Molecular Mechanism of Inhibition of Polysialyltransferase Domain (PSTD) by Heparin

: Polysialic acid (polySia) is a unique carbohydrate polymer produced on the neuronal cell adhesion molecule (NCAM) in many cancer cells. It strongly correlates with the migration and invasion of tumor cells and aggressive, metastatic disease, and poor clinical prognosis in the clinic. Its synthesis is catalyzed by two polysialyltransferases (polySTs), ST8SiaIV (PST) and ST8SiaII (STX). Therefore, selective inhibition of polySTs presents a therapeutic opportunity to inhibit tumor invasion and metastasis due to NCAM polysialylation. It has been proposed that NCAM polysialylation could be inhibited by two types of heparin inhibitors, low molecular heparin (LMWH) and heparin tetrasaccharide (DP4). This review summarizes the interactions between Polysialyltransferase Domain (PSTD) in ST8SiaIV and CMP-Sia, and between the PSTD and polySia; and how LMWH and DP4 inhibit these interactions. Our NMR studies indicate that LMWH is a more effective inhibitor than DP4 for inhibition of NCAM polysialylation. The NMR identification of heparin-binding sites in the PSTD may provide insight into the design of specific inhibitors of polysialylation.

New Partners Identified by Mass Spectrometry Assay Reveal Functions of NCAM2 in Neural Cytoskeleton Organization

Neuronal cell adhesion molecule 2 (NCAM2) is a membrane protein with an important role in the morphological development of neurons. In the cortex and the hippocampus, NCAM2 is essential for proper neuronal differentiation, dendritic and axonal outgrowth and synapse formation. However, little is known about NCAM2 functional mechanisms and its interactive partners during brain development. Here we used mass spectrometry to study the molecular interactome of NCAM2 in the second postnatal week of the mouse cerebral cortex. We found that NCAM2 interacts with >100 proteins involved in numerous processes, including neuronal morphogenesis and synaptogenesis. We validated the most relevant interactors, including Neurofilaments (NEFs), Microtubule-associated protein 2 (MAP2), Calcium/calmodulin kinase II alpha (CaMKIIα), Actin and Nogo. An in silico analysis of the cytosolic tail of the NCAM2.1 isoform revealed specific phosphorylation site motifs with a putative affinity for some of these interactors. Our results expand the knowledge of NCAM2 interactome and confirm the key role of NCAM2 in cytoskeleton organization, neuronal morphogenesis and synaptogenesis. These findings are of interest in explaining the phenotypes observed in different pathologies with alterations in the NCAM2 gene.

Ezrin interacts with the tumor suppressor CHL1 and promotes neuronal differentiation of human neuroblastoma

In a previous study, we demonstrated that CHL1, the neuronal cell adhesion molecule close homolog of L1, acts as a tumor suppressor in human neuroblastoma (NB), a still highly lethal childhood malignancy, influencing its differentiation and proliferation degree. Here we found that ezrin, one of the ERM (ezrin, radixin, moesin) proteins involved in cytoskeleton organization, strongly interacts with CHL1. The low expression of EZRIN, as well as the low expression of CHL1 and of the neuronal differentiation marker MAP2, correlates with poor outcome in NB patients. Knock-down of ezrin in HTLA-230 cell line induces neurite retraction, enhances cell proliferation and migration, and triggers anchorage-independent growth, with effects very similar to those already obtained by CHL1 silencing. Furthermore, lack of ezrin inhibits the expression of MAP2 and of the oncosuppressor molecule p53, whereas it enhances MAPK activation, all typical features of tumor aggressiveness. As already described, CHL1 overexpression in IMR-32 cell line provokes an opposite trend, but the co-silencing of ezrin reduces these effects, confirming the hypothesis that CHL1 acts in close connection with ezrin. Overall, our data show that ezrin reinforces the differentiating and oncosuppressive functions of CHL1, identifying this ERM protein as a new targetable molecule for NB therapy.

Investigation of anti-IgLON5-induced neurodegenerative changes in human neurons

Anti-IgLON5 disease is a progressive neurological disorder, associated with autoantibodies against a neuronal cell adhesion molecule, IgLON5. In human post-mortem brain tissue neurodegeneration and accumulation of phosphorylated-Tau is found. Whether IgLON5 antibodies induce neurodegeneration or neurodegeneration provokes an immune response remains to be elucidated. To clarify this, we exposed human stem cell derived neurons to patient anti-IgLON5 antibodies.Human neural stem cells were differentiated for 14-48 days, and exposed from day 9-14, or day 13-48 to either 1) IgG from a patient with confirmed anti-IgLON5 antibodies; 2) IgG from a healthy person or left untreated. Electrical neuronal activity was quantified using a multi electrode array. Cultures were immunostained for β-tubulin III, phosphorylated-Tau and counterstained with DAPI. Other cultures were immunostained for synaptic proteins PSD95 and synaptophysin. Lactate dehydrogenase release and nuclei morphology analyses were performed to assess cell viability.Spike rates and quantity of synaptic clusters were significantly reduced in anti-IgLON5 exposed cultures, and the proportion of cells with degenerative appearance was significantly higher for the anti-IgLON5 IgG group. Furthermore, the content of cells displaying accumulation of phosphorylated-Tau was higher for anti-IgLON5 exposed cultures. After five weeks, cultures exposed to anti-IgLON5 IgG contained significantly more dying cells.In conclusion, exposure of human neurons to pathological anti-IgLON5 antibodies cause functional impairment, synaptic and neurodegenerative changes, ultimately leading to cell death. This supports autoantibodies as a causative factor responsible for the neurodegenerative changes seen in patients with anti-IgLON5 disease and mimics pathological observations.