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

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

Cloning and expression of an alpha-2,8-polysialyltransferase (STX) from Xenopus laevis

Polysialic acid (polySia) is a carbohydrate structure found on neural cell adhesion molecules (N-CAM). Two polysialyltransferases (polySiaTs) that catalyze synthesis of polySia have been described, and designated PST-1/PST/ST8SiaIV and STX/ST8SiaII. We cloned a polySiaT (xSTX) from a nonmammalian vertebrate, Xenopus laevis . xSTX had 80% amino acid similarity to the rat STX. This clone induced polySia expression when transfected into polySia-negative COS-1 cells. Northern blot analysis of whole embryos at different stages of development revealed that xSTX mRNA was most abundantly expressed in premetamorphic stages. The relative level of xSTX and N-CAM mRNAs was also examined and found to change in parallel to the extent of polysialylation on N-CAM. In adult tissues, the expression of xSTX mRNA was restricted to brain, eye and heart, which also expressed polySia. These results suggest that xSTX is the major enzyme responsible for the synthesis of polysialylated N-CAM in embryos at certain stages of development and also in adult tissues.      

Enzyme-dependent variations in the polysialylation of the neural cell adhesion molecule (NCAM) in vivo

Polysialic acid (polySia), an alpha2,8-linked polymer of N-acetylneuraminic acid, represents an essential regulator of neural cell adhesion molecule (NCAM) functions. Two polysialyltransferases, ST8SiaII and ST8SiaIV, account for polySia synthesis, but their individual roles in vivo are still not fully understood. Previous in vitro studies defined differences between the two enzymes in their usage of the two NCAM N-glycosylation sites affected and suggested a synergistic effect. Using mutant mice, lacking either enzyme, we now assessed in vivo the contribution of ST8SiaII and ST8SiaIV to polysialylation of NCAM. PolySia-NCAM was isolated from mouse brains and trypsinized, and polysialylated glycopeptides as well as glycans were analyzed in detail. Our results revealed an identical glycosylation and almost complete polysialylation of N-glycosylation sites 5 and 6 in polySia-NCAM irrespective of the enzyme present. The same sets of glycans were substituted by identical numbers of polySia chains in vivo, the length distribution of which, however, differed with the enzyme setting. Expression of ST8SiaIV alone led to higher amounts of short polySia chains and gradual decrease with length, whereas exclusive action of ST8SiaII evoked a slight reduction in long polySia chains only. These variations were most pronounced at N-glycosylation site 5, whereas the polysialylation pattern at N-glycosylation site 6 did not differ between NCAM from wild-type and ST8SiaII- or ST8SiaIV-deficient mice. Thus, our fine structure analyses suggest a comparable quality of polysialylation by ST8SiaII and ST8SiaIV and a distinct synergistic action of the two enzymes in the synthesis of long polySia chains at N-glycosylation site 5 in vivo.     

Differential biosynthesis of polysialic or disialic acid Structure by ST8Sia II and ST8Sia IV

ST8Sia II (STX) and ST8Sia IV (PST) are polysialic acid (polySia) synthases that catalyze polySia formation of neural cell adhesion molecule (NCAM) in vivo and in vitro. It still remains unclear how these structurally similar enzymes act differently in vivo. In the present study, we performed the enzymatic characterization of ST8Sia II and IV; both ST8Sia II and IV have pH optima of 5.8-6.1 and have no requirement of metal ions. Because the pH dependence of ST8Sia II and IV enzyme activities and the pK profile of His residues are similar, we hypothesized that a histidine residue would be involved in their catalytic activity. There is a conserved His residue (cf. His(348) in ST8Sia II and His(331) in ST8Sia IV, respectively) within the sialyl motif VS in all sialyltransferase genes cloned to date. Mutant ST8Sia II and IV enzymes in which this His residue was changed to Lys showed no detectable enzyme activity, even though they were folded correctly and could bind to CDP-hexanolamine, suggesting the importance of the His residue for their catalytic activity. Next, the degrees of polymerization of polySia in NCAM catalyzed by ST8Sia II and IV were compared. ST8Sia IV catalyzed larger polySia formation of NCAM than ST8Sia II. We also analyzed the (auto)polysialylated enzymes themselves. Interestingly, when ST8Sia II or IV itself was sialylated under conditions for polysialylation, the disialylated compound was the major product, even though polysialylated compounds were also observed. These results suggested that both ST8Sia II and IV catalyze polySia synthesis toward preferred acceptor substrates such as NCAM, whereas they mainly catalyze disialylation, similarly to ST8Sia III, toward unfavorable substrates such as enzyme themselves.     

