Polysialic Acid Biosynthesis Research Articles

Terminal-Enhanced Polymerization in the Biosynthesis of Polysialic Acid

Plasmids are commonly used tools in microbiology and molecular biology and have important and wide-ranging applications in the study of gene function, protein expression, and compound synthesis. The complex relationship between necessary antibiotic addition, compatibility between multiple plasmids, and the growth burden of host bacteria has plagued the wider use of compatibility plasmids. In this study, we constructed the terminal polymerization pathway of PSA by exogenously expressing the neuA, neuD, and neuS genes after the knockdown of Eschesrichia coli BL21 (DE3). Duet series vectors were utilized to regulate the expression level of neuA, neuD, and neuS genes to study the gene expression level, plasmid copy number growth burden, pressure of antibiotic addition, stability of compatible plasmids, and the level of expression stability of exogenous genes, as well as the effect on the biological reaction process. The results showed that the three genes, neuA, neuD, and neuS, were enhanced in the recombinant strain E. coli NA-05, with low copy, medium copy, and high copy, respectively. The effect of PSA synthesis under standard antibiotic pressure was remarkable. The results of this thesis suggest the use of a Duet series of compatible expression vectors to achieve the stable existence and co-expression of multiple genes in recombinant bacteria, which is a good reason for further research.

Attenuation of Polysialic Acid Biosynthesis in Cells by the Small Molecule Inhibitor 8-Keto-sialic acid

Sialic acids are key mediators of cell function, particularly with regard to cellular interactions with the surrounding environment. Reagents that modulate the display of specific sialyl glycoforms at the cell surface would be useful biochemical tools and potentially allow for therapeutic intervention in numerous challenging chronic diseases. While multiple strategies are being explored for the control of cell surface sialosides, none that shows high selectivity between sialyltransferases or that targets a specific sialyl glycoform has yet to emerge. Here, we describe a strategy to block the formation of α2,8-linked sialic acid chains (oligo- and polysialic acid) through the use of 8-keto-sialic acid as a chain-terminating metabolic inhibitor that, if incorporated, cannot be elongated. 8-Keto-sialic acid is nontoxic at effective concentrations and serves to block polysialic acid synthesis in cancer cell lines and primary immune cells, with minimal effects on other sialyl glycoforms.

Polysialic acid biosynthesis and production in Escherichia coli: current state and perspectives.

Polysialic acid (PSA) is a unique polysaccharide that plays critical roles in many bioprocesses, which makes it useful in a wide range of biomedical applications. The increased demand for PSA has led to considerable efforts to improve its production using bacteria, such as Escherichia coli. Bioprocess optimization and metabolic engineering have allowed the efficient production of PSA. This review aims to summarize the metabolism of PSA with an emphasis on the importance of the key pathway components. In addition, this review provides an update on state of the art PSA production using E. coli with a special emphasis on strategies of strain engineering and process development for the enhanced production of PSA.

Function of KpsS, KpsC, and NeuE in Escherichia coli K1 Polysialic Acid Biosynthesis

Pathogenic Escherichia coli are often encapsulated with acidic polysaccharides such as polysialic acid. We have shown that four genes, kpsS, kpsC, neuE and neuS, in the E. coli K1 gene cluster are essential for initiation and elongation in E. coli K1 polysialic acid biosynthesis. NeuS is a polysialyltransferase. A previous report demonstrated that homologues of kpsC and kpsS gene products in N. meningitidis are cytidine 5′‐monophosphate‐2‐keto‐3‐deoxyoctulosonate (CMP‐KDO) transferases. These enzymes are involved in the biosynthesis of the putative glycolipid terminus of terminus of polysialic acid. Our objective in this report is to determine the function of KpsC, KpsS, and NeuE of E. coli in initiation of polysialic acid synthesis. We have successfully expressed in a soluble form and purified KpsC, KpsS, and NeuE of E. coli K1 to near homogeneity. This is the first time these full length membrane proteins have been expressed in a soluble form. We used HPLC and TLC methods to demonstrate that E. coli K1 KpsS and KpsC can transfer KDO to a fluorescence glycolipid acceptor substrate. The fluorescent products of KpsS and KpsC were isolated and used to investigate the function of the NeuE in polysialic acid biosynthesis.

