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• W2086522105 abstract "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. In this work, we highlight the importance of an FN1 acidic patch in polyST recognition and also reveal that the polySTs are required to interact with sequences in the Ig5 domain for polysialylation to occur. We find that features of the Ig5 domain of the olfactory cell adhesion molecule (OCAM) are responsible for its lack of polysialylation. Specifically, two basic OCAM Ig5 residues (Lys and Arg) found near asparagines equivalent to those carrying the polysialylated N-glycans in NCAM substantially decrease or eliminate polysialylation when used to replace the smaller and more neutral residues (Ser and Asn) in analogous positions in NCAM Ig5. This decrease in polysialylation does not reflect altered glycosylation but instead is correlated with a decrease in polyST-NCAM binding. In addition, inserting non-conserved OCAM sequences into NCAM Ig5, including an “extra” N-glycosylation site, decreases or completely blocks NCAM polysialylation. Taken together, these results indicate that the polySTs not only recognize an acidic patch in the FN1 domain of NCAM but also must contact sequences in the Ig5 domain for polysialylation of Ig5 N-glycans to occur.Background: Recognition of the protein substrate is the first step in polysialylation of its glycans.Results: Residues in the OCAM Ig5 domain are non-permissive for its polysialylation.Conclusion: The polysialyltransferases interact with residues in both the Ig5 and FN1 domains of NCAM to allow its polysialylation.Significance: A two-domain polysialyltransferase recognition site may be required for all polysialylated proteins. 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. In this work, we highlight the importance of an FN1 acidic patch in polyST recognition and also reveal that the polySTs are required to interact with sequences in the Ig5 domain for polysialylation to occur. We find that features of the Ig5 domain of the olfactory cell adhesion molecule (OCAM) are responsible for its lack of polysialylation. Specifically, two basic OCAM Ig5 residues (Lys and Arg) found near asparagines equivalent to those carrying the polysialylated N-glycans in NCAM substantially decrease or eliminate polysialylation when used to replace the smaller and more neutral residues (Ser and Asn) in analogous positions in NCAM Ig5. This decrease in polysialylation does not reflect altered glycosylation but instead is correlated with a decrease in polyST-NCAM binding. In addition, inserting non-conserved OCAM sequences into NCAM Ig5, including an “extra” N-glycosylation site, decreases or completely blocks NCAM polysialylation. Taken together, these results indicate that the polySTs not only recognize an acidic patch in the FN1 domain of NCAM but also must contact sequences in the Ig5 domain for polysialylation of Ig5 N-glycans to occur. Background: Recognition of the protein substrate is the first step in polysialylation of its glycans. Results: Residues in the OCAM Ig5 domain are non-permissive for its polysialylation. Conclusion: The polysialyltransferases interact with residues in both the Ig5 and FN1 domains of NCAM to allow its polysialylation. Significance: A two-domain polysialyltransferase recognition site may be required for all polysialylated proteins. Proteins that co-translationally enter the endoplasmic reticulum and progress through the secretory pathway often become glycosylated on N- or O-glycans. Glycosylation allows proper protein folding and stability and can confer distinct functional properties to a protein (1.Varki A. Biological roles of oligosaccharides. All of the theories are correct.Glycobiology. 1993; 3: 97-130Crossref PubMed Scopus (4979) Google Scholar, 2.Haltiwanger R.S. Lowe J.B. Role of glycosylation in development.Annu. Rev. Biochem. 