Identification of a buried ß-strand as a novel disease-related motif in the human polysialyltransferases.

The polysialyltransferases ST8SIA2 and ST8SIA4 and their product, polysialic acid (polySia), are known to be related to cancers and mental disorders. ST8SIA2 and ST8SIA4 have conserved amino acid (AA) sequence motifs essential for the synthesis of the polySia structures on the neural cell adhesion molecule. To search for a new motif in the polysialyltransferases, we adopted the in silico Individual Meta Random Forest program that can predict disease-related AA substitutions. The Individual Meta Random Forest program predicted a new eight-amino-acids sequence motif consisting of highly pathogenic AA residues, thus designated as the pathogenic (P) motif. A series of alanine point mutation experiments in the pathogenic motif (P motif) showed that most P motif mutants lost the polysialylation activity without changing the proper enzyme expression levels or localization in the Golgi. In addition, we evaluated the enzyme stability of the P motif mutants using newly established calculations of mutation energy, demonstrating that the subtle change of the conformational energy regulates the activity. In the AlphaFold2 model, we found that the P motif was a buried ß-strand underneath the known surface motifs unique to ST8SIA2 and ST8SIA4. Taken together, the P motif is a novel buried ß-strand that regulates the full activity of polysialyltransferases from the inside of the molecule.

Comprehensive Analysis of Oligo/Polysialylglycoconjugates in Cancer Cell Lines.

In cancer cells, cell-surface sialylation is altered, including a change in oligo/polysialic acid (oligo/polySia) structures. Since they are unique and rarely expressed in normal cells, oligo/polySia structures may serve as promising novel biomarkers and targets for therapies. For the diagnosis and treatment of the disease, a precise understanding of the oligo/polySia structures in cancer cells is necessary. In this study, flow cytometric analysis and gene expression datasets were obtained from sixteen different cancer cell lines. These datasets demonstrated the ability to predict glycan structures and their sialylation status. Our results also revealed that sialylation patterns are unique to each cancer cell line. Thus, we can suggest promising combinations of antibody and cancer cell for glycan prediction. However, the precise prediction of minor glycans need to be further explored.

The α2,8-sialyltransferase 6 (St8sia6) localizes in the ER and enhances the anchorage-independent cell growth in cancer.

Sialylation, the final stage of post-translational modification of proteins, is achieved in the Golgi apparatus and is related to the malignant phenotype of cancer. Disialylation of ganglioside (GD3) by St8sia1 and polysialylation by St8sia2 and 4 have been shown to be related to malignant phenotypes; however, di/oligosialylation by St8sia6 is still unknown. In this study, we analyzed the malignant phenotype of St8sia6 and found that upregulation of St8sia6 in melanoma B16 cells increased anchorage-independent cell growth, which was not due to sialic acid cleavage by a sialidase. Moreover, unlike other sialyltransferases, St8sia6 localized to the endoplasmic reticulum (ER). We found that the localization to the Golgi apparatus could be regulated by swapping experiments using St8sia2; however, the malignant phenotype did not change. These data demonstrate that the enhancement of anchorage-independent cell growth by St8sia6 is not due to its localization of ER, but is due to the expression of the protein itself.

Analysis of biochemical features of ST8 α-N-acetyl-neuraminide α2,8-sialyltransferase (St8sia) 5 isoforms.

Gangliosides are important components of the membrane and are involved in many biological activities. St8sia5 is an α2,8-sialyltransferase involved in ganglioside synthesis, and has three isoforms. In this study, we analyzed the features of three isoforms, St8sia5-S, -M, and -L that had not been analyzed, and found that only St8sia5-L was localized in the Golgi, while the majority of St8sia5-M and -S were localized in the ER. The localization of Golgi of St8sia5 depended on the stem region. In addition, the incorporation of exogenous GD3 was upregulated only in St8sia5-L expressing cells. Taken together, the localization of St8sia5 is important for the activity of the enzyme.

Human-specific microglial Siglec-11 transcript variant has the potential to affect polysialic acid-mediated brain functions at a distance.

CD33-related Siglecs are often found on innate immune cells and modulate their reactivity by recognition of sialic acid-based "self-associated molecular patterns" and signaling via intracellular tyrosine-based cytosolic motifs. Previous studies have shown that Siglec-11 specifically binds to the brain-enriched polysialic acid (polySia/PSA) and that its microglial expression in the brain is unique to humans. Furthermore, human microglial Siglec-11 exists as an alternate splice form missing the exon encoding the last (fifth) Ig-like C2-set domain of the extracellular portion of the protein, but little is known about the functional consequences of this variation. Here, we report that the recombinant soluble human microglial form of Siglec-11 (hSiglec-11(4D)-Fc) binds endogenous and immobilized polySia better than the tissue macrophage form (hSiglec-11(5D)-Fc) or the chimpanzee form (cSiglec-11(5D)-Fc). The Siglec-11 protein is also prone to aggregation, potentially influencing its ligand-binding ability. Additionally, Siglec-11 protein can be secreted in both intact and proteolytically cleaved forms. The microglial splice variant has reduced proteolytic release and enhanced incorporation into exosomes, a process that appears to be regulated by palmitoylation of cysteines in the cytosolic tail. Taken together, these data demonstrate that human brain specific microglial hSiglec-11(4D) has different molecular properties and can be released on exosomes and/or as proteolytic products, with the potential to affect polySia-mediated brain functions at a distance.

