The Minimal Structural Domains Required for Neural Cell Adhesion Molecule Polysialylation by PST/ST8Sia IV and STX/ST8Sia II

Close, Brett E.; Mendiratta, Shalu; Geiger, Kristin M.; Broom, Lucy J.; Ho, Li Lun; Colley, Karen J.

doi:10.1074/jbc.m305390200

Cited by 53 publications

(68 citation statements)

References 73 publications

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“…The Ig5FN1 module of NCAM is a well-characterized target for polysialylation (27). Thus, this module was used as a reporter for in planta polysialylation.…”

Section: Human Polysialyltransferases Are Functional When Transientlymentioning

confidence: 99%

Engineering of complex protein sialylation in plants

Kallolimath

Castilho

Strasser

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Sialic acids (Sias) are abundant terminal modifications of protein-linked glycans. A unique feature of Sia, compared with other monosaccharides, is the formation of linear homo-polymers, with its most complex form polysialic acid (polySia). Sia and polySia mediate diverse biological functions and have great potential for therapeutic use. However, technological hurdles in producing defined protein sialylation due to the enormous structural diversity render their precise investigation a challenge. Here, we describe a plant-based expression platform that enables the controlled in vivo synthesis of sialylated structures with different interlinkages and degree of polymerization (DP). The approach relies on a combination of stably transformed plants with transient expression modules. By the introduction of multigene vectors carrying the human sialylation pathway into glycosylation-destructed mutants, transgenic plants that sialylate glycoproteins in α2,6- or α2,3-linkage were generated. Moreover, by the transient coexpression of human α2,8-polysialyltransferases, polySia structures with a DP >40 were synthesized in these plants. Importantly, plant-derived polySia are functionally active, as demonstrated by a cell-based cytotoxicity assay and inhibition of microglia activation. This pathway engineering approach enables experimental investigations of defined sialylation and facilitates a rational design of glycan structures with optimized biotechnological functions.

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“…The Ig5FN1 module of NCAM is a well-characterized target for polysialylation (27). Thus, this module was used as a reporter for in planta polysialylation.…”

Section: Human Polysialyltransferases Are Functional When Transientlymentioning

confidence: 99%

Engineering of complex protein sialylation in plants

Kallolimath

Castilho

Strasser

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…1), is polysialylated exclusively on O-glycans (38). We also showed that replacing the acidic patch with either alanine or arginine residues substantially decreases or eliminates NCAM7 polysialylation, although replacing the ␣-helix has no effect (40).…”

Section: Fn1 Qvq Sequence Plays a Role In Polyst Positioning For Ig5 mentioning

confidence: 73%

“…It was first observed on salmonid egg polysialoglycoprotein, PSGP (49), and later found on neuropilin-2, CD36, unidentified proteins in the RBL-1 rat basophilic leukemia cell line, and several NCAM mutants, such as those lacking the FN1 ␣-helix or QVQ sequences, and the truncated NCAM protein NCAM7 (29,30,38,40,50). In addition, we frequently observe a small amount of O-glycan polysialylation in full-length NCAM (see Fig.…”

Section: Discussionmentioning

confidence: 94%

“…The majority of polysialic acid is added to N-glycans on Asn 449 (Asn5) and Asn 478 (Asn6), the fifth and sixth N-glycosylation sites located on Ig5 (37). We demonstrated that a truncated NCAM140 protein consisting of Ig5-FN1-TM-tail is fully polysialylated when co-expressed with a polyST, whereas an Ig5-TM-tail protein remains unpolysialylated (38). In addition, deletion of the FN1 domain from fulllength NCAM eliminates Ig5 N-glycan polysialylation (39).…”

Section: The Neural Cell Adhesion Molecule (Ncam)mentioning

confidence: 96%

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Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