2004; 73: 491-537Crossref PubMed Scopus (645) Google Scholar). For example, the homophilic interactions of the neural cell adhesion molecule (NCAM) mediate cell-cell interactions, and the presence of long chains of polysialic acid (polySia) 3The abbreviations used are: polySiapolysialic acidpolySTpolysialyltransferaseFN1 and FN2first and second fibronectin type III repeat of NCAM, respectively. on NCAM disrupts these and other interactions to promote cell migration and signaling (3.Johnson C.P. Fujimoto I. Rutishauser U. Leckband D.E. Direct evidence that neural cell adhesion molecule (NCAM) polysialylation increases intermembrane repulsion and abrogates adhesion.J. Biol. Chem. 2005; 280: 137-145Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 4.Hildebrandt H. Mühlenhoff M. Weinhold B. Gerardy-Schahn R. Dissecting polysialic acid and NCAM functions in brain development.J. Neurochem. 2007; 103: 56-64Crossref PubMed Scopus (119) Google Scholar, 5.Rutishauser U. Polysialic acid in the plasticity of the developing and adult vertebrate nervous system.Nat. Rev. Neurosci. 2008; 9: 26-35Crossref PubMed Scopus (478) Google Scholar, 6.Rutishauser U. Polysialic acid and the regulation of cell interactions.Curr. Opin. Cell Biol. 1996; 8: 679-684Crossref PubMed Scopus (146) Google Scholar). polysialic acid polysialyltransferase first and second fibronectin type III repeat of NCAM, respectively. NCAM is heavily polysialylated during embryonic development and early post-natal growth (7.Nakayama J. Angata K. Ong E. Katsuyama T. Fukuda M. Polysialic acid, a unique glycan that is developmentally regulated by two polysialyltransferases, PST and STX, in the central nervous system. From biosynthesis to function.Pathol. Int. 1998; 48: 665-677Crossref PubMed Scopus (64) Google Scholar). Mouse knock-out studies demonstrated that polysialylation is absolutely required to down-regulate the adhesive properties of NCAM during nervous system development (8.Weinhold B. Seidenfaden R. Röckle I. Mühlenhoff M. Schertzinger F. Conzelmann S. Marth J.D. Gerardy-Schahn R. Hildebrandt H. Genetic ablation of polysialic acid causes severe neurodevelopmental defects rescued by deletion of the neural cell adhesion molecule.J. Biol. Chem. 2005; 280: 42971-42977Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). In adults, polysialylated NCAM is restricted to specific regions of the brain, such as the hippocampus and olfactory bulb, where it has roles in synaptic plasticity and general cell migration (5.Rutishauser U. Polysialic acid in the plasticity of the developing and adult vertebrate nervous system.Nat. Rev. Neurosci. 2008; 9: 26-35Crossref PubMed Scopus (478) Google Scholar). Importantly, re-expression of polysialylated NCAM is associated with the growth and invasiveness of several cancers, including neuroblastoma, small cell lung carcinoma, and Wilms tumor (9.Hildebrandt H. Becker C. Glüer S. Rösner H. Gerardy-Schahn R. Rahmann H. Polysialic acid on the neural cell adhesion molecule correlates with expression of polysialyltransferases and promotes neuroblastoma cell growth.Cancer Res. 1998; 58: 779-784PubMed Google Scholar, 10.Komminoth P. Roth J. Lackie P.M. Bitter-Suermann D. Heitz P.U. Polysialic acid of the neural cell adhesion molecule distinguishes small cell lung carcinoma from carcinoids.Am. J. Pathol. 1991; 139: 297-304PubMed Google Scholar, 11.Roth J. Brada D. Blaha I. Ghielmini C. Bitter-Suermann D. Komminoth P. Heitz P.U. Evaluation of polysialic acid in the diagnosis of Wilms' tumor. A comparative study on urinary tract tumors and non-neuroendocrine tumors.Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 1988; 56: 95-102Crossref PubMed Scopus (12) Google Scholar, 12.Roth J. Zuber C. Wagner P. Blaha I. Bitter-Suermann D. Heitz P.U. Presence of the long chain form of polysialic acid of the neural cell adhesion molecule in Wilms' tumor. Identification of a cell adhesion molecule as an oncodevelopmental antigen and implications for tumor histogenesis.Am. J. Pathol. 1988; 133: 227-240PubMed Google Scholar, 13.Scheidegger E.P. Lackie P.M. Papay J. Roth J. In vitro and in vivo growth of clonal sublines of human small cell lung carcinoma is modulated by polysialic acid of the neural cell adhesion molecule.Lab. Invest. 