Environmental enrichment rescues memory in mice deficient for the polysialytransferase ST8SiaIV.

The neural cell adhesion molecule NCAM and its association with the polysialic acid (PSA) are believed to contribute to brain structural plasticity that underlies memory formation. Indeed, the attachment of long chains of PSA to the glycoprotein NCAM down-regulates its adhesive properties by altering cell-cell interactions. In the brain, the biosynthesis of PSA is catalyzed by two polysialyltransferases, which are differentially regulated during lifespan. One of them, ST8SiaIV (PST), is predominantly expressed during adulthood whereas the other one, ST8SiaII (STX), dominates during embryonic and post-natal development. To understand the role played by ST8SiaIV during learning and memory and its underlying hippocampal plasticity, we used knockout mice deleted for the enzyme ST8SiaIV (PST-ko mice). At adult age, PST-ko mice show a drastic reduction of PSA-NCAM expression in the hippocampus and intact hippocampal adult neurogenesis. We found that these mice display impaired long-term but not short-term memory in both, spatial and non-spatial behavioral tasks. Remarkably, memory deficits of PST-ko mice were abolished by exposure to environmental enrichment that was also associated with an increased number of PSA-NCAM expressing new neurons in the dentate gyrus of these mice. Whether the presence of a larger pool of immature, likely plastic, new neurons favored the rescue of long-term memory in PST-ko mice remains to be determined. Our findings add new evidence to the role played by PSA in memory consolidation. They also suggest that PSA synthesized by PST critically controls the tempo of new neurons maturation in the adult hippocampus.

Sialic Acids in the Brain: Gangliosides and Polysialic Acid in Nervous System Development, Stability, Disease, and Regeneration

Every cell in nature carries a rich surface coat of glycans, its glycocalyx, which constitutes the cell's interface with its environment. In eukaryotes, the glycocalyx is composed of glycolipids, glycoproteins, and proteoglycans, the compositions of which vary among different tissues and cell types. Many of the linear and branched glycans on cell surface glycoproteins and glycolipids of vertebrates are terminated with sialic acids, nine-carbon sugars with a carboxylic acid, a glycerol side-chain, and an N-acyl group that, along with their display at the outmost end of cell surface glycans, provide for varied molecular interactions. Among their functions, sialic acids regulate cell-cell interactions, modulate the activities of their glycoprotein and glycolipid scaffolds as well as other cell surface molecules, and are receptors for pathogens and toxins. In the brain, two families of sialoglycans are of particular interest: gangliosides and polysialic acid. Gangliosides, sialylated glycosphingolipids, are the most abundant sialoglycans of nerve cells. Mouse genetic studies and human disorders of ganglioside metabolism implicate gangliosides in axon-myelin interactions, axon stability, axon regeneration, and the modulation of nerve cell excitability. Polysialic acid is a unique homopolymer that reaches >90 sialic acid residues attached to select glycoproteins, especially the neural cell adhesion molecule in the brain. Molecular, cellular, and genetic studies implicate polysialic acid in the control of cell-cell and cell-matrix interactions, intermolecular interactions at cell surfaces, and interactions with other molecules in the cellular environment. Polysialic acid is essential for appropriate brain development, and polymorphisms in the human genes responsible for polysialic acid biosynthesis are associated with psychiatric disorders including schizophrenia, autism, and bipolar disorder. Polysialic acid also appears to play a role in adult brain plasticity, including regeneration. Together, vertebrate brain sialoglycans are key regulatory components that contribute to proper development, maintenance, and health of the nervous system.

Polysialic and colanic acids metabolism in Escherichia coli K92 is regulated by RcsA and RcsB

We have shown previously that Escherichia coli K92 produces two different capsular polymers known as CA (colanic acid) and PA (polysialic acid) in a thermoregulated manner. The complex Rcs phosphorelay is largely related to the regulation of CA synthesis. Through deletion of rscA and rscB genes, we show that the Rcs system is involved in the regulation of both CA and PA synthesis in E. coli K92. Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C. Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant. Under this condition, expression of the nan operon responsible for PA catabolism was greatly reduced. Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression. Finally, our results revealed that RcsB controlled the expression of several genes (dsrA, rfaH, h-ns and slyA) involved in the thermoregulation of CA and PA synthesis, indicating that RcsB is part of a complex regulatory mechanism governing the surface appearance in E. coli.