The polysialyltransferase ST8Sia II/STX: posttranslational processing and role of autopolysialylation in the polysialylation of neural cell adhesion molecule.

The presence of alpha2,8-linked polysialic acid on the neural cell adhesion molecule (NCAM) is known to modulate cell interactions during development and oncogenesis. Two enzymes, the alpha2,8-polysialyltransferases ST8Sia IV()/PST and ST8Sia II()/STX are responsible for the polysialylation of NCAM. We previously reported that both ST8Sia IV/PST and ST8Sia II/STX enzymes are themselves modified by alpha2,8-linked polysialic acid chains, a process called autopolysialylation. In the case of ST8Sia IV/PST, autopolysialylation is not required for enzymatic activity. However, whether the autopolysialylation of ST8Sia II/STX is required for its ability to polysialylate NCAM is unknown. To understand how autopolysialylation impacts ST8Sia II/STX enzymatic activity, we employed a mutagenesis approach. We found that ST8Sia II/STX is modified by six Asn-linked oligosaccharides and that polysialic acid is distributed among the oligosaccharides modifying Asn 89, 219, and 234. Coexpression of a nonautopolysialylated ST8Sia II/STX mutant with NCAM demonstrated that autopolysialylation is not required for ST8Sia II/STX polysialyltransferase activity. In addition, catalytically active, nonautopolysialylated ST8Sia II/STX does not polysialylate any endogenous COS-1 cell proteins, highlighting the protein specificity of polysialylation. Furthermore, immunoblot analysis of NCAM polysialylation by autopolysialylated and nonautopolysialylated ST8Sia II/STX suggests that the NCAM is polysialylated to a higher degree by autopolysialylated ST8Sia II/STX. Therefore, we conclude that autopolysialylation of ST8Sia II/STX, like that of ST8Sia IV/PST, is not required for, but does enhance, NCAM polysialylation.     

Involvement of the alpha2,8-polysialyltransferases II/STX and IV/PST in the biosynthesis of polysialic acid chains on the O-linked glycoproteins in rainbow trout ovary

Polysialoglycoprotein (PSGP) in salmonid fish egg is a unique glycoprotein bearing alpha2,8-linked polysialic acid (polySia) on its O-linked glycans. Biosynthesis of the polySia chains is developmentally regulated and only occurs at later stage of oogenesis. Two alpha2,8-polysialyltransferases (alpha2,8-polySTs), PST (ST8Sia IV) and STX (ST8Sia II), responsible for the biosynthesis of polySia on N-glycans of glycoproteins, are known in mammals. However, nothing has been known about which alpha2,8-polySTs are involved in the biosynthesis of polySia on O-linked glycans in any glycoproteins. We thus sought to identify cDNA encoding the alpha2,8-polyST involved in polysialylation of PSGP. A clone for PST orthologue, rtPST, and two clones for the STX orthologue, rtSTX-ov and rtSTX-em, were identified in rainbow trout. The deduced amino acid sequence of rtPST shows a high identity (72-77%) to other vertebrate PSTs, while that of rtSTX-ov shows 92% identity with rtSTX-em and a significant identity (63-76%) to other vertebrate STXs. The rtPST exhibited the in vivo alpha2,8-polyST activity, although its in vitro activity was low. However, the rtSTXs showed no in vivo and very low in vitro activities. Interestingly, co-existence of rtPST and rSTX-ov in the reaction mixture synergistically enhanced the alpha2,8-polyST activity. During oogenesis, rtPST was constantly expressed, while the expression of rtSTX-ov was not increased until polySia chain is abundantly biosynthesized in the later stage. rtSTX-em was not expressed in ovary. These results suggest that the enhanced expression of rtSTX-ov under the co-expression with rtPST may be important for the biosynthesis of polySia on O-linked glycans of PSGP.     

Polysialic acid is associated with sodium channels and the neural cell adhesion molecule N-CAM in adult rat brain.

