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

Abstract

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...

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

“…We found that when either the FN1 ␣-helix or QVQ sequence of NCAM was replaced with alanines, the polySTs continued to recognize the mutant proteins but added polysialic acid to O-glycans on FN1 rather than N-glycans on Ig5 (39,42), suggesting that these sequences play a role in positioning the polySTs for N-glycan polysialylation. The importance of the FN1 acidic patch varies in N-glycan and O-glycan polysialylation.…”

“…Molecular modeling and structural analysis of NCAM FN1 showed two novel features not seen in other fibronectin type III repeats, a surface acidic patch composed of Asp 506 , Asp 520 , Glu 521 , and Glu 523 and an ␣-helix linking strands 4 and 5 of the FN1 ␤ sandwich (39,40). Two other unique FN1 sequences, 510 PYS 512 and 516 QVQ 518 (QVQ), were also identified (42). The role of all four sequences in NCAM polysialylation was analyzed by mutagenesis and the creation of chimeric proteins.…”

“…The role of all four sequences in NCAM polysialylation was analyzed by mutagenesis and the creation of chimeric proteins. Although the PYS sequence was shown to be required for the maintenance of a conformation of the FN1 domain that enhanced the biosynthesis of sialylated O-glycans and promoted O-glycan polysialylation, the acidic surface patch, ␣-helix, and QVQ sequence were shown to play roles in polyST recognition and positioning (39,40,42).…”

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

“…Although the FN1 domain of OCAM could partially replace that of NCAM to support polyST recognition and polysialylation, and N-glycans were present in appropriate locations on the OCAM Ig5 domain, other sequences in the OCAM Ig5 domain served to block a stable interaction and OCAM polysialylation (28,29). In addition, the FN1 domain of OCAM had a weak polyST recognition site with only one of the three acidic patch residues found in the NCAM FN1 present; inserting the other two acidic residues substantially increased OCAM polysialylation when the blocking sequences in the OCAM Ig5 domain were removed (28).…”

“…At 80 -90% confluence, cells were transfected using 3 g of V5-or Fc-tagged NRP cDNA and ST8SiaIV-Myc cDNA (cloning of ST8SiaIV cDNA into the pcDNA3.1 Myc/HisB expression was described previously (29)) and 30 l of Lipofectin in 3 ml of Opti-MEM I and incubated at 37°C in a 5% CO 2 incubator, according to the manufacturer's protocol. After a 6-h incubation, 7 ml of DMEM, 10% FBS, 1% pencillin/streptomycin was added to each plate and incubated for an additional 18 -24 h.…”