Abstract 4410: Inhibition of cell surface polysialic acid biosynthesis modulates tumor cell migration.

Abstract Polysialic acid (polySia) is a linear alpha-2,8-linked carbohydrate homopolymer of up to 100 sialic acid residues. It is characteristically re-expressed on the surface of NCAM (neuronal cell adhesion molecule) in many cancer cells where it modulates tumour dissemination. PolySia-NCAM expression is strongly associated with poor clinical prognosis and correlates with aggressive/invasive disease in small cell lung cancer, pancreatic cancer, neuroblastoma and many other tumours principally of neural crest origin [1]. SiRNA knockdown of polysialyltransferases (polySTs) ST8SiaII and ST8SiaIV, the enzymes responsible for polysialylation of neural cell adhesion molecule (NCAM), has been shown to abolish cell migration in tumour cells [2]. Besides brain regions with persistent neuronal plasticity, polySia is essentially absent from the adult body. Its exclusive synthesis by two polySTs, ST8SiaIV and particularly ST8SiaII, present a novel, selective, highly attractive but largely unexplored therapeutic opportunity [1]. We have embarked on a programme of design and synthesis of novel polyST inhibitors, and have established a screening cascade to determine enzyme inhibition and effects on polySia-expressing cells in vitro. Using endoneuraminidase, an enzyme that specifically cleaves polySia chains from NCAM, we can assess the rate and extent to which polySia growth is inhibited in the presence of agent in cellular systems. In addition, an in vitro scratch assay was established to evaluate modulation of cell migration using a transfected isogenic cell line system (C6-STX: polySia +, ST8SiaII + / C6-WT: polySia -, ST8SiaII - [3]) and neuroblastoma cell lines known to express polySia-NCAM, ST8SiaIV and ST8SiaII (SHSY-5Y and IMR-32). We have identified compounds, including ICT-3067, ICT-3128 and ICT-3147, which lead to a significant reduction in cell surface polySia-NCAM and that modulate tumour cell migration in in vitro. We have also identified a number of key modifications to CMP-sialic acid precursor molecules which dramatically decrease cell migration in ST8SiaII-expressing cells, through modulation of polySia assembly. It is also noteworthy that the potency of these compounds has been increased with simple chemical modifications, resulting in greater lipophilicity. In summary, a number of efficient modulators of polySia assembly and their efficacy in reducing cell migration is described. This points to the potential of the polySia biosynthesis pathway, and in particular the polySTs, as attractive therapeutic targets in metastatic tumours. [1] Falconer, R.A. et al., Curr. Cancer Drug Targets, 2012, 12, 925-939; [2] Schreiber et al., Gastroenterology, 2008, 134, 1555-1566; [3] Suzuki, M. et al., Glycobiology, 2005, 15, 887-894. Citation Format: Bradley R. Springett, Yousef M.J. Al-Saraireh, Virginie Viprey, Mark Sutherland, Melanie Begouin, Matthew Northrop, Rida Saeed, Paul M. Loadman, Laurence H. Patterson, Steven D. Shnyder, Robert A. Falconer. Inhibition of cell surface polysialic acid biosynthesis modulates tumor cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4410. doi:10.1158/1538-7445.AM2013-4410

The Polysialyltransferases Interact with Sequences in Two Domains of the Neural Cell Adhesion Molecule to Allow Its Polysialylation

The neural cell adhesion molecule (NCAM) is the major substrate for the polysialyltransferases (polySTs), ST8SiaII/STX and ST8SiaIV/PST. The polysialylation of NCAM N-glycans decreases cell adhesion and alters signaling. Previous work demonstrated that the first fibronectin type III repeat (FN1) of NCAM is required for polyST recognition and the polysialylation of the N-glycans on the adjacent Ig5 domain. In this work, we highlight the importance of an FN1 acidic patch in polyST recognition and also reveal that the polySTs are required to interact with sequences in the Ig5 domain for polysialylation to occur. We find that features of the Ig5 domain of the olfactory cell adhesion molecule (OCAM) are responsible for its lack of polysialylation. Specifically, two basic OCAM Ig5 residues (Lys and Arg) found near asparagines equivalent to those carrying the polysialylated N-glycans in NCAM substantially decrease or eliminate polysialylation when used to replace the smaller and more neutral residues (Ser and Asn) in analogous positions in NCAM Ig5. This decrease in polysialylation does not reflect altered glycosylation but instead is correlated with a decrease in polyST-NCAM binding. In addition, inserting non-conserved OCAM sequences into NCAM Ig5, including an "extra" N-glycosylation site, decreases or completely blocks NCAM polysialylation. Taken together, these results indicate that the polySTs not only recognize an acidic patch in the FN1 domain of NCAM but also must contact sequences in the Ig5 domain for polysialylation of Ig5 N-glycans to occur.

