Engineering polysialic acid on Schwann cells using polysialyltransferase gene transfer or purified enzyme exposure for spinal cord injury transplantation.

Polysialic acid (PolySia) is a critical post-translational modification on the neural cell adhesion molecule (NCAM, a.k.a., CD56), important for cell migration and axon growth during nervous system development, plasticity and repair. PolySia induction on Schwann cells (SCs) enhances their migration, axon growth support and ability to improve functional recovery after spinal cord injury (SCI) transplantation. In the current investigation two methods of PolySia induction on SCs, lentiviral vector transduction of the mouse polysialytransferase gene ST8SIA4 (LV-PST) or enzymatic engineering with a recombinant bacterial PST (PSTNm), were examined comparatively for their effects on PolySia induction, SC migration, the innate immune response and axon growth after acute SCI. PSTNm produced significant PolySia induction and a greater diversity of surface molecule polysialylation on SCs as evidenced by immunoblot. In the scratch wound assay, PSTNm was superior to LV-PST in the promotion of SC migration and gap closure. At 24 h after SCI transplantation, PolySia induction on SCs was most pronounced with LV-PST. Co-delivery of PSTNm with SCs, but not transient cell exposure, led to broader induction of PolySia within the injured spinal cord due to polysialylation upon both host cells and transplanted SCs. The innate immune response after SCI, measured by CD68 immunoreactivity, was similar among PolySia induction methods. LV-PST or PSTNm co-delivery with SCs provided a similar enhancement of SC migration and axon growth support above that of unmodified SCs. These studies demonstrate that LV-PST and PSTNm provide comparable acute effects on SC polysialation, the immune response and neurorepair after SCI.

Apoptosis of human breast carcinoma cells in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and glycolipid: Glycosyltransferases.

Apoptosis of human breast carcinoma cells (SKBR-3, MCF-7, and MDA-468) has been observed after treatment of these cells with anti-cancer drug cis-platin and glycosphingolipid biosynthesis inhibitor L- and D-PPMP, respectively. These drugs initiated apoptosis in a dose-dependent manner as measured by phenotypic morphological changes, by binding of a fluorescent phophatidyl serine-specific dye (PSS-380) onto the outer leaflet of the cell membranes, and by activation of caspases, -3, -8, and -9. It was observed that in two hours very little apoptotic process had started but predominant biochemical changes occurred after 6 h. DNA degradation started after 24 hours of drug treatment. However, very little is known about the stability of the ';Replication Complexes'' during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during its replication, repair, and recombination processes. Previously, DNA helicase-III was characterized as a component of the replication complexes isolated from embryonic chicken brains as well as breast and colon carcinoma cells. Helicase activities were measured by a novel method (ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of the nicked ACT-DNA, by determining values obtained from +/- aphidicolin-treated incubation mixtures. In all three breast carcinoma cell lines, a common trend was observed: a decrease of activities of DNA polymerase-alpha and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 alpha2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a alpha2-3 sialyltransferase) was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin.

Cytidine 5'-monophosphate (CMP)-induced structural changes in a multifunctional sialyltransferase from Pasteurella multocida.

Sialyltransferases catalyze reactions that transfer a sialic acid from CMP-sialic acid to an acceptor (a structure terminated with galactose, N-acetylgalactosamine, or sialic acid). They are key enzymes that catalyze the synthesis of sialic acid-containing oligosaccharides, polysaccharides, and glycoconjugates that play pivotal roles in many critical physiological and pathological processes. The structures of a truncated multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1), in the absence and presence of CMP, have been determined by X-ray crystallography at 1.65 and 2.0 A resolutions, respectively. The Delta24PmST1 exists as a monomer in solution and in crystals. Different from the reported crystal structure of a bifunctional sialyltransferase CstII that has only one Rossmann domain, the overall structure of the Delta24PmST1 consists of two separate Rossmann nucleotide-binding domains. The Delta24PmST1 structure, thus, represents the first sialyltransferase structure that belongs to the glycosyltransferase-B (GT-B) structural group. Unlike all other known GT-B structures, however, there is no C-terminal extension that interacts with the N-terminal domain in the Delta24PmST1 structure. The CMP binding site is located in the deep cleft between the two Rossmann domains. Nevertheless, the CMP only forms interactions with residues in the C-terminal domain. The binding of CMP to the protein causes a large closure movement of the N-terminal Rossmann domain toward the C-terminal nucleotide-binding domain. Ser 143 of the N-terminal domain moves up to hydrogen-bond to Tyr 388 of the C-terminal domain. Both Ser 143 and Tyr 388 form hydrogen bonds to a water molecule, which in turn hydrogen-bonds to the terminal phosphate oxygen of CMP. These interactions may trigger the closure between the two domains. Additionally, a short helix near the active site seen in the apo structure becomes disordered upon binding to CMP. This helix may swing down upon binding to donor CMP-sialic acid to form the binding pocket for an acceptor.