We have studied alpha 2,8-linked polysialic acid (polySia) and the neural cell adhesion molecule (N-CAM) in the adult rat brain by immunohistochemistry and Western blot analysis. Both molecules were widely distributed but not ubiquitous. Various brain regions showed colocalization of polySia and N-CAM. Strong immunoreactivity for polySia was seen in regions which were negative for N-CAM, such as the main and accessory olfactory bulbs. Immunohistochemical evidence for the heterogeneity of polySia expression in different brain regions was confirmed by immunoblotting. We present evidence that N-CAM is not the only polySia bearing protein in adult rat brain. Specifically, immunoprecipitation using the polySia-specific monoclonal antibody mAb 735 precipitated not only N-CAM isoforms carrying polySia, but also the sodium channel alpha subunit. Immunoblotting using sodium channel alpha subunit antibody (SP20) revealed a smear from 250 kDa upwards. PolySia removal using an endoneuraminidase specific for alpha 2,8-linked polysialic acid of 8 or more residues long, reduced this smear to a single band at 250 kDa. Thus both N-CAM and sodium channels carry homopolymers of alpha 2,8-linked polysialic acid in adult rat brain.     

Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide acceptors by three distinct alpha 2,8-sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III

Polysialylated neural cell adhesion molecule (NCAM) is thought to play a critical role in neural development. Polysialylation of NCAM was shown to be achieved by two alpha2,8-polysialyltransferases, ST8Sia IV (PST) and ST8Sia II (STX), which are moderately related to another alpha2,8-sialyltransferase, ST8Sia III. Here we describe that all three alpha2,8-sialyltransferases can utilize oligosaccharides as acceptors but differ in the efficiency of adding polysialic acid on NCAM. First, we found that ST8Sia III can form polysialic acid on the enzyme itself (autopolysialylation) but not on NCAM. These discoveries prompted us to determine if ST8Sia IV and ST8Sia II share the property of ST8Sia III in utilizing low molecular weight oligosaccharides as acceptors. By using a newly established method, we found that ST8Sia IV, ST8Sia II, and ST8Sia III all add oligosialic and polysialic acid on various sialylated N-acetyllactosaminyl oligosaccharides, including NCAM N-glycans, fetuin N-glycans, synthetic sialylated N-acetyllactosamines, and on alpha(2)-HS-glycoprotein. Our results also showed that monosialyl and disialyl N-acetyllactosamines can serve equally as an acceptor, suggesting that no initial addition of alpha2,8-sialic acid is necessary for the action of polysialyltransferases. Polysialylation of NCAM by ST8Sia IV and ST8Sia II is much more efficient than polysialylation of N-glycans isolated from NCAM. Moreover, ST8Sia IV and ST8Sia II catalyze polysialylation of NCAM much more efficiently than ST8Sia III. These results suggest that no specific acceptor recognition is involved in polysialylation of low molecular weight sialylated oligosaccharides, whereas the enzymes exhibit pronounced acceptor specificities if glycoproteins are used as acceptors.     

Developmental profile of neural cell adhesion molecule glycoforms with a varying degree of polymerization of polysialic acid chains

More precise information on the degree of polymerization (DP) of polysialic acid (polySia) chains expressed on neural cell adhesion molecule (NCAM) and its developmental stage-dependent variation are considered important in understanding the mechanism of regulated polysialylation and fine-tuning of NCAM-mediated cell adhesion by polySia. In this paper, first we performed a kinetic study of acid-catalyzed hydrolysis of polySia and report our findings that (a) in (-->8Neu5Ac alpha 2-->)(n)-->8Neu5Ac alpha 2-->3Gal beta 1-->R, the proximal Neu5Ac residue alpha 2-->3 linked to Gal is cleaved about 2.5-4 times faster than the alpha 2-->8 linkages and (b) in contrary to general belief that alpha 2-->8 linkages in polySia are extremely labile, the kinetic consideration showed that they are not so unstable, and every ketosidic bond is hydrolyzed at the same rate. These findings are the basis of our strategy for DP analysis of polySia on NCAM. Second, using the recently developed method that provides base-line resolution of oligo/polySia from DP 2 to >80 with detection thresholds of 1.4 fmol per resolved peak, we have determined the DP of polySia chains expressed in embryonic chicken brains at different developmental stages. Our results support the presence of numerous NCAM glycoforms differing in DPs of oligo/polySia chains and a delicate change in their distribution during development.     