Polysialic Acid: Versatile Modification of NCAM, SynCAM 1 and Neuropilin-2

The glycan polysialic acid is well-known as a unique posttranslational modification of the neural cell adhesion molecule NCAM. Despite remarkable acceptor specificity, however, a few other proteins can be targets of polysialylation. Here, we recapitulate the biosynthesis of polysialic acid by the two polysialyltransferases ST8SIA2 and ST8SIA4 and highlight the increasing evidence that variation in the human ST8SIA2 gene is linked to schizophrenia and possibly other neuropsychiatric disorders. Moreover, we summarize the knowledge on the role of NCAM polysialylation in brain development gained by the analysis of NCAM- and polysialyltransferase-deficient mouse models. The last part of this review is focused on recent advances in identifying SynCAM 1 and neuropilin-2 as novel acceptors of polysialic acid in NG2 cells of the perinatal brain and in dendritic cells of the immune system, respectively.

Impact of structural aberrancy of polysialic acid and its synthetic enzyme ST8SIA2 in schizophrenia

Psychiatric disorders are a group of human diseases that impair higher cognitive functions. Whole-genomic analyses have recently identified susceptibility genes for several psychiatric disorders, including schizophrenia. Among the genes reported to be involved in psychiatric disorders, a gene encoding a polysialyltransferase involved in the biosynthesis of polysialic acid (polySia or PSA) on cell surfaces has attracted attention for its potential role in emotion, learning, memory, circadian rhythm, and behaviors. PolySia is a unique polymer that spatio-temporally modifies neural cell adhesion molecule (NCAM) and is predominantly found in embryonic brains, although it persists in areas of the adult brain where neural plasticity, remodeling of neural connections, or neural generation is ongoing, such as the hippocampus, subventricular zone (SVZ), thalamus, prefrontal cortex, and amygdala. PolySia is thought to be involved in the regulation of cell-cell interactions; however, recent evidence suggests that it is also involved in the functional regulation of ion channels and neurologically active molecules, such as Brain-derived neurotrophic factor (BDNF), FGF2, and dopamine (DA) that are deeply involved in psychiatric disorders. In this review, the possible involvement of polysialyltransferase (ST8SIA2/ST8SiaII/STX/Siat8B) and its enzymatic product, polySia, in schizophrenia is discussed.

204 Modulation of Tumour Cell Migration by Inhibition of Polysialic Acid Biosynthesis

204 Modulation of Tumour Cell Migration by Inhibition of Polysialic Acid Biosynthesis

The Biosynthesis of Polysialic Acid: A Developmentally Regulated, Anti‐Adhesive Glycan

Polysialic acid is a unique glycan that modulates cell adhesion and signaling, and is critical for brain development, neuronal regeneration, and cancer invasiveness. Polysialic acid is found on a limited number of glycoproteins suggesting that polysialylation is a protein‐specific modification that requires an initial polysialyltransferase (polyST)‐substrate protein‐protein interaction. Previously we found that acidic residues on the surface of the first fibronectin type III repeat of NCAM are required for the polysialylation of the N‐glycans on its adjacent immunoglobulin domain. Using competition and polysialylation assays we have identified a polyST polybasic region that is involved in substrate recognition. Truncated, catalytically inactive ST8Sia IV (PST) proteins containing this region are effective inhibitors of NCAM polysialylation. Replacing Arg82 and Arg93 in region substantially decreases NCAM polysialylation by PST without equally compromising enzyme activity, and also eliminates the ability of a catalytically inactive PST mutant (H331K) to block NCAM polysialylation. In contrast, replacing only Arg82 eliminates the ability of PST to polysialylate SynCAM 1 and neuropilin‐2. These results suggest that the polySTs may use distinct but overlapping basic sites to recognize different substrates. Supported by NIH RO1 GM063843 (KJC) and NIH F31 GM096739 (JLZ).

