Genome-scale functional genomics screening highlights genes impacting protein fucosylation in Chinese hamster ovary cells.

N-linked glycosylation is a common post-translational modification that has various effects on multiple types of proteins. The extent to which an N-linked glycoprotein is modified and the identity of glycans species involved is of great interest to the biopharmaceutical industry, since glycosylation can impact the efficacy and safety of therapeutic monoclonal antibodies (mAbs). mAbs lacking core fucose, for example, display enhanced clinical efficacy through increased antibody-dependent cellular cytotoxicity. We performed a genome-wide CRISPR knockout screen in Chinese hamster ovary (CHO) cells, the workhorse cell culture system for industrial production of mAbs, aimed at identifying novel regulators of protein fucosylation. Using a lectin binding assay, we identified 224 gene perturbations that significantly alter protein fucosylation, including well-known glycosylation genes. This functional genomics framework could readily be extended and applied to study the genetic pathways involved in regulation of other glycoforms. We hope this resource will provide useful guidance toward the development of next generation CHO cell lines and mAb therapeutics.

Novel method to specifically determine the structures of non-N297 glycans in IgGs.

Monoclonal antibodies comprise a major class of biologic therapeutics and are also extensively studied in immunology. Given the importance of glycans on antibodies, fluorescent labeling of enzymatically released glycans and their LC/MS analysis is routinely used for in-depth characterization of antibody glycosylation. In this technical note, we propose a method for facile characterization of glycans in the variable region of antibodies using sequential enzymatic digests with Endoglycosidase-S2 and RapidTM Peptide-N-Glycosidase-F followed by labeling with a fluorescent dye carrying an NHS-carbamate moiety. The results and proposed mechanism also suggest that the choice of glycosidases along with the labeling chemistry is critical for accurate glycan analysis for a desired application.

Noncatalytic Endosialidase Enables Surface Capture of Small-Cell Lung Cancer Cells Utilizing Strong Dendrimer-Mediated Enzyme-Glycoprotein Interactions.

Enumeration of circulating tumor cells (CTCs) of small-cell lung cancer (SCLC) patients has been shown to predict the disease progress and long-term survival. Most CTC detection methods rely on epithelial surface markers, such as epithelial cell adhesion molecule (EpCAM). However, this marker in SCLC is reported to be often downregulated after a variety of phenotypic changes, which impairs the reliability of EpCAM-based CTC detections. In this regard, the development of an alternative CTC detection method involving different CTC surface markers is in demand. In this study, we evaluated, for the first time to our knowledge, the feasibility of detecting SCLC CTCs using a noncatalytic endosialidase (EndoN Trap, EndoNt). This noncatalytic enzyme was chosen due to its high affinity to polysialic acid (polySia), a cell-surface glycan, that is highly expressed by SCLC tissue. Furthermore, this enzyme-based system was integrated into our dendrimer-mediated CTC capture platform to further enhance the capture efficiency via multivalent binding. We found that the EndoNt-immobilized surfaces could specifically capture polySia-positive SCLC cells and the binding between SCLC cells and EndoNt surfaces was further stabilized by dendrimer-mediated multivalent binding. When compared to the EpCAM-based capture, EndoNt significantly improved the capture efficiency of polySia-positive SCLC cells under flow due to its higher binding affinity (lower dissociation rate constants). These findings suggest that this enzyme-based CTC capture strategy has the potential to be used as a superior alternative to the commonly used EpCAM-based methods, particularly for those types of cancer that overexpress polySia.

Autopolysialylation of polysialyltransferases is required for polysialylation and polysialic acid chain elongation on select glycoprotein substrates.

Polysialic acid (polySia) is a large glycan polymer that is added to some glycoproteins by two polysialyltransferases (polySTs), ST8Sia-II and ST8Sia-IV. As polySia modulates cell adhesion and signaling, immune cell function, and tumor metastasis, it is of interest to determine how the polySTs recognize their select substrates. We have recently identified residues within the ST8Sia-IV polybasic region (PBR) that are required for neural cell adhesion molecule (NCAM) recognition and subsequent polysialylation. Here, we compared the PBR sequence requirements for NCAM, neuropilin-2 (NRP-2), and synaptic cell adhesion molecule 1 (SynCAM 1) for polysialylation by their respective polySTs. We found that the polySTs use unique but overlapping sets of PBR residues for substrate recognition, that the NCAM-recognizing PBR sites in ST8Sia-II and ST8Sia-IV include homologous residues, but that the ST8Sia-II site is larger, and that fewer PBR residues are involved in NRP-2 and SynCAM 1 recognition than in NCAM recognition. Noting that the two sites for ST8Sia-IV autopolysialylation flank the PBR, we evaluated the role of PBR residues in autopolysialylation and found that the requirements for polyST autopolysialylation and substrate polysialylation overlap. These data together with the evaluation of the polyST autopolysialylation mechanism enabled us to further identify PBR residues potentially playing dual roles in substrate recognition and in polySia chain polymerization. Finally, we found that ST8Sia-IV autopolysialylation is required for NRP-2 polysialylation and that ST8Sia-II autopolysialylation promotes the polymerization of longer polySia chains on SynCAM 1, suggesting a critical role for polyST autopolysialylation in substrate selection and polySia chain elongation.

The Polybasic Region of the Polysialyltransferase ST8Sia-IV Binds Directly to the Neural Cell Adhesion Molecule, NCAM.

Polysialic acid (polySia) is a unique post-translational modification found on a small set of mammalian glycoproteins. Composed of long chains of α2,8-linked sialic acid, this large, negatively charged polymer attenuates protein and cell adhesion and modulates signaling mediated by its carriers and proteins that interact with these carriers. PolySia is crucial for the proper development of the nervous system and is upregulated during tissue regeneration and in highly invasive cancers. Our laboratory has previously shown that the neural cell adhesion molecule, NCAM, has an acidic surface patch in its first fibronectin type III repeat (FN1) that is critical for the polysialylation of N-glycans on the adjacent immunoglobulin domain (Ig5). We have also identified a polysialyltransferase (polyST) polybasic region (PBR) that may mediate substrate recognition. However, a direct interaction between the NCAM FN1 acidic patch and the polyST PBR has yet to be demonstrated. Here, we have probed this interaction using isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. We observe direct and specific binding between FN1 and the PBR peptide that is dependent upon acidic residues in FN1 and basic residues of the PBR. NMR titration experiments verified the role of the FN1 acidic patch in the recognition of the PBR and suggest a conformational change of the Ig5-FN1 linker region following binding of the PBR to the acidic patch. Finally, mutation of residues identified by NMR titration experiments impacts NCAM polysialylation, supporting their mechanistic role in protein-specific polysialylation.

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 ß1-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.

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

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 ∼50% 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 membrane-associated or soluble. Evaluation of the soluble chimeric proteins demonstrated that the meprin A5 antigen-µ 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.