Comparative Studies of Polysialic Acids Derived from Five Different Vertebrate Brains.

Polysialic acid (polySia/PSA) is a linear homopolymer of sialic acid (Sia) that primarily modifies the neural cell adhesion molecule (NCAM) in mammalian brains. PolySia-NCAM not only displays an anti-adhesive function due to the hydration effect, but also possesses a molecule-retaining function via a direct binding to neurologically active molecules. The quality and quantity of polySia determine the function of polySia-NCAM and are considered to be profoundly related to the maintenance of normal brain functions. In this study, to compare the structures of polySia-NCAM in brains of five different vertebrates (mammals, birds, reptiles, amphibians, and fish), we adopted newly developed combinational methods for the analyses. The results revealed that the structural features of polySia considerably varied among different species. Interestingly, mice, as a mammal, possess eminently distinct types of polySia, in both quality and quantity, compared with those possessed by other animals. Thus, the mouse polySia is of larger quantities, of longer and more diverse chain lengths, and of a larger molecular size with higher negative charge, compared with polySia of other species. These properties might enable more advanced brain function. Additionally, it is suggested that the polySia/Sia ratio, which likely reflects the complexity of brain function, can be used as a new promising index to evaluate the intelligence of different vertebrate brains.

Combinational Analyses with Multiple Methods Reveal the Existence of Several Forms of Polysialylated Neural Cell Adhesion Molecule in Mouse Developing Brains.

Polysialic acid (polySia/PSA) is an anionic glycan polymer of sialic acid, and it mostly modifies the neural cell adhesion molecule (NCAM) in mammalian brains. Quality and quantity of the polySia of the polySia-NCAM is spatio-temporally regulated in normal brain development and functions, and their impairments are reported to be related to diseases, such as psychiatric disorders and cancers. Therefore, precise understanding of the state of polySia-NCAM structure would lead to the diagnosis of diseases for which their suitable evaluation methods are necessary. In this study, to develop these evaluation methods, structures of polySia-NCAM from mouse brains at six different developmental stages were analyzed by several conventional and newly developed methods. Integrated results of these experiments clearly demonstrated the existence of different types of polySia-NCAMs in developing brains. In addition, combinational analyses were shown to be useful for precise understanding of the quantity and quality of polySia, which can provide criteria for the diagnosis of diseases.

Acute stress-induced change in polysialic acid levels mediated by sialidase in mouse brain.

Stress is an important environmental factor influencing human behaviour and causing several mental disorders. Alterations in the structure of polysialic acid (polySia/PSA) due to genetic alterations in ST8SIA2, which encodes a polySia-synthesizing enzyme, are related to certain mental disorders. However, whether stress as an environmental factor leads to changes in polySia structure is unknown. Here we studied the effects of acute stress on polySia expression and found reductions in both the quantity and quality of polySia in the olfactory bulb and prefrontal cortex, even with short-term exposure to acute stress. The use of inhibitors for sialidase, microglia and astrocytes revealed that these declines were due to a transient action of sialidase from microglia and astrocytes in the olfactory bulb and prefrontal cortex, respectively. These data suggest that sialidase dynamically regulates polySia expression in a brain region-specific manner.

Mental disorders and an acidic glycan-from the perspective of polysialic acid (PSA/polySia) and the synthesizing enzyme, ST8SIA2.

Mental disorders, such as schizophrenia, bipolar disorder, and autism spectrum disorder, are challenging to manage, worldwide. Understanding the molecular mechanisms underlying these disorders is essential and required. Studies investigating such molecular mechanisms are well performed and important findings are accumulating apace. Based on the fact that these disorders are due in part to the accumulation of genetic and environmental risk factors, consideration of multi-molecular and/or multi-system dependent phenomena might be important. Acidic glycans are an attractive family of molecules for understanding these disorders, because impairment of the fine-tuned glycan system affects a large number of molecules that are deeply involved in normal brain function. One of the candidates of this important family of glycan epitopes in the brain is polysialic acid (PSA/polySia). PSA is a well-known molecule because of its role as an oncodevelopmental antigen and is also widely used as a marker of adult neurogenesis. Recently, several reports have suggested that PSA and PSA-related genes are associated with multiple mental disorders. The relationships among PSA, PSA-related genes, and mental disorders are reviewed here.

Chlorpromazine Increases the Expression of Polysialic Acid (PolySia) in Human Neuroblastoma Cells and Mouse Prefrontal Cortex.