Functional and Structural Characterization of Polysaccharide Co-polymerase Proteins Required for Polymer Export in ATP-binding Cassette Transporter-dependent Capsule Biosynthesis Pathways

Neisseria meningitidis serogroup B and Escherichia coli K1 bacteria produce a capsular polysaccharide (CPS) that is composed of α2,8-linked polysialic acid (PSA). Biosynthesis of PSA in these bacteria occurs via an ABC (ATP-binding cassette) transporter-dependent pathway. In N. meningitidis, export of PSA to the surface of the bacterium requires two proteins that form an ABC transporter (CtrC and CtrD) and two additional proteins, CtrA and CtrB, that are proposed to form a cell envelope-spanning export complex. CtrA is a member of the outer membrane polysaccharide export (OPX) family of proteins, which are proposed to form a pore to mediate export of CPSs across the outer membrane. CtrB is an inner membrane protein belonging to the polysaccharide co-polymerase (PCP) family. PCP proteins involved in other bacterial polysaccharide assembly systems form structures that extend into the periplasm from the inner membrane. There is currently no structural information available for PCP or OPX proteins involved in an ABC transporter-dependent CPS biosynthesis pathway to support their proposed roles in polysaccharide export. Here, we report cryo-EM images of purified CtrB reconstituted into lipid bilayers. These images contained molecular top and side views of CtrB and showed that it formed a conical oligomer that extended ∼125 Å from the membrane. This structure is consistent with CtrB functioning as a component of an envelope-spanning complex. Cross-complementation of CtrA and CtrB in E. coli mutants with defects in genes encoding the corresponding PCP and OPX proteins show that PCP-OPX pairs require interactions with their cognate partners to export polysaccharide. These experiments add further support for the model of an ABC transporter-PCP-OPX multiprotein complex that functions to export CPS across the cell envelope.

Temperature has reciprocal effects on colanic acid and polysialic acid biosynthesis in E. coli K92

Escherichia coli K92 is an opportunistic pathogen bacterium able to produce polysialic acid (PA) capsules when grows at 37 degrees C. PA polysaccharides are cell-associated homopolymers tailored from acid sialic monomers that function as virulence factors in different neuroinvasive diseases caused by certain Enterobacteriaceae. Conversely, when grows at 19 degrees C (restrictive conditions), PA synthesis was negligible, whereas in such condition, a slimy substance started to be accumulated in the culture broths. Analysis by uronic acids colorimetric determinations, gas chromatography-mass spectrometry, and Fourier transform infrared spectroscopy allowed the isolation and identification of mucoid substance as colanic acid (CA). CA is a heteropolymer containing glucose, galactose, fucose, and glucuronic acid as monomers which seems to be involved in the protection of this bacterium against environment assaults. The study of physicochemical conditions required for CA synthesis revealed that in E. coli K92, nutrient (carbon and nitrogen sources) modulates CA production, reaching the maximal values when glucose and proline were as carbon and nitrogen sources, respectively. Furthermore, we have found that E. coli K92 is able to produce CA at all temperatures tested (from 42 degrees C to 15 degrees C), whereas PA synthesis only occurred when bacteria were cultured at temperatures higher than 25 degrees C. Additionally, genetic engineering approaches revealed that the CA cluster including several genes required for synthesis was placed into a DNA fragment of 100 kb using polymerase chain reaction methodology.

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.

Polysialic acid bioengineering of neuronal cells by N-acyl sialic acid precursor treatment

The inherent promiscuity of the polysialic acid (PSA) biosynthetic pathway has been exploited by the use of exogenous unnatural sialic acid precursor molecules to introduce unnatural modifications into cellular PSA, and has found applications in nervous system development and tumor vaccine studies. The sialic acid precursor molecules N-propionyl- and N-butanoyl-mannosamine (ManPr, ManBu) have been variably reported to affect PSA biosynthesis ranging from complete inhibition to de novo production of modified PSA, thus illustrating the need for further investigation into their effects. In this study, we have used a monoclonal antibody (mAb) 13D9, specific to both N-propionyl-PSA and N-butanoyl-PSA (NPrPSA and NBuPSA), together with flow cytometry, to study precursor-treated tumor cells and NT2 neurons at different stages of their maturation. We report that both ManPr and ManBu sialic acid precursors are metabolized and the resultant unnatural sialic acids are incorporated into de novo surface sialylglycoconjugates in murine and human tumor cells and, for the first time, in human NT2 neurons. Furthermore, neither precursor treatment deleteriously affected endogenous PSA expression; however, with NT2 cells, PSA levels were naturally downregulated as a function of their maturation into polarized neurons independent of sialic acid precursor treatment.