Experimental approaches to interfere with the polysialylation of the neural cell adhesion molecule in vitro and in vivo

Sialic acid (Sia) is expressed as terminal sugar in many glycoconjugates and plays an important role during development and regeneration. Addition of homopolymers of Sia (polysialic acid; polySia/PSA) is a unique and highly regulated post-translational modification of the neural cell adhesion molecule (NCAM). The presence of polySia affects NCAM-dependent cell adhesion and plays an important role during brain development, neural regeneration, and plastic processes including learning and memory. PolySia-NCAM is expressed on several neuroendocrine tumors of high malignancy and correlates with poor prognosis. Two closely related enzymes, the polysialyltransferases ST8SiaII and ST8SiaIV, catalyze the biosynthesis of polySia. This review summarizes recent knowledge on Sia biosynthesis and the correlation between Sia biosynthesis and polysialylation of NCAM and report on approaches to modify the degree of polySia on NCAM in vitro and in vivo. First, we describe the inhibition of polysialylation of NCAM in ST8SiaII-expressing cells using synthetic Sia precursors. Second, we demonstrate that the key enzyme of the Sia biosynthesis (UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase) regulates and limits the synthesis of polySia by controlling the cellular Sia concentration.     

ST8Sia II and ST8Sia IV polysialyltransferases exhibit marked differences in utilizing various acceptors containing oligosialic acid and short polysialic acid. The basis for cooperative polysialylation by two enzymes

Polysialylation of the neural cell adhesion molecule (NCAM) is thought to play a critical role in neural development. Two polysialyltransferases, ST8Sia II and ST8Sia IV, play dominant roles in polysialic acid synthesis on NCAM. However, the individual roles and mechanisms by which these two enzymes form large amounts of polysialic acid on NCAM were heretofore unknown. Previous studies indicate that ST8Sia IV forms more highly polysialylated N-glycans on NCAM than ST8Sia II in vitro. In the present study, we first demonstrated that a combination of ST8Sia II and ST8Sia IV cooperatively polysialylated NCAM, resulting in NCAM N-glycans containing more, and thus longer, polysialic acid than when the enzymes were used individually. There was also an increase in polysialylated NCAM when we used ST8Sia II and ST8Sia IV sequentially, whereas there appeared to be a subtle increase when the enzymes were used in the reverse order. Furthermore, ST8Sia IV was able to add polysialic acid to oligosialylated oligosaccharides and unpolysialylated antennas in N-glycans attached to NCAM, even when polysialic acid was attached to at least one of the other antennas. By contrast, ST8Sia II added little polysialic acid to the same acceptors. On the other hand, neither ST8Sia II nor ST8Sia IV could add polysialic acid to a polysialylated antenna of NCAM N-glycans. These combined results indicate that the synergistic effect of ST8Sia II and ST8Sia IV is caused by: 1) the ability of ST8Sia IV to add polysialic acid to oligosialic acid formed by ST8Sia II, 2) the potential of ST8Sia IV to act on more antennas of N-glycans than ST8Sia II, and 3) the ability of ST8Sia II and ST8Sia IV in combination to act on the fifth and sixth N-glycosylation sites of NCAM.     

Polysialic acid profiles of mice expressing variant allelic combinations of the polysialyltransferases ST8SiaII and ST8SiaIV

The post-translational modification of the neural cell adhesion molecule (NCAM) by polysialic acid (polySia) represents a remarkable example of dynamic modulation of homo- and heterophilic cell interactions by glycosylation. The synthesis of this unique carbohydrate polymer depends on the polysialyltransferases ST8SiaII and ST8SiaIV. Aiming to understand in more detail the contributions of ST8SiaII and ST8SiaIV to polySia biosynthesis in vivo, we used mutant mouse lines that differ in the number of functional polysialyltransferase alleles. The 1,2-diamino-4,5-methylenedioxybenzene method was used to qualitatively and quantitatively assess the polySia patterns. Similar to the wild-type genotype, long polySia chains (>50 residues) were detected in all genotypes expressing at least one functional polysialyltransferase allele. However, variant allelic combinations resulted in distinct alterations in the total amount of poly-Sia; the relative abundance of long, medium, and short polymers; and the ratio of polysialylated to non-polysialylated NCAM. In ST8SiaII-null mice, 45% of the brain NCAM was non-polysialylated, whereas a single functional allele of ST8SiaII was sufficient to polysialylate approximately 90% of the NCAM pool. Our data reveal a complex polysialylation pattern and show that, under in vivo conditions, the coordinated action of ST8SiaII and ST8SiaIV is crucial to fine-tune the amount and structure of polySia on NCAM.     

