Karen J. Colley

Hyposialylation of Integrins Stimulates the Activity of Myeloid Fibronectin Receptors

Despite numerous reports suggesting that β1 integrin receptors undergo differential glycosylation, the potential role of N-linked carbohydrates in modulating integrin function has been largely ignored. In the present study, we find that β1 integrins are differentially glycosylated during phorbol ester (PMA)-stimulated differentiation of myeloid cells along the monocyte/macrophage lineage. PMA treatment of two myeloid cell lines, U937 and THP-1, induces a down-regulation in expression of the ST6Gal I sialyltransferase. Correspondingly, the β1 integrin subunit becomes hyposialylated, suggesting that the β1 integrin is a substrate for this enzyme. The expression of hyposialylated β1 integrin isoforms is temporally correlated with enhanced binding of myeloid cells to fibronectin, and, importantly, fibronectin binding is inhibited when the Golgi disrupter, brefeldin A, is used to block the expression of the hyposialylated form. Consistent with the observation that cells with hyposialylated integrins are more adhesive to fibronectin, we demonstrate that the enzymatic removal of sialic acid residues from purified α5β1integrins stimulates fibronectin binding by these integrins. These data support the hypothesis that unsialylated β1 integrins are more adhesive to fibronectin, although desialylation of α5 subunits could also contribute to increased fibronectin binding. Collectively our results suggest a novel mechanism for regulation of the β1 integrin family of cell adhesion receptors.

A Novel α-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. This repeat cannot be replaced by other fibronectin type III repeats. We solved the crystal structure of human NCAM FN1 and found that, in addition to a unique acidic surface patch, it possesses a novel α-helix that links strands 4 and 5 of its β-sandwich structure. Replacement of the α-helix did not eliminate polysialyltransferase recognition, but shifted the addition of polysialic acid from the N-glycans modifying the adjacent immunoglobulin domain to O-glycans modifying FN1. Other experiments demonstrated that replacement of residues in the acidic surface patch alter the polysialylation of both N- and O-glycans in the same way, while the α-helix is only required for the polysialylation of N-glycans. Our data are consistent with a model in which the FN1 α-helix is involved in an Ig5-FN1 interaction that is critical for the correct positioning of Ig5 N-glycans for polysialylation.

Polysialyltransferase-1 autopolysialylation is not requisite for polysialylation of neural cell adhesion molecule.

Polysialyltransferase-1 (PST; ST8Sia IV) is one of the α2,8-polysialyltransferases responsible for the polysialylation of the neural cell adhesion molecule (NCAM). The presence of polysialic acid on NCAM has been shown to modulate cell-cell and cell-matrix interactions. We previously reported that the PST enzyme itself is modified by α2,8-linked polysialic acid chainsin vivo. To understand the role of autopolysialylation in PST enzymatic activity, we employed a mutagenesis approach. We found that PST is modified by five Asn-linked oligosaccharides and that the vast majority of the polysialic acid is found on the oligosaccharide modifying Asn-74. In addition, the presence of the oligosaccharide on Asn-119 appeared to be required for folding of PST into an active enzyme. Co-expression of the PST Asn mutants with NCAM demonstrated that autopolysialylation is not required for PST polysialyltransferase activity. Notably, catalytically active, non-autopolysialylated PST does not polysialylate any endogenous COS-1 cell proteins, highlighting the protein specificity of polysialylation. Immunoblot analyses of NCAM polysialylation by polysialylated and non-autopolysialylated PST suggests that the NCAM is polysialylated to a higher degree by autopolysialylated PST. We conclude that autopolysialylation of PST is not required for, but does enhance, NCAM polysialylation.

Polysialic acid: Biosynthesis, novel functions and applications

Abstract 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. Studies show that mammalian polysialyltransferases employ a unique recognition mechanism to limit the addition of polySia to a select group of proteins. PolySia has long been considered an anti-adhesive molecule, and its impact on cell adhesion and signaling attributed directly to this property. However, recent studies have shown that polySia specifically binds neurotrophins, growth factors, and neurotransmitters and that this binding depends on chain length. This work highlights the importance of considering polySia quality and quantity, and not simply its presence or absence, as its various roles are explored. The capsular polySia of neuroinvasive bacteria allows these organisms to evade the host immune response. While this “stealth” characteristic has made meningitis vaccine development difficult, it has also made polySia a worthy replacement for polyetheylene glycol in the generation of therapeutic proteins with low immunogenicity and improved circulating half-lives. Bacterial polysialyltransferases are more promiscuous than the protein-specific mammalian enzymes, and new studies suggest that these enzymes have tremendous therapeutic potential, especially for strategies aimed at neural regeneration and tissue repair.