Valproic acid modulates NCAM polysialylation and polysialyltransferase mRNA expression in human tumor cells.

Polysialic acid (PSA) is a dynamically regulated carbohydrate modification of the neural cell adhesion molecule NCAM, which has been linked to cancer development and dissemination. Two enzymes, the polysialyltransferases ST8SiaIV and ST8SiaII, are known to be involved in the polysialylation of NCAM. The antiepileptic drug valproic acid (VPA) is associated with anti-cancer activity. In this study, VPA blocked the adhesion of several neuroectodermal tumor cell lines to human umbilical vein endothelial cells. Furthermore, VPA induced intracellular PSA accumulation and enhanced expression of PSA-NCAM on the cell surface. Using a semiquantitative RT-PCR strategy, VPA was shown to up-regulate ST8SiaIV mRNA, whereas ST8SiaII mRNA was down-regulated by this compound. Our data indicate that increased expression of ST8SiaIV enables accelerated polysialylation of NCAM, which might be coupled to a loss of adhesive functions of tumor cells.

Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase.

The present study investigated the mechanism underlying alterations of cell surface sugar chains of Jurkat cells by inducing apoptosis with etoposide, an inhibitor of topoisomerase II. Within 3 h of etoposide treatment, flowcytometric analysis revealed a decrease in Maackia amurensis agglutinin recognized alpha2,3-linked sialic acid moieties and an increase in Ricinus communis agglutinin recognized galactose. The results suggested that asialo-sugar chains on glycoconjugates were rapidly induced on the etoposide-treated cell surface. To clarify the desialylation mechanism, we studied alpha2,3-sialyltransferase mRNA expression and the activity of sialidase on the cell surface during etoposide-induced apoptosis. The expression of hST3Gal III and hST3Gal IV mRNAs were down-regulated and sialidase activity on the cell surface increased threefold within 2 h of etoposide treatment. Moreover, the decrease in alpha2,3-linked sialic acid levels was significantly suppressed in the presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, an inhibitor of sialidase. These results suggested that activation or exposure of sialidase on the cell surface was induced by etoposide treatment and was the main cause of the decrease in sialic acids.

Alcohol and molecular regulation of protein glycosylation and function.

Chronic alcohol exposure leads to the appearance of carbohydrate-deficient transferrin (CDT), a N-glycosylated protein and sialic acid-deficient apolipoprotein E (apoE), an O-glycosylated protein. We show that chronic ethanol treatment destabilizes sialyltransferase (ST) mRNA resulting in a concomitant decreased steady-state level of ST mRNA. As a result, alcohol markedly decreases the hepatic synthetic rate of ST. This leads to impaired sialylation of transferrin and apoE. Consequently, apoE content in plasma high-density lipoproteins (HDL) is decreased. ApoE plays a significant role in the delivery of HDL cholesterol to the liver via apo B/E receptor, a process called reverse cholesterol transport (RCT). Desialylation of apoE results in its decreased association with HDL. Thus, the dissociation constant of HDL for binding to sialo-apoE is 90 +/- 35 nM, whereas that for desialo-apoE is 1010 +/- 250 nM. More importantly, the uptake of labeled cholesterol by human HepG2 cells is decreased by 30-40% from reconstituted HDL particles (rHDL)-containing desialo-apoE compared to rHDL with sialo-apoE. We conclude that chronic alcohol exposure down-regulates the expression of sialyltransferase genes resulting in impaired sialylation of apoE. This leads to its decreased binding to plasma HDL and thereby, impairs the RCT function of HDL.

Developmental expression and characterization of the alpha2,8-polysialyltransferase activity in embryonic chick brain.

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.

The hydrocortisone-induced transcriptional down-regulation of beta-galactoside alpha2,6-sialyltransferase in the small intestine of suckling rats is suppressed by mifepristone (RU-38.486).

The progressive loss of sialic acids of the brush-border membrane glycoproteins is one of the major biochemical changes which occur in the rat small intestine during the transition from suckling to weaning, and this process is speeded up by an injection of glucocorticoids to the suckling animals. We used the rat liver beta-galactoside alpha2,6-sialyltransferase (ST6(N), EC 2.4.99.1) cDNA as a probe to examine the mRNA level of this enzyme in the small intestine of both suckling (13-day-old) and weaned (25-day-old) rats. In the ileum of suckling rats, the ST6(N) mRNA level was about four times higher than in the jejunum, whereas the membrane-bound enzyme activity was less than two times higher. In comparison with the controls, hydrocortisone treatment significantly decreased the level of this transcript and of the corresponding enzyme activity in both segments of the small intestine of suckling rats. Additionally, the antiglucocorticoid mifepristone (RU-38.486) suppressed the effect of hydrocortisone. The expression of ST6(N) mRNA in the small intestine of weaned (25-day-old) rats was several times lower than that in suckling (13-day-old) rats, and was unresponsive to hydrocortisone as well as to mifepristone. These results indicate that the glucocorticoid-induced transcriptional down-regulation of ST6(N) expression in the small intestine of suckling rats is mediated via the glucocorticoid receptor pathway, and support the notion that alterations in sialylation of brush-border membrane glycoconjugates occurring upon weaning are the result of a lower expression of ST6(N).