1994; 70: 95-106PubMed Google Scholar). PolySia is also up-regulated during neuronal regeneration, where it serves to promote the extension and repair of damaged neurons (reviewed in Ref. 5.Rutishauser U. Polysialic acid in the plasticity of the developing and adult vertebrate nervous system.Nat. Rev. Neurosci. 2008; 9: 26-35Crossref PubMed Scopus (478) Google Scholar). Conversely, decreased expression of polysialylated NCAM is associated with certain neuropsychiatric disorders, including schizophrenia (14.Barbeau D. Liang J.J. Robitalille Y. Quirion R. Srivastava L.K. Decreased expression of the embryonic form of the neural cell adhesion molecule in schizophrenic brains.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 2785-2789Crossref PubMed Scopus (285) Google Scholar). Studies by Sato, Kitajima, and colleagues (15.Isomura R. Kitajima K. Sato C. Structural and functional impairments of polysialic acid by a mutated polysialyltransferase found in schizophrenia.J. Biol. Chem. 2011; 286: 21535-21545Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 16.Kanato Y. Kitajima K. Sato C. Direct binding of polysialic acid to a brain-derived neurotrophic factor depends on the degree of polymerization.Glycobiology. 2008; 18: 1044-1053Crossref PubMed Scopus (111) Google Scholar, 17.Ono S. Hane M. Kitajima K. Sato C. Novel regulation of fibroblast growth factor 2 (FGF2)-mediated cell growth by polysialic acid.J. Biol. Chem. 2012; 287: 3710-3722Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) suggest why this might be the case. They were the first to demonstrate that polySia binds neurotrophic factors, neurotransmitters, and growth factors, and their work suggests that the presence of polySia can modulate signaling by these molecules. In addition, these investigators showed that a single point mutation in the polysialyltransferase ST8SiaII/STX in schizophrenic patients leads to a decrease in enzyme activity and the polymerization of shorter polySia chains on NCAM (15.Isomura R. Kitajima K. Sato C. Structural and functional impairments of polysialic acid by a mutated polysialyltransferase found in schizophrenia.J. Biol. Chem. 2011; 286: 21535-21545Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). This change in polySia chain length decreases its ability to bind brain-derived neurotrophic factor and dopamine, two factors whose action is impaired in psychiatric disorders. The polysialyltransferases (polySTs), ST8SiaII/STX and ST8SiaIV/PST, are responsible for the synthesis of polySia chains on the termini of N- or O-glycans on a very small number of mammalian proteins (7.Nakayama J. Angata K. Ong E. Katsuyama T. Fukuda M. Polysialic acid, a unique glycan that is developmentally regulated by two polysialyltransferases, PST and STX, in the central nervous system. From biosynthesis to function.Pathol. Int. 1998; 48: 665-677Crossref PubMed Scopus (64) Google Scholar, 18.Eckhardt M. Mühlenhoff M. Bethe A. Koopman J. Frosch M. Gerardy-Schahn R. Molecular characterization of eukaryotic polysialyltransferase-1.Nature. 1995; 373: 715-718Crossref PubMed Scopus (266) Google Scholar, 19.Kojima N. Yoshida Y. Tsuji S. A developmentally regulated member of the sialyltransferase family (ST8Sia II, STX) is a polysialic acid synthase.FEBS Lett. 1995; 373: 119-122Crossref PubMed Scopus (79) Google Scholar, 20.Scheidegger E.P. Sternberg L.R. Roth J. Lowe J.B. A human STX cDNA confers polysialic acid expression in mammalian cells.J. Biol. Chem. 1995; 270: 22685-22688Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The only polyST substrates identified to date are NCAM, CD36 receptor in human milk (21.Yabe U. Sato C. Matsuda T. Kitajima K. Polysialic acid in human milk. CD36 is a new member of mammalian polysialic acid-containing glycoprotein.J. Biol. Chem. 2003; 278: 13875-13880Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar), α-subunit of the voltage-dependent sodium channel (22.Zuber C. Lackie P.M. Catterall W.A. Roth J. Polysialic acid is associated with sodium channels and the neural cell adhesion