Foley

Swartzentruber

Thompson

et al. 2010

Journal of Biological Chemistry

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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 ␣-helix that play roles in NCAM polysialylation. Here, we characterize the contribution of two additional FN1 sequences, Pro The neural cell adhesion molecule (NCAM)2 is a member of the immunoglobulin superfamily. It is expressed on the cell surface where it engages in homophilic and heterophilic interactions, resulting in cell adhesion and modulation of signal transduction cascades (reviewed in Refs. 1, 2). In the developing embryo and neonate, NCAM is modified by the addition of long chains of ␣2,8-polysialic acid (3, 4). The presence of this highly hydrated and negatively charged carbohydrate polymer disrupts overall cell adhesion and allows cell migration (5-7). Polysialylation is catalyzed by the polysialyltransferases (polySTs), . NCAM polysialylation is critical during embryogenesis and early postnatal development. Mice that are null for both polySTs are born at Mendelian ratios but have severe neuronal defects, fail to thrive, and the majority die within 4 weeks of birth (12). Simultaneous deletion of NCAM and the polySTs rescues the lethal phenotype, indicating that polysialic acid is required to down-regulate the adhesive properties of NCAM during development (12). In addition, using varying allelic combinations of ST8SiaIV, ST8SiaII, and NCAM, Hildebrandt et al. (13) demonstrated that an aberrant increase in the level of unpolysialylated NCAM caused defects in brain connectivity in postnatal day 1 mice.NCAM is mainly unpolysialylated in the adult brain (3, 4). However, polysialylated NCAM does persist in specific regions of the central nervous system, including the olfactory bulb and hippocampus, where it functions in cell migration, synaptic plasticity, and repair (14 -17). Highly polysialylated NCAM is also re-expressed in several cancers, including neuroblastoma, glioma, small cell and non-small cell lung tumors, and Wilms' tumor, where it has been suggested to promote cancer invasiveness (18 -23). Recently, polysialylated NCAM has been shown to enhance metastasis of a murine model of lung cancer (24) and has been implicated in colorectal carcinoma progression (25). In addition, polysialic acid has been demonstrated to be involved in hematopoietic development (26,27). For example, ST8SiaIV Ϫ/Ϫ mice have reduced thymocyte numbers due to fewer progenitor cells mobilizing to the thymus (27).Although NCAM is by far the most abundant and well documented polysialylated protein, it is striking that only six other polysialylated glycoproteins have been identified. These are the ␣ subu...

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“…Using NCAM as a model substrate, we showed that the first fibronectin type III repeat (FN1) is required for the polysialylation of N-glycans in the adjacent immunoglobulin domain (Ig5) (24). A three-residue acidic patch on the surface of the FN1 domain plays a primary role in NCAM recognition, binding, and polysialylation by ST8SiaIV (25,27).…”

Section: Polysialic Acid (Polysia)mentioning

confidence: 99%

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

Bhide

Fernandes

Colley

2016

Journal of Biological Chemistry

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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. To our surprise, we found that membrane-associated neuropilin-1 is polysialylated at ϳ50% of the level of neuropilin-2 but not polysialylated when it lacks its cytoplasmic tail and transmembrane region and is secreted from the cell. This was not the case for neuropilin-2, which is polysialylated when either membraneassociated or soluble. Evaluation of the soluble chimeric proteins demonstrated that the meprin A5 antigen-tyrosine phosphatase (MAM) domain and the O-glycan-containing linker region of neuropilin-2 are necessary and sufficient for its polysialylation and serve as better recognition and acceptor sites in the polysialylation process than those regions of neuropilin-1. In addition, specific acidic residues on the surface of the MAM domain are critical for neuropilin-2 polysialylation. Based on these data and pull-down experiments, we propose a model where ST8SiaIV recognizes and docks on an acidic surface of the neuropilin-2 MAM domain to polysialylate O-glycans on the adjacent linker region. These results together with those related to neural cell adhesion molecule polysialylation establish a paradigm for the process of protein-specific polysialylation. Polysialic acid (polySia)2 is a glycopolymer consisting of 8 to Ͼ100 ␣2,8-linked sialic acid residues (1). It is synthesized on the N-linked or O-linked glycans of a very specific set of cell surface proteins by two Golgi-localized polysialyltransferases (polySTs), ST8SiaII and ST8SiaIV (2, 3). Polysialylated proteins include the neural cell adhesion molecule (NCAM) (4), neuropilin-2 (NRP-2) (5), synaptic cell adhesion molecule 1 (6), the CD36 scavenger receptor in human milk (7), the ␣ subunit of the voltage-dependent sodium channel (8), and the polySTs themselves (9).PolySia has been shown to be crucially important for the development of the nervous system, synaptic plasticity and cell migration in the adult nervous system, and the regeneration of damaged nerves and tissues, and it is also up-regulated in many types of late stage cancers, where it is suggested to promote cancer metastasis (reviewed in Refs. 2, 3, and 10). Notably, work by Tanaka et al. (11) demonstrates that a substantial proportion of late stage non-small cell lung cancers express polySia, but not NCAM, suggesting that other substrates are polysialylated in these cancers. These functions of polySia are due to its abilities to serve as an anti-adhesive, a reservoir for biologically significant ligands, and a signaling modulator (reviewed ...

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The Minimal Structural Domains Required for Neural Cell Adhesion Molecule Polysialylation by PST/ST8Sia IV and STX/ST8Sia II

Cited by 53 publications

References 73 publications

Engineering of complex protein sialylation in plants

Engineering of complex protein sialylation in plants

Sequences from the First Fibronectin Type III Repeat of the Neural Cell Adhesion Molecule Allow O-Glycan Polysialylation of an Adhesion Molecule Chimera

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

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