Dynamic Glycoprotein Hyposialylation Promotes Chemotherapy Evasion and Metastatic Seeding of Quiescent Circulating Tumor Cell Clusters in Breast Cancer.

Unlabelled: Most circulating tumor cells (CTC) are detected as single cells, whereas a small proportion of CTCs in multicellular clusters with stemness properties possess 20- to 100-times higher metastatic propensity than the single cells. Here we report that CTC dynamics in both singles and clusters in response to therapies predict overall survival for breast cancer. Chemotherapy-evasive CTC clusters are relatively quiescent with a specific loss of ST6GAL1-catalyzed α2,6-sialylation in glycoproteins.

Noncatalytic Endosialidase Enables Surface Capture of Small-Cell Lung Cancer Cells Utilizing Strong Dendrimer-Mediated Enzyme-Glycoprotein Interactions.

Enumeration of circulating tumor cells (CTCs) of small-cell lung cancer (SCLC) patients has been shown to predict the disease progress and long-term survival. Most CTC detection methods rely on epithelial surface markers, such as epithelial cell adhesion molecule (EpCAM). However, this marker in SCLC is reported to be often downregulated after a variety of phenotypic changes, which impairs the reliability of EpCAM-based CTC detections.

Autopolysialylation of polysialyltransferases is required for polysialylation and polysialic acid chain elongation on select glycoprotein substrates.

Polysialic acid (polySia) is a large glycan polymer that is added to some glycoproteins by two polysialyltransferases (polySTs), ST8Sia-II and ST8Sia-IV. As polySia modulates cell adhesion and signaling, immune cell function, and tumor metastasis, it is of interest to determine how the polySTs recognize their select substrates. We have recently identified residues within the ST8Sia-IV polybasic region (PBR) that are required for neural cell adhesion molecule (NCAM) recognition and subsequent polysialylation.

The Polybasic Region of the Polysialyltransferase ST8Sia-IV Binds Directly to the Neural Cell Adhesion Molecule, NCAM.

Polysialic acid (polySia) is a unique post-translational modification found on a small set of mammalian glycoproteins. Composed of long chains of α2,8-linked sialic acid, this large, negatively charged polymer attenuates protein and cell adhesion and modulates signaling mediated by its carriers and proteins that interact with these carriers. PolySia is crucial for the proper development of the nervous system and is upregulated during tissue regeneration and in highly invasive cancers.

Sialylation of N-glycans: mechanism, cellular compartmentalization and function.

Sialylated N-glycans play essential roles in the immune system, pathogen recognition and cancer. This review approaches the sialylation of N-glycans from three perspectives. The first section focuses on the sialyltransferases that add sialic acid to N-glycans.

Sequence Requirements for Neuropilin-2 Recognition by ST8SiaIV and Polysialylation of Its O-Glycans.

Polysialic acid is an oncofetal glycopolymer, added to the glycans of a small group of substrates, that controls cell adhesion and signaling. One of these substrates, neuropilin-2, is a VEGF and semaphorin co-receptor that is polysialylated on its O-glycans in mature dendritic cells and macrophages by the polysialyltransferase ST8SiaIV. To understand the biochemical basis of neuropilin-2 polysialylation, we created a series of domain swap chimeras with sequences from neuropilin-1, a protein for which polysialylation had not been previously reported.

Polysialic acid: biosynthesis, novel functions and applications.

As an anti-adhesive, a reservoir for key biological molecules, and a modulator of signaling, polysialic acid (polySia) is critical for nervous system development and maintenance, promotes cancer metastasis, tissue regeneration and repair, and is implicated in psychiatric diseases. In this review, we focus on the biosynthesis and functions of mammalian polySia, and the use of polySia in therapeutic applications. PolySia modifies a small subset of mammalian glycoproteins, with the neural cell adhesion molecule, NCAM, serving as its major carrier.

Glycobiology: drifting toward polymer perfection.

The addition of polysialic acid to proteins and cells is emerging as a promising therapeutic strategy. Polysialyltransferases synthesize polymers of widely varying lengths not optimal for therapeutic reagents, but the development of enzyme variants using neutral genetic drift offers a new way to overcome this problem.

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.

Sequences prior to conserved catalytic motifs of polysialyltransferase ST8Sia IV are required for substrate recognition.

Polysialic acid on the neural cell adhesion molecule (NCAM) modulates cell-cell adhesion and signaling, is required for proper brain development, and plays roles in neuronal regeneration and the growth and invasiveness of tumor cells. Evidence indicates that NCAM polysialylation is highly protein-specific, requiring an initial polysialyltransferase-NCAM protein-protein interaction. Previous work suggested that a polybasic region located prior to the conserved polysialyltransferase catalytic motifs may be involved in NCAM recognition, but not overall enzyme activity (Foley, D.

Sequences at the interface of the fifth immunoglobulin domain and first fibronectin type III repeat of the neural cell adhesion molecule are critical for its polysialylation.