molecule N-CAM in adult rat brain.J. Biol. Chem. 1992; 267: 9965-9971Abstract Full Text PDF PubMed Google Scholar), neuropilin-2 (NRP-2) (23.Curreli S. Arany Z. Gerardy-Schahn R. Mann D. Stamatos N.M. Polysialylated neuropilin-2 is expressed on the surface of human dendritic cells and modulates dendritic cell-T lymphocyte interactions.J. Biol. Chem. 2007; 282: 30346-30356Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar), SynCAM 1 (24.Galuska S.P. Rollenhagen M. Kaup M. Eggers K. Oltmann-Norden I. Schiff M. Hartmann M. Weinhold B. Hildebrandt H. Geyer R. Mühlenhoff M. Geyer H. Synaptic cell adhesion molecule SynCAM 1 is a target for polysialylation in postnatal mouse brain.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 10250-10255Crossref PubMed Scopus (133) Google Scholar), and the polySTs themselves (autopolysialylation) (25.Close B.E. Colley K.J. In vivo autopolysialylation and localization of the polysialyltransferases PST and STX.J. Biol. Chem. 1998; 273: 34586-34593Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26.Mühlenhoff M. Eckhardt M. Bethe A. Frosch M. Gerardy-Schahn R. Autocatalytic polysialylation of polysialyltransferase-1.EMBO J. 1996; 15: 6943-6950Crossref PubMed Scopus (96) Google Scholar). Overwhelming evidence from our laboratory indicates that polysialylation is a protein-specific glycosylation event in which the polySTs initially recognize protein determinants on their glycoprotein substrates prior to modifying substrate glycans (27.Close B.E. Mendiratta S.S. Geiger K.M. Broom L.J. Ho L.L. Colley K.J. The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II.J. Biol. Chem. 2003; 278: 30796-30805Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 28.Mendiratta S.S. Sekulic N. Hernandez-Guzman F.G. Close B.E. Lavie A. Colley K.J. A novel α-helix in the first fibronectin type III repeat of the neural cell adhesion molecule is critical for N-glycan polysialylation.J. Biol. Chem. 2006; 281: 36052-36059Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 29.Mendiratta S.S. Sekulic N. Lavie A. Colley K.J. 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.J. Biol. Chem. 2005; 280: 32340-32348Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 30.Thompson M.G. Foley D.A. Swartzentruber K.G. Colley K.J. 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.J. Biol. Chem. 2011; 286: 4525-4534Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). This protein specificity was first suggested by the observation that N-glycans attached to NCAM are far more efficiently polysialylated in vitro than free glycans enzymatically released from NCAM (31.Angata K. Suzuki M. McAuliffe J. Ding Y. Hindsgaul O. Fukuda M. Differential biosynthesis of polysialic acid on neural cell adhesion molecule (NCAM) and oligosaccharide acceptors by three distinct α2,8-sialyltransferases, ST8Sia IV (PST), ST8Sia II (STX), and ST8Sia III.J. Biol. Chem. 2000; 275: 18594-18601Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 32.Kojima N. Tachida Y. Yoshida Y. Tsuji S. Characterization of mouse ST8Sia II (STX) as a neural cell adhesion molecule-specific polysialic acid synthase. Requirement of core α1,6-linked fucose and a polypeptide chain for polysialylation.J. Biol. Chem. 1996; 271: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). NCAM is the major carrier of polySia. There are three main isoforms of NCAM, which arise from alternative splicing of a single gene. NCAM-140 and NCAM-180 are type I transmembrane proteins that differ in the length of their cytosolic tail, whereas NCAM-120 is linked to the membrane via a glycosylphosphatidylinositol anchor (33.Cunningham B.A. Hemperly J.J. Murray B.A. Prediger E.A. Brackenbury R. Edelman G.M. Neural cell adhesion molecule. Structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing.Science. 