The expression of Galβ1,4GlcNAc α2,6 sialyltransferase and α2,6-linked sialoglycoconjugates in human brain tumors

Abstract CMP-NeuAc: Galβ1,4GlcNAc α2,6 sialyltransferase (α2,6-ST) [EC 2.4.99.1] is developmentally regulated, shows a high degree of tissue specificity, and appears to play a role in oncogenic transformation and metastasis. In the present study, we have performed the first detailed analysis of the expression of α2,6-ST and α2,6-linked sialoglycoconjugates in human brain tumors. We used a polyclonal, monospecific anti-rat α2,6-ST antibody and the α2,6-linked sialic acid-specific lectin, Sambucus nigra agglutinin (SNA) for histochemical studies, and a human a2,6-ST-specific cDNA probe for Northern analysis. Meningiomas, chordomas and craniopharyngiomas frequently expressed α2,6-ST and α2,6-linked sialoglycoconjugates. Among the different meningioma subtypes, meningothelial meningiomas stained more strongly with both anti-α2,6-ST antibody and SNA than the fibroblastic and anaplastic meningiomas. On the other hand, all tumors of glial origin and medulloblastomas were virtually devoid of either α2,6-ST or α2,6-linked sialoglycoconjugate expression. Moreover, very weak to negligible expression of both α2,6-ST and α2,6-linked sialoglycoconjugates was observed in brain metastases. In conclusion, α2,6-ST and α2,6-linked sialoglycoconjugate expression is associated with non-neuroectodermal epithelial-like tumors.

Structural Basis for the Polysialylation of the Neural Cell Adhesion Molecule

Polysialic acid is a unique glycan polymer composed of long chains of α2, 8-linked sialic acid residues; it is found on a small subset of mammalian proteins. The neural cell adhesion molecule, NCAM, is the most abundant polysialylated protein in mammalian cells. The presence of polysialic acid on NCAM has been demonstrated to decrease cell adhesion, and it is critical for a variety of processes including brain development, synaptic plasticity, axon guidance and pathfinding, neurite outgrowth, and general cell migration. Polysialic acid is also expressed on the surface of several highly metastatic cancers and has been implicated in cancer cell growth and invasiveness. This review will focus on the protein specificity of the polysialylation of NCAM by summarizing the current information on the sequence and structural requirements for NCAM recognition, and polysialylation by the polysialyltransferases, ST8Sia IV/PST and ST8Sia II/STX.

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 α-helix in the first fibronectin type III repeat of NCAM are required for the polysialylation of N-glycans on the adjacent immunoglobulin domain. Inspection of ST8Sia IV sequences revealed two conserved polybasic regions that might interact with the NCAM acidic patch or the growing polysialic acid chain. One is the previously identified polysialyltransferase domain (Nakata, D., Zhang, L., and Troy, F. A. (2006) Glycoconj. J. 23, 423–436). The second is a 35-amino acid polybasic region that contains seven basic residues and is equidistant from the large sialyl motif in both polysialyltransferases. We replaced these basic residues to evaluate their role in enzyme autopolysialylation and NCAM-specific polysialylation. We found that replacement of Arg276/Arg277 or Arg265 in the polysialyltransferase domain of ST8Sia IV decreased both NCAM polysialylation and autopolysialylation in parallel, suggesting that these residues are important for catalytic activity. In contrast, replacing Arg82/Arg93 in ST8Sia IV with alanine substantially decreased NCAM-specific polysialylation while only partially impacting autopolysialylation, suggesting that these residues may be particularly important for NCAM polysialylation. Two conserved negatively charged residues, Glu92 and Asp94, surround Arg93. Replacement of these residues with alanine largely inactivated ST8Sia IV, whereas reversing these residues enhanced enzyme autopolysialylation but significantly reduced NCAM polysialylation. In sum, we have identified selected amino acids in this conserved polysialyltransferase polybasic region that are critical for the protein-specific polysialylation of NCAM.