Structural characterization of sialic acid synthase by electrospray mass spectrometry—a tetrameric enzyme composed of dimeric dimers

Sialic acid synthase (NeuB) encoded by the neuB gene catalyzes the condensation of N-acetylmannosamine and phospho(enol)pyruvate to form N-acetylneuraminic acid. The enzyme is essential for the biosynthesis of polysialic acid, a capsular sugar polymer functioning as a virulent factor and antiphagocytic barrier. This report demonstrates the first characterization on the quaternary structure of NeuB from Escherichia coli (EcNeuB) and Streptococcus agalactiae (SaNeuB) by nanoflow electrospray ionization mass spectrometry (ESI-MS). Under non-denaturing conditions, Tris buffer was observed to induce a higher ratio of tetramer/dimer of NeuB in the ESI mass spectra, providing supportive evidence for the existence of a "structurally-specific" tetramer. The instrument parameters were found to significantly affect the ratio of detected tetramer/dimer in ESI mass spectra. The harshest conditions, including high desolvation voltages and pressure in the collision cell, led to enhanced detection of the 160 kDa tetramer. The prevalence of dimeric form is likely the cause in loss of tetramer stability in gas-phase arising from insufficient collisional cooling, which implies an asymmetric assembly, possibly composed of dimeric dimers. Most interestingly, the hypothesis was further supported by chemical cross-linking of SaNeuB, in which the reaction of shorter linker yielded mainly the dimer whereas that of longer linker produced both dimer and tetramer. Furthermore, the ESI-MS analysis can reflect dramatic change of pH-dependent quaternary structure in association with enzyme activity, suggesting the tetrameric form may be the primary species responsible for the enzyme catalysis.

Production of polysialic acid from fed-batch fermentation with pH control

During batch fermentation pH affects the production of biomass and polysialic acid in Escherichia coli K235-WXJ4. When the pH of the medium was maintained at 6.4 during the stationary phase, polysialic acid production was prolonged and the specific rate of polysialic acid biosynthesis was higher than when the fermentation process was not controlled for pH. Controlling the pH during fed-batch fermentation of polysialic acid increased the yield of dry cell weight and polysialic acid, which reached 10.16 and 2606 g/l, respectively.

Molecular Defects That Cause Loss of Polysialic Acid in the Complementation Group 2A10

Polysialic acid (PSA) is a dynamically regulated posttranslational modification of the neural cell adhesion molecule (NCAM), which modulates NCAM binding functions. PSA biosynthesis is catalyzed by two polysialyltransferases, ST8SiaII and ST8SiaIV. The catalytic mechanisms of these enzymes are unknown. In Chinese hamster ovary cells, ST8SiaIV is responsible for PSA expression. In the complementation group 2A10, the ST8SiaIV gene is disrupted. Investigating the molecular defects in this complementation group, seven clones with missense mutations in ST8SiaIV were found. Mutations cause replacement of amino acids that are highly conserved in alpha2,8-sialyltransferases. To verify the physiological relevance of identified mutations, identical amino acid substitutions were introduced into epitope-tagged variants of hamster ST8SiaIV and murine ST8SiaII and recombinant proteins were tested in vivo and in vitro. None of these constructs reconstituted PSA synthesis in 2A10 cells, although the proteins were expressed and with the exception of the cysteine variants ST8SiaIV-C356F and ST8SiaII-C371F correctly targeted to the Golgi apparatus. Interestingly, two mutations (ST8SiaIV-R277G and -M333V and the corresponding mutants ST8SiaII-R292G and -M348V) could be partially rescued if tested in vitro. Although these mutants were negative for autopolysialylation, partial reconstitution of both auto- and NCAM polysialylation was achieved in the presence of NCAM. The data presented in this study suggest a functional link between auto- and NCAM polysialylation.