1987; 236: 799-806Crossref PubMed Scopus (872) Google Scholar). The three isoforms share a common extracellular structure consisting of five immunoglobulin (Ig) domains and two fibronectin type III repeats (FN1 and FN2). The sites of polysialylation are found on the fifth and sixth N-glycosylation sites, located on Ig5 (ASN5 and ASN6) (34.Nelson R.W. Bates P.A. Rutishauser U. Protein determinants for specific polysialylation of the neural cell adhesion molecule.J. Biol. Chem. 1995; 270: 17171-17179Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). We have previously demonstrated that FN1 is required for the polysialylation of ASN5 and ASN6 (27.Close B.E. Mendiratta S.S. Geiger K.M. Broom L.J. Ho L.L. Colley K.J. The minimal structural domains required for neural cell adhesion molecule polysialylation by PST/ST8Sia IV and STX/ST8Sia II.J. Biol. Chem. 2003; 278: 30796-30805Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 29.Mendiratta S.S. Sekulic N. Lavie A. Colley K.J. 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.J. Biol. Chem. 2005; 280: 32340-32348Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). In fact, deletion of the FN1 domain prevents both polyST-NCAM interaction and NCAM polysialylation (30.Thompson M.G. Foley D.A. Swartzentruber K.G. Colley K.J. 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.J. Biol. Chem. 2011; 286: 4525-4534Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Furthermore, we have identified specific residues in NCAM FN1, including a key surface acidic patch, that have roles in polyST recognition and/or positioning and binding (28.Mendiratta S.S. Sekulic N. Hernandez-Guzman F.G. Close B.E. Lavie A. Colley K.J. A novel α-helix in the first fibronectin type III repeat of the neural cell adhesion molecule is critical for N-glycan polysialylation.J. Biol. Chem. 2006; 281: 36052-36059Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 29.Mendiratta S.S. Sekulic N. Lavie A. Colley K.J. 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.J. Biol. Chem. 2005; 280: 32340-32348Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 30.Thompson M.G. Foley D.A. Swartzentruber K.G. Colley K.J. 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.J. Biol. Chem. 2011; 286: 4525-4534Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 35.Foley D.A. Swartzentruber K.G. Thompson M.G. Mendiratta S.S. Colley K.J. Sequences from the first fibronectin type III repeat of the neural cell adhesion molecule allow O-glycan polysialylation of an adhesion molecule chimera.J. Biol. Chem. 2010; 285: 35056-35067Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The olfactory cell adhesion molecule, OCAM, is mainly expressed in neural tissue such as the brain, olfactory epithelium, and the retina (36.Paoloni-Giacobino A. Chen H. Antonarakis S.E. Cloning of a novel human neural cell adhesion molecule gene (NCAM2) that maps to chromosome region 21q21 and is potentially involved in Down syndrome.Genomics. 1997; 43: 43-51Crossref PubMed Scopus (59) Google Scholar, 37.Yoshihara Y. Kawasaki M. Tamada A. Fujita H. Hayashi H. Kagamiyama H. Mori K. OCAM. A new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons.J. Neurosci. 1997; 17: 5830-5842Crossref PubMed Google Scholar). Like NCAM, OCAM has been shown to form homophilic trans interactions (37.Yoshihara Y. Kawasaki M. Tamada A. Fujita H. Hayashi H. Kagamiyama H. Mori K. OCAM. A new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons.J. Neurosci. 1997; 17: 5830-5842Crossref PubMed Google Scholar). Mouse knock-out studies have suggested a role for OCAM in intraglomerular compartmental organization (38.Walz A. Mombaerts P. Greer C.A. Treloar H.B. Disrupted compartmental organization of axons and dendrites within olfactory glomeruli of mice deficient in the olfactory cell adhesion molecule, OCAM.Mol. Cell. Neurosci. 2006; 32: 1-14Crossref PubMed Scopus (42) Google Scholar). Recently, it has been demonstrated that OCAM is highly expressed in androgen-dependent prostrate cancer cell lines and estrogen-dependent breast cancer cell lines, whereas it is expressed only at low levels in normal prostrate cell lines (39.Takahashi S. Kato K. Nakamura K. Nakano R. Kubota K. Hamada H. Neural cell adhesion molecule 2 as a target molecule for prostate and breast cancer gene therapy.Cancer Sci. 2011; 102: 808-814Crossref PubMed Scopus (16) Google Scholar). There are two isoforms of OCAM, a transmembrane and glycosylphosphatidylinositol-anchored form, and they both share the same extracellular domain as NCAM, with five Ig domains and two fibronectin type III repeats (37.Yoshihara Y. Kawasaki M. Tamada A. Fujita H. Hayashi H. Kagamiyama H. Mori K. OCAM. A new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons.J. Neurosci. 