Polysialyltransferases: major players in polysialic acid synthesis on the neural cell adhesion molecule

Polysialic acid is a unique carbohydrate composed of a linear homopolymer of alpha2,8-linked sialic acid, and is mainly attached to the fifth immunoglobulin-like domain of the neural cell adhesion molecule (NCAM) via a typical N-linked glycan in vertebrate neural system. Polysialic acid plays critical roles in neural development by modulating adhesive property of NCAM such as neural cell migration, neurite outgrowth, neural pathfinding, and synaptogenesis. The expression of polysialic acid is temporally and spatially regulated during neural development. Polysialylation of NCAM is catalyzed by two polysialyltransferases, ST8Sia II (STX) and ST8Sia IV (PST), which belong to the family of six genes encoding alpha 2,8-sialyltransferases. ST8Sia II and IV are expressed differentially in tissue-specific and cell-specific manners, and they apparently have distinct roles in development and organogenesis. The presence of polysialic acid is always associated with expression of ST8Sia II and/or IV, suggesting that ST8Sia II and IV are the key enzymes that control the expression of polysialic acid. Both ST8Sia II and IV can transfer multiple alpha 2,8-linked sialic acid residues to an acceptor N-glycan containing a NeuNAc alpha 2-->3 (or 6) Gal beta 1-->4GlcNAc beta 1-->R structure without participation of other enzymes. The two enzymes differently but cooperatively act on NCAM and the amount of polysialic acid synthesized by both enzymes together is greater than that synthesized by either enzyme alone. The polysialyltransferases are thus important regulators in polysialic acid synthesis and contribute to neural development in the vertebrate.     

Polysialylated neuropilin-2 is expressed on the surface of human dendritic cells and modulates dendritic cell-T lymphocyte interactions

Polysialic acid (PSA) is a unique linear homopolymer of alpha2,8-linked sialic acid that has been identified as a posttranslational modification on only five mammalian proteins. Studied predominantly on neural cell adhesion molecule (NCAM) during development of the vertebrate nervous system, PSA modulates cell interactions mediated by NCAM and other adhesion molecules. An isoform of NCAM (CD56) on natural killer (NK) cells is the only protein known to be polysialylated in cells of the immune system, yet the function of PSA in NK cells remains unclear. We show here that neuropilin-2 (NRP-2), a receptor for the semaphorin and vascular endothelial growth factor families in neurons and endothelial cells, respectively, is expressed on the surface of human dendritic cells and is polysialylated. Expression of NRP-2 is up-regulated during dendritic cell maturation, coincident with increased expression of ST8Sia IV, one of the key enzymes of PSA biosynthesis, and with the appearance of PSA on the cell surface. PSA on NRP-2 is resistant to digestion with peptide N-glycosidase F but is sensitive to release under alkaline conditions, suggesting that PSA chains are added to O-linked glycans of NRP-2. Removal of polysialic acid from the surface of dendritic cells or binding of NRP-2 with specific IgG promoted dendritic cell-induced activation and proliferation of T lymphocytes. Thus, this newly recognized polysialylated protein on the surface of dendritic cells influences dendritic cell-T lymphocyte interactions through one or more of its distinct extracellular domains.     

The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II

A limited number of mammalian proteins are modified by polysialic acid, with the neural cell adhesion molecule (NCAM) being the most abundant of these. We hypothesize that polysialylation is a protein-specific glycosylation event and that an initial protein-protein interaction between polysialyltransferases and glycoprotein substrates mediates this specificity. To evaluate the regions of NCAM required for recognition and polysialylation by PST/ST8Sia IV and STX/ST8Sia II, a series of domain deletion proteins were generated, co-expressed with each enzyme, and their polysialylation analyzed. A protein consisting of the fifth immunoglobulin-like domain (Ig5), which contains the reported sites of polysialylation, and the first fibronectin type III repeat (FN1) was polysialylated by both enzymes, whereas a protein consisting of Ig5 alone was not polysialylated by either enzyme. This demonstrates that the Ig5 domain of NCAM and FN1 are sufficient for polysialylation, and suggests that the FN1 may constitute an enzyme recognition and docking site. Two other NCAM mutants, NCAM-6 (Ig1-5) and NCAM-7 (FN1-FN2), were weakly polysialylated by PST/ST8Sia IV, suggesting that a weaker enzyme recognition site may exist within the Ig domains, and that glycans in the FN region are polysialylated. Further analysis indicated that O-linked oligosaccharides in NCAM-7, and O-linked and N-linked glycans in full-length NCAM, are polysialylated when these proteins are co-expressed with the polysialyltransferases in COS-1 cells. Our data support a model in which the polysialyltransferases bind to the FN1 of NCAM to polymerize polysialic acid chains on appropriately presented glycans in adjacent regions.     