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. Included in the discussion is a description of these enzymes’ glycan acceptors, conserved domain organization and sequences, molecular structure and catalytic mechanism. In addition, we discuss the protein interactions underlying the polysialylation of a select group of adhesion and signaling molecules. In the second section, the biosynthesis of sialic acid, CMP-sialic acid and sialylated N-glycans is discussed, with a special emphasis on the compartmentalization of these processes in the mammalian cell. The sequences and mechanisms maintaining the sialyltransferases and other glycosylation enzymes in the Golgi are also reviewed. In the final section, we have chosen to discuss processes in which sialylated glycans, both N- and O-linked, play a role. The first part of this section focuses on sialic acid-binding proteins including viral hemagglutinins, Siglecs and selectins. In the second half of this section, we comment on the role of sialylated N-glycans in cancer, including the roles of β1-integrin and Fas receptor N-glycan sialylation in cancer cell survival and drug resistance, and the role of these sialylated proteins and polysialic acid in cancer metastasis.

Sialyltransferase Isoforms Are Phosphorylated in the Cis-medial Golgi on Serine and Threonine Residues in Their Luminal Sequences

ST6Gal-I (α2,6-sialyltransferase) is expressed as two isoforms, STTyr and STCys, which exhibit differences in catalytic activity, trafficking through the secretory pathway, and proteolytic processing and secretion. We have found that the ST6Gal-I isoforms are phosphorylated on luminal Ser and Thr residues. Immunoprecipitation of 35S- and32P-labeled proteins expressed in COS-1 cells suggests that the STTyr isoform is phosphorylated to a greater extent than the STCys isoform. Analysis of domain deletion mutants revealed that STTyr is phosphorylated on stem and catalytic domain amino acids, whereas STCys is phosphorylated on catalytic domain amino acids. An endoplasmic reticulum retained/retrieved chimeric Iip33-ST protein demonstrates drastically lower phosphorylation than does the wild type STTyrisoform. This suggests that the bulk of the ST6Gal-I phosphorylation is occurring in the Golgi. Treatment of cells with the ionophore monensin does not significantly block phosphorylation of the STTyrisoform, suggesting that phosphorylation is occurring in the cis-medial Golgi prior to the monensin block. This study demonstrates the presence of kinase activities in the cis-medial Golgi and the substantial phosphorylation of the luminal sequences of a glycosyltransferase.

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

Background: The polysialyltransferase, polysialylate, selects a group of proteins. Results: Substrate recognition and polysialylation are reduced when basic residues in a noncatalytic region of ST8SiaIV/PST are replaced. Conclusion: Specific residues in a polysialyltransferase polybasic region are critical for substrate recognition. Significance: Understanding the mechanism of protein-specific polysialylation will allow for the modulation of this process during development and disease. 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. A., Swartzentruber, K. G., and Colley, K. J. (2009) J. Biol. Chem. 284, 15505–15516). Here, we employ a competition assay to evaluate the role of this region in substrate recognition. We find that truncated, catalytically inactive ST8SiaIV/PST proteins that include the polybasic region, but not those that lack this region, compete with endogenous ST8SiaIV/PST and reduce NCAM polysialylation in SW2 small cell lung carcinoma cells. Replacing two polybasic region residues, Arg82 and Arg93, eliminates the ability of a full-length, catalytically inactive enzyme (PST H331K) to compete with SW2 cell ST8SiaIV/PST and block NCAM polysialylation. Replacing these residues singly or together in ST8SiaIV/PST substantially reduces or eliminates NCAM polysialylation, respectively. In contrast, replacing Arg82, but not Arg93, substantially reduces the ability of ST8SiaIV/PST to polysialylate neuropilin-2 and SynCAM 1, suggesting that Arg82 plays a general role in substrate recognition, whereas Arg93 specifically functions in NCAM recognition. Taken together, our results indicate that the ST8SiaIV/PST polybasic region plays a critical role in substrate recognition and suggest that different combinations of basic residues may mediate the recognition of distinct substrates.