1997; 17: 5830-5842Crossref PubMed Google Scholar, 40.Alenius M. Bohm S. Identification of a novel neural cell adhesion molecule-related gene with a potential role in selective axonal projection.J. Biol. Chem. 1997; 272: 26083-26086Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Interestingly, OCAM Ig5 has consensus N-glycosylation sites at the same positions as ASN5 and ASN6 of NCAM (ASN6 and ASN7 in OCAM). Although the expression pattern of OCAM can overlap with that of NCAM, OCAM is not polysialylated (37.Yoshihara Y. Kawasaki M. Tamada A. Fujita H. Hayashi H. Kagamiyama H. Mori K. OCAM. A new member of the neural cell adhesion molecule family related to zone-to-zone projection of olfactory and vomeronasal axons.J. Neurosci. 1997; 17: 5830-5842Crossref PubMed Google Scholar). Recently, we generated a chimeric protein in which the FN1 domain of NCAM was replaced with that of OCAM (35.Foley D.A. Swartzentruber K.G. Thompson M.G. Mendiratta S.S. Colley K.J. Sequences from the first fibronectin type III repeat of the neural cell adhesion molecule allow O-glycan polysialylation of an adhesion molecule chimera.J. Biol. Chem. 2010; 285: 35056-35067Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). We anticipated that this chimera would not be polysialylated and that we would be able to reconstitute polysialylation by inserting specific NCAM FN1 residues. Surprisingly, ST8SiaIV/PST could polysialylate the NCAM-OCAM chimera, indicating that OCAM FN1 is recognized by the polySTs, enabling them to polysialylate glycans located on NCAM Ig5. The polysialylation of the chimera was not as efficient as NCAM polysialylation, suggesting that although ST8SiaIV/PST can engage OCAM FN1, recognition/binding is not optimal (35.Foley D.A. Swartzentruber K.G. Thompson M.G. Mendiratta S.S. Colley K.J. Sequences from the first fibronectin type III repeat of the neural cell adhesion molecule allow O-glycan polysialylation of an adhesion molecule chimera.J. Biol. Chem. 2010; 285: 35056-35067Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). These results suggested that other factors either within FN1 itself or adjacent domains of NCAM could have a role in stabilizing polyST recognition and binding. In this study, we performed domain swap experiments and compared the sequences and structures of NCAM and OCAM Ig5 domains to evaluate the factors preventing OCAM polysialylation and, conversely, the requirements for NCAM polysialylation. We found that replacing the OCAM Ig5 domain with NCAM Ig5 allows polysialylation of an OCAM-NCAM chimera, suggesting that aspects of OCAM Ig5 were preventing its polysialylation. To this end, we found that an “extra” N-glycan located within OCAM Ig5 did prevent the polysialylation of NCAM when present, but it is not the only factor preventing OCAM polysialylation. We also found that two large basic residues located near OCAM ASN6 and ASN7, which are replaced by smaller more neutral residues in NCAM Ig5, function to block polysialylation when inserted into NCAM Ig5. The presence of these large basic residues in NCAM does not alter the glycosylation pattern of Ig5 but instead serves to decrease polyST-substrate binding. These results suggest that the polySTs make contact with residues in both the FN1 and Ig5 domains in the process of Ig5 N-glycan polysialylation. Tissue culture materials, including Dulbecco's modified Eagle's medium (DMEM), Opti-MEM I, Lipofectin, Lipofectamine 2000, and fetal bovine serum (FBS) were purchased from Invitrogen. Oligonucleotides, restriction enzymes, PCR supermix, and anti-V5 epitope tag antibody were also obtained from