Polysialic acid is an anti-adhesive glycan that modifies a select group of mammalian proteins. The primary substrate of the polysialyltransferases (polySTs) is the neural cell adhesion molecule (NCAM). Polysialic acid negatively regulates cell adhesion, is required for proper brain development, and is expressed in specific areas of the adult brain where it promotes on-going cell migration and synaptic plasticity.

Sequences from the first fibronectin type III repeat of the neural cell adhesion molecule allow O-glycan polysialylation of an adhesion molecule chimera.

Polysialic acid is a developmentally regulated, anti-adhesive polymer that is added to N-glycans on the fifth immunoglobulin domain (Ig5) of the neural cell adhesion molecule (NCAM). We found that the first fibronectin type III repeat (FN1) of NCAM is required for the polysialylation of N-glycans on the adjacent Ig5 domain, and we proposed that the polysialyltransferases recognize specific sequences in FN1 to position themselves for Ig5 N-glycan polysialylation. Other studies identified a novel FN1 acidic surface patch and α-helix that play roles in NCAM polysialylation.

Structure and mutagenesis of neural cell adhesion molecule domains: evidence for flexibility in the placement of polysialic acid attachment sites.

The addition of alpha2,8-polysialic acid to the N-glycans of the neural cell adhesion molecule, NCAM, is critical for brain development and plays roles in synaptic plasticity, learning and memory, neuronal regeneration, and the growth and invasiveness of cancer cells. Our previous work indicates that the polysialylation of two N-glycans located on the fifth immunoglobulin domain (Ig5) of NCAM requires the presence of specific sequences in the adjacent fibronectin type III repeat (FN1). To understand the relationship of these two domains, we have solved the crystal structure of the NCAM Ig5-FN1 tandem.

Identification of sequences in the polysialyltransferases ST8Sia II and ST8Sia IV that are required for the protein-specific polysialylation of the neural cell adhesion molecule, NCAM.

The polysialyltransferases ST8Sia II and ST8Sia IV polysialylate the glycans of a small subset of mammalian proteins. Their most abundant substrate is the neural cell adhesion molecule (NCAM). An acidic surface patch and a novel alpha-helix in the first fibronectin type III repeat of NCAM are required for the polysialylation of N-glycans on the adjacent immunoglobulin domain.

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.

A novel alpha-helix in the first fibronectin type III repeat of the neural cell adhesion molecule is critical for N-glycan polysialylation.

Polysialic acid is a developmentally regulated, anti-adhesive glycan that is added to the neural cell adhesion molecule, NCAM. Polysialylated NCAM is critical for brain development and plays roles in synaptic plasticity, axon guidance, and cell migration. The first fibronectin type III repeat of NCAM, FN1, is necessary for the polysialylation of N-glycans on the adjacent immunoglobulin domain.

The CMP-sialic acid transporter is localized in the medial-trans Golgi and possesses two specific endoplasmic reticulum export motifs in its carboxyl-terminal cytoplasmic tail.

The addition of sialic acid to glycoproteins and glycolipids requires Golgi sialyltransferases to have access to their glycoconjugate substrates and nucleotide sugar donor, CMP-sialic acid. CMP-sialic acid is transported into the lumen of the Golgi complex through the CMP-sialic acid transporter, an antiporter that also functions to transport CMP into the cytosol. We localized the transporter using immunofluorescence and deconvolution microscopy to test the prediction that it is broadly distributed across the Golgi stack to serve the many sialyltransferases involved in glycoconjugate sialylation.

Specific amino acids in the first fibronectin type III repeat of the neural cell adhesion molecule play a role in its recognition and polysialylation by the polysialyltransferase ST8Sia IV/PST.

Polysialic acid is an anti-adhesive protein modification that promotes cell migration and the plasticity of cell interactions. Because so few proteins carry polysialic acid, we hypothesized that polysialylation is a protein-specific event and that a specific polysialyltransferase-substrate interaction is the basis of this specificity. The major substrate for the polysialyltransferases is the neural cell adhesion molecule, NCAM.

Multiple signals are required for alpha2,6-sialyltransferase (ST6Gal I) oligomerization and Golgi localization.

A single amino acid difference in the catalytic domain of two isoforms of the alpha2,6-sialyltransferase (ST6Gal I) leads to differences in their trafficking, processing, and oligomerization. The STtyr isoform is transiently localized in the Golgi and is ultimately cleaved and secreted, whereas the STcys isoform is stably localized in the Golgi and is not cleaved and secreted. The stable localization of STcys is correlated with its enhanced ability to oligomerize.

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.

The two rat alpha 2,6-sialyltransferase (ST6Gal I) isoforms: evaluation of catalytic activity and intra-Golgi localization.

alpha2,6-Sialyltransferase (ST6Gal I) functions in the Golgi to terminally sialylate the N-linked oligosaccharides of glycoproteins. Interestingly, rat ST6Gal I is expressed as two isoforms, STtyr and STcys, that differ by a single amino acid in their catalytic domains. In this article, our goal was to evaluate more carefully possible differences in the catalytic activity and intra-Golgi localization of the two isoforms that had been suggested by earlier work.