The neural cell adhesion molecule (NCAM) is modified by polysialic acid (polySia or PSA) in embryonic brains. In adult brains, polySia modification of NCAM is only observed in restricted areas where neural plasticity, remodeling of neural connections, or neural generation is ongoing although the amount of NCAM remains unchanged. Impairments of the polySia-expression and several single nucleotide polymorphisms (SNPs) of the polysialyltransferase (polyST) ST8SIA2 gene are reported to be associated with schizophrenia and bipolar disorder. Chlorpromazine (CPZ) is well-known as an agent for treating schizophrenia, and our hypothesis is that CPZ may affect the polySia expression or the gene expression of polySTs or NCAM. To test this hypothesis, we analyzed the effects of CPZ on the expression of polySia-NCAM on human neuroblastoma cell line, IMR-32 cells, by immunochemical and chemical methods. Interestingly, the cell surface expression of polySia, especially those with lower chain lengths, was significantly increased on the CPZ-treated cells, while mRNAs for polySTs and NCAM, and the amounts of total polySia-NCAM remained unchanged. The addition of brefeldin A, an inhibitor of endocytosis, suppressed the CPZ-induced cell surface polySia expression. In addition, polySia-NCAM was also observed in the vesicle compartment inside the cell. All these data suggest that the level of cell surface expression of polySia in IMR-32 is highly regulated and that CPZ changes the rate of the recycling of polySia-NCAM, leading to the up-regulation of polySia-NCAM on the cell surface. We also analyzed the effect of CPZ on polySia-expression in various brain regions in adult mice and found that CPZ only influenced the total amounts of polySia-NCAM in prefrontal cortex. These results suggest a brain-region-specific effect of CPZ on the expression of total polySia in mouse brain. Collectively, anti-schizophrenia agent CPZ consistently up-regulates the expression polySia at both cellular and animal levels.

Effects of intronic single nucleotide polymorphisms (iSNPs) of a polysialyltransferase, ST8SIA2 gene found in psychiatric disorders on its gene products.

Polysialic acid (polySia) is a linear homopolymer of sialic acid and mainly modifies neural cell adhesion molecule. PolySia plays important roles in synapse formation, learning and memory, social behavior and is associated with several diseases. Gene analyses of one of the biosynthetic enzymes for polySia, ST8SIA2, have revealed that several SNPs and genetic variations in the ST8SIA2 gene are associated with several psychiatric disorders; however, the mechanisms underlying these associations remain unknown. Here, we analyzed the effects of two iSNPs of ST8SIA2, rs2168351 and rs3784730, which are associated with bipolar disorder and autism spectrum disorder, respectively, on the expression of mRNA, ST8SIA2 and its final product, polySia in mouse neuroblastoma and human adenocarcinoma cell lines. We found that both iSNPs affected the expression of pre-mRNA and mRNA of ST8SIA2, and altered the cellular levels of ST8SIA2 and polySia. Taken together, these results indicate that impairment of the regulated expression of ST8SIA2 and the resulting downstream effects on gene products by these two iSNPs contribute to the development of these psychiatric disorders.

Relationship between ST8SIA2, polysialic acid and its binding molecules, and psychiatric disorders.

Polysialic acid (polySia, PSA) is a unique and functionally important glycan, particularly in vertebrate brains. It is involved in higher brain functions such as learning, memory, and social behaviors. Recently, an association between several genetic variations and single nucleotide polymorphisms (SNPs) of ST8SIA2/STX, one of two polysialyltransferase genes in vertebrates, and psychiatric disorders, such as schizophrenia (SZ), bipolar disorder (BD), and autism spectrum disorder (ASD), was reported based on candidate gene approaches and genome-wide studies among normal and mental disorder patients. It is of critical importance to determine if the reported mutations and SNPs in ST8SIA2 lead to impairments of the structure and function of polySia, which is the final product of ST8SIA2. To date, however, only a few such forward-directed studies have been conducted. In addition, the molecular mechanisms underlying polySia-involved brain functions remain unknown, although polySia was shown to have an anti-adhesive effect. In this report, we review the relationships between psychiatric disorders and polySia and/or ST8SIA2, and describe a new function of polySia as a regulator of neurologically active molecules, such as brain-derived neurotrophic factor (BDNF) and dopamine, which are deeply involved in psychiatric disorders. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc.

Rapid Trimming of Cell Surface Polysialic Acid (PolySia) by Exovesicular Sialidase Triggers Release of Preexisting Surface Neurotrophin.

As acidic glycocalyx on primary mouse microglial cells and a mouse microglial cell line Ra2, expression of polysialic acid (polySia/PSA), a polymer of the sialic acid Neu5Ac (N-acetylneuraminic acid), was demonstrated. PolySia is known to modulate cell adhesion, migration, and localization of neurotrophins mainly on neural cells. PolySia on Ra2 cells disappeared very rapidly after an inflammatory stimulus. Results of knockdown and inhibitor studies indicated that rapid surface clearance of polySia was achieved by secretion of endogenous sialidase Neu1 as an exovesicular component. Neu1-mediated polySia turnover was accompanied by the release of brain-derived neurotrophic factor normally retained by polySia molecules. Introduction of a single oxygen atom change into polySia by exogenous feeding of the non-neural sialic acid Neu5Gc (N-glycolylneuraminic acid) caused resistance to Neu1-induced polySia turnover and also inhibited the associated release of brain-derived neurotrophic factor. These results indicate the importance of rapid turnover of the polySia glycocalyx by exovesicular sialidases in neurotrophin regulation.