Polysialyltransferase-1 autopolysialylation is not requisite for polysialylation of neural cell adhesion molecule

Polysialyltransferase-1 (PST; ST8Sia IV) is one of the alpha2, 8-polysialyltransferases responsible for the polysialylation of the neural cell adhesion molecule (NCAM). The presence of polysialic acid on NCAM has been shown to modulate cell-cell and cell-matrix interactions. We previously reported that the PST enzyme itself is modified by alpha2,8-linked polysialic acid chains in vivo. To understand the role of autopolysialylation in PST enzymatic activity, we employed a mutagenesis approach. We found that PST is modified by five Asn-linked oligosaccharides and that the vast majority of the polysialic acid is found on the oligosaccharide modifying Asn-74. In addition, the presence of the oligosaccharide on Asn-119 appeared to be required for folding of PST into an active enzyme. Co-expression of the PST Asn mutants with NCAM demonstrated that autopolysialylation is not required for PST polysialyltransferase activity. Notably, catalytically active, non-autopolysialylated PST does not polysialylate any endogenous COS-1 cell proteins, highlighting the protein specificity of polysialylation. Immunoblot analyses of NCAM polysialylation by polysialylated and non-autopolysialylated PST suggests that the NCAM is polysialylated to a higher degree by autopolysialylated PST. We conclude that autopolysialylation of PST is not required for, but does enhance, NCAM polysialylation.     

An ultrasensitive chemical method for polysialic acid analysis

An ultrasensitive method for analysis of polysialic acid (polySia) chains, using fluorescence-assisted high-performance liquid chromatography was developed. The new method is a substantial improvement of our earlier method in which the reducing terminal Sia residues of a homologous series of oligo/polySia hydrolytically released during derivatization reaction were simultaneously labeled with a fluorogenic reagent, 1,2-diamino-4,5-methylenedioxybenzene (DMB) in situ. We first studied extensively the stability of oligo/polySia in the acid (0.02 M trifluoracetic acid) used for 1,2-diamino-4,5-methylenedioxybenzene derivatization under various conditions of reaction time and temperature, analyzing the hydrolytic products by high-performance anion exchange chromatography with pulsed electrochemical detection (HPAEC-PED). Then we optimized the reaction conditions to minimize degradation of the parent polySia while maintaining high derivatization rate. Using a DNAPac PA-100 column rather than a MonoQ column, baseline resolution of polySia peaks up to DP 90 with a detection threshold of 1.4 femtomol per resolved peak was achieved. The new method was used to analyze the degree of polymerization of a polySia-containing glycopeptide fraction derived from embryonic chicken brain, and the results were compared with those obtained by HPAEC-PED.     

Polysialyltransferase ST8Sia II (STX) polysialylates all of the major isoforms of NCAM and facilitates neurite outgrowth

The neural cell adhesion molecule (NCAM) has different isoforms due to different sizes in its polypeptide and plays a significant role in neural development. In neural development, the function of NCAM is modified by polysialylation catalyzed by two polysialyltransferases, ST8Sia II and ST8Sia IV. Previously, it was reported by others that ST8Sia II polysialylates only transmembrane isoforms of the NCAM, such as NCAM-140 and NCAM-180, but not NCAM-120 and NCAM-125 anchored by a glycosylphosphotidylinositol. In the present study, we first discovered that ST8Sia II polysialylates all isoforms of the NCAM examined, and we demonstrated that polysialylation of NCAM expressed on 3T3 cells facilitates neurite outgrowth regardless of isoforms of NCAM, where polysialic acid is attached. We then show that neurite outgrowth is significantly facilitated only when polysialylated NCAM is present in cell membranes. Moreover, the soluble NCAM coated on plates did not have an effect on neurite outgrowth exerted by soluble L1 adhesion molecule coated on plates. These results, taken together, indicate that ST8Sia II plays critical roles in modulating the function of all major isoforms of NCAM. The results also support previous studies showing that a signal cascade initiated by NCAM differs from that initiated by L1 molecule.