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 Promotes Early Neurodevelopment and Cognition in Postnatal Piglets by Upregulating the BDNF Signaling Pathway and Polysialylation.

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.

3D structural conformation and functional domains of polysialyltransferase ST8Sia IV required for polysialylation of neural cell adhesion molecules.

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.

Development of new population-averaged standard templates for spatial normalization and segmentation of MR images for postnatal piglet brains.

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 <0.25 cm, validating the sensitivity and resolution of the template. 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.

Lactoferrin up-regulates intestinal gene expression of brain-derived neurotrophic factors BDNF, UCHL1 and alkaline phosphatase activity to alleviate early weaning diarrhea in postnatal piglets.

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<.05). Piglets in the high and low Lf group had 30% and 7% larger jejunal crypts compared with the control group (P<.05). Escherichia coli 16S rRNA copy number per gram of ascending colon contents was significantly reduced (P=.001), while the copy number of Bifidobacteria and Lactobacillus spp. was not affected. In addition, Lf increased intestinal alkaline phosphatase activity (P<.05) and delayed the onset of food transitional diarrhea, reducing its frequency and duration (P<.05). The incidence of diarrhea in the high and low Lf groups was decreased 54% and 15%, respectively, compared with the control group (P=.035). In summary, these findings provide new evidence that dietary Lf supplementation up-regulated gene expression of BDNF and UCHL1, decreased the colon microbiota of E. coli, improved gut maturation and reduced early weaning diarrhea in piglets. The molecular basis underlying these findings suggests that Lf may enhance gut development and immune function by providing new insight into the gut-brain-microbe axis that has not been previously reported.

Expression of polysialylated neural cell adhesion molecules on adult stem cells after neuronal differentiation of inner ear spiral ganglion neurons.

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, ß-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.

LC-MS/MS quantification of N-acetylneuraminic acid, N-glycolylneuraminic acid and ketodeoxynonulosonic acid levels in the urine and potential relationship with dietary sialic acid intake and disease in 3- to 5-year-old children.

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< 0·05), but not in 3-year-old children; (4) Sia intake levels at breakfast and lunch were approximately 2·5 and 0·16 mg Sia/kg body weight; and (5) there was no significant correlation between dietary Sia intake levels and urinary Sia levels. Urinary Sia levels varied with age and time of day, but did not correlate with Sia intake in 3- to 5-year-old children. The difference in urinary Sia levels in children of different age groups suggests that the metabolism and utilisation rates of dietary Sia are age dependent.

Evaluation of dietary intake of lactating women in China and its potential impact on the health of mothers and infants.

BACKGROUND: Optimal nutrition for lactating mothers is importance for mother and infants' health and well-being. We determined the nutrient intake and dietary changes during the first 3-month of lactation, and its potential effect on health and disease risk. METHOD: Personal interviews were conducted to collect a 24 h diet recall questionnaire from 199 healthy lactating women in the postpartum days 2, 7, 30, 90 and healthy 58 non-pregnant women served as the controls. RESULTS: We found in lactating women (1) the mean daily energy and carbohydrate intake was lower than that of the Chinese Recommended Nutrient Intake (RNI, 2600 Kcal, 357.5 ~ 422.5 g) by 11% ~ 17% and 33% ~ 49%, respectively; (2) the fat intake increased from 3% to 13%, which was 9 ~ 77% higher than the RNI (57 ~ 86.7 g); (3) the protein intake exceeded the RNI of 85 g by 32 ~ 53%; (4) the total calories consumed from carbohydrate (39%-44%), fat (34% ~ 42%) and protein (20%-23%) failed to meet Chinese RNI (5) the intake of vitamin C, B1, folate, zinc, dietary fiber, and calcium was 5% ~ 73% lower than the RNI while vitamin B2, B3, E, iron and selenium intake was 20% to 3 times higher than the RNI. Nutrient intake in the control group was lower for all nutrients than the recommended RNI. CONCLUSION: Lactating women on a self-selected diet did not meet the Chinese RNI for many important micronutrients, which may influence the nutritional composition of breast milk and thus impact the potential health of mothers and infants. RNI should consider the regional dietary habits and culture. A single national RNI is not applicable for all of China. Nutritional education into the community is needed.

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

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

Molecular basis for polysialylation: a novel polybasic polysialyltransferase domain (PSTD) of 32 amino acids unique to the alpha 2,8-polysialyltransferases is essential for polysialylation.

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.

Structural relationships in small molecule interactions governing gas-phase enantioselectivity and zwitterionic formation.

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.

NMR studies on how the binding complex of polyisoprenol recognition sequence peptides and polyisoprenols can modulate membrane structure.

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.

Degree of polymerization (DP) of polysialic acid (polySia) on neural cell adhesion molecules (N-CAMS): development and application of a new strategy to accurately determine the DP of polySia chains on N-CAMS.

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.

NMR study of the preferred membrane orientation of polyisoprenols (dolichol) and the impact of their complex with polyisoprenyl recognition sequence peptides on membrane structure.

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.