Regulation of glycolipid synthesis in HL-60 cells by antisense oligodeoxynucleotides to glycosyltransferase sequences: effect on cellular differentiation.

Treatment of the human promyelocytic leukemia cell line HL-60 with antisense oligodeoxynucleotides to UDP-N-acetylgalactosamine:beta-1,4-N-acetylgalactosaminyl-transferase (GM2-synthase; EC 2.4.1.92) and CMP-sialic acid:alpha-2,8-sialyltransferase (GD3-synthase; EC 2.4.99.8) sequences effectively down-regulated the synthesis of more complex gangliosides in the ganglioside synthetic pathways after GM3, resulting in a remarkable increase in endogenous GM3 with concomitant decreases in more complex gangliosides. The treated cells underwent monocytic differentiation as judged by morphological changes, adherent ability, and nitroblue tetrazolium staining. These data provide evidence that the increased endogenous ganglioside GM3 may play an important role in regulating cellular differentiation and that the antisense DNA technique proves to be a powerful tool in manipulating glycolipid synthesis in the cell.

Effect of interferon alpha IIb on the activity of sialyltransferase in testis homogenates of adult rats.

Interferon alpha IIb was injected to adult male rats at doses ranging from 10,000 to 200,000 units. Animals were dissected at intervals of 12 h, 24 h, and 5 days. The activity of the enzyme sialyltransferase in testis homogenates was estimated. In the majority of experiments enzyme activity decreased in comparison to controls.

Double immunofluorescent staining of alpha 2,6 sialyltransferase and beta 1,4 galactosyltransferase in monensin-treated cells: evidence for different Golgi compartments?

Beta 1,4 galactosyl- and alpha 2,6 sialyltransferase (gal-T EC 2.4.1.22 and sialyl-T EC 2.4.99.1) sequentially elongate and terminate complex N-glycan chains of glycoproteins. Both enzymes reside in trans Golgi cisternae; their ultrastructural relationship, however, is unknown. To delineate their respective Golgi compartment(s) we conducted a double label immunofluorescent study by conventional and confocal laser scanning microscopy in HepG2, HeLa, and other cells in presence of Golgi-disturbing agents. Polyclonal, peptide-specific antibodies to human sialyl-T expressed as a beta-galactosidase-sialyl-T fusion protein in E. coli were developed and applied together with mABs to human milk gal-T. In untreated HepG2 and HeLa cells Golgi morphology identified by immunofluorescent labeling of sialyl-T and gal-T, respectively, was nearly identical. Treatment of cells with brefeldin A (BFA) led to rapid and coordinated disappearance of immunostaining of both enzymes; after BFA washout, vesicular structures reappeared which first stained for gal-T followed by sialyl-T; in the reassembled Golgi apparatus sialyl-T and gal-T were co-localized again. In contrast, monensin treatment produced a reversible swelling and scattering of gal-T positive Golgi elements while sialyl-T positive structures showed little change. Treatment with nocodazole led to dispersal of Golgi elements in which gal-T and sialyl-T remained co-localized. Treatment with chloroquine affected Golgi structures less than monensin and led to condensation of gal-T positive and to slight enlargement of sialyl-T positive structures. Sequential recovery from BFA of gal-T and sialyl-T and their segregation by monensin suggest that these enzymes are targeted to different Golgi subcompartments.

Regulation of sialylation of intestinal brush-border enzymes and of sialyltransferase activity in organ cultures by dexamethasone.

To investigate the direct effect of glucocorticoids on sialylation of intestinal brush-border hydrolases, explants of fetal and suckling rat intestine were maintained in serum-free or serum-containing organ culture with or without dexamethasone (Dx). Glucoamylase and dipeptidyl peptidase IV developed in organ culture from 18-day-old fetuses persisted in highly sialylated forms for 8 days irrespective of Dx presence, parallel in vivo development leading to less sialylated forms at the age of 6 days. In postnatal cultures the Dx-stimulated glucoamylase appeared in a new highly sialylated form never seen after the hormone application in vivo. These findings are in agreement with the elevation of bound sialyltransferase (ST) of cultured intestine in protein-free media. In serum-containing medium Dx stimulated the formation and release of soluble ST into the culture medium.