Invitrogen. The cDNA for human NCAM-140 was a gift from Dr. Nancy Kedersha (Brigham and Women's Hospital, Boston, MA). The cDNA for mouse OCAM was kindly provided by Dr Yoshihiro Yoshihara (RIKEN Brain Science Institute, Wako, Saitama, Japan). The cDNA for human ST8SiaIV/PST was obtained from Dr. Minoru Fukuda (Burnham Institute, La Jolla, CA). The QuikChangeTM site-directed mutagenesis kit and Pfu DNA polymerase were purchased from Stratagene. DNA purification kits were purchased from Qiagen. Protein A-Sepharose was purchased from GE Healthcare. T4 DNA ligase was obtained from New England Biolabs. Precision Plus ProteinTM standard was purchased from Bio-Rad. Nitrocellulose membranes were purchased from Schleicher & Schuell. Horseradish peroxidase (HRP)-conjugated and fluorescein isothiocyanate (FITC)-conjugated secondary antibodies were obtained from Jackson Laboratories. Supersignal West Pico chemiluminescence reagent was obtained from Pierce. Other chemicals and reagents were purchased from Sigma and Fisher. The full-length mouse OCAM sequence was PCR-amplified using PCR supermix and the following primers: 5′-AAGCTTGTCCTGAACATGAGCCTCCTCC-3′ and 5′-TCTAGATGCCTTTATGTCATCTTCTTTAGACTGG-3′. These primers specifically introduced a HindIII and XbaI site at the 5′- and 3′-ends of the amplified OCAM sequence, respectively. The OCAM PCR product and empty pcDNA3.1 V5/HisB expression vector were digested with HindIII and XbaI. After gel purification, the OCAM PCR product was ligated into the expression vector. A frameshift mutation, introduced during cloning near the XbaI site, was corrected by mutagenesis using the following primers: 5′-GATGACATAAAGGCAGGTCTAGAGGGCCCGC-3′ and 5′-GCGGGCCCTCTAGACCTGCCTTTATGTCATC-3′. To generate the chimeric proteins, BamHI and XbaI restriction sites, flanking the Ig5, FN1, or Ig5-FN1 domains, were inserted into the full-length OCAM or NCAM cDNAs by site-directed mutagenesis, and the domains were subsequently removed by restriction enzyme digestion. The OCAM FN1 domain, NCAM Ig5 domain, NCAM FN1 domain, or NCAM Ig5-FN1 domain was PCR-amplified using the following primers that inserted BamHI and XbaI sites at the 5′- and 3′-ends of the cDNAs, respectively: 5′-GGATCCGATGTCCCCTCTAGTCCCCATG-3′/5′-TCTAGAGGCTCACGGACTGGCAGTGTC-3′, 5′-GGATCCTATGCCCCAAAGCTACAGGGC-3′/5′-TCTAGAGCTGCTTGAACAAGGATGAATTC-3′, 5′-GGATCCGACACCCCCTCTTCACCATCC-3′/5′-TCTAGAGCTTCCCCTTGGACTGGCTGCGTC-3′, and 5′-GGATCCTATGCCCCAAAGCTACAGGGC-3′/5′-TCTAGAGCTTCCCCTTGGACTGGCTGCGTC-3′. The PCR products were cut with BamHI and XbaI and ligated in frame into NCAM lacking FN1, OCAM lacking Ig5, OCAM lacking FN1, and OCAM lacking Ig5-FN1, to generate NCAM-OCAM FN1, OCAM-NCAM Ig5, OCAM-NCAM FN1, and OCAM-NCAM Ig5-FN1, respectively. The BamHI and XbaI restriction sites flanking the Ig5 or FN1 domains were removed from all chimeras by site-directed mutagenesis. Mutagenesis reactions were performed using the Stratagene QuikChangeTM site-directed mutagenesis kit according to the manufacturer's protocol. The primers used are listed in supplemental Table 1. Mutations were confirmed by DNA sequencing performed by the DNA Sequencing Facility of the Research Resources Center at the University of Illinois (Chicago, IL). COS-1 cells maintained in DMEM, 10% FBS were plated on 12-mm glass coverslips in 24-well plates and grown at 37 °C in 5% CO2. Cells in each well were transfected using 3 μl of Lipofectin and 0.5 μg of NCAM, OCAM, or chimeric" @default.
• W2086522105 created "2016-06-24" @default.
• W2086522105 creator A5058855517 @default.
• W2086522105 creator A5062580593 @default.
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• W2086522105 date "2013-03-01" @default.
• W2086522105 modified "2023-10-18" @default.
• W2086522105 title "The Polysialyltransferases Interact with Sequences in Two Domains of the Neural Cell Adhesion Molecule to Allow Its Polysialylation" @default.
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• W2086522105 doi "https://doi.org/10.1074/jbc.m112.438374" @default.
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