Jeong-Yeh Yang

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1).

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the β-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED β-strands and the other involving GFCC′C″ β-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC′C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions.

Expression system for structural and functional studies of human glycosylation enzymes

Vertebrate glycoproteins and glycolipids are synthesized in complex biosynthetic pathways localized predominantly within membrane compartments of the secretory pathway. The enzymes that catalyze these reactions are exquisitely specific, yet few have been extensively characterized because of challenges associated with their recombinant expression as functional products. We used a modular approach to create an expression vector library encoding all known human glycosyltransferases, glycoside hydrolases, and sulfotransferases, as well as other glycan-modifying enzymes. We then expressed the enzymes as secreted catalytic domain fusion proteins in mammalian and insect cell hosts, purified and characterized a subset of the enzymes, and determined the structure of one enzyme, the sialyltransferase ST6GalNAcII. Many enzymes were produced at high yields and at similar levels in both hosts, but individual protein expression levels varied widely. This expression vector library will be a transformative resource for recombinant enzyme production, broadly enabling structure-function studies and expanding applications of these enzymes in glycochemistry and glycobiology.

Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway.

Immunoglobulin G (IgG) is a major effector molecule of the human immune response, and aberrations in IgG glycosylation are linked to various diseases. However, the molecular mechanisms underlying protein glycosylation are still poorly understood. We present a data-driven approach to infer reactions in the IgG glycosylation pathway using large-scale mass-spectrometry measurements. Gaussian graphical models are used to construct association networks from four cohorts. We find that glycan pairs with high partial correlations represent enzymatic reactions in the known glycosylation pathway, and then predict new biochemical reactions using a rule-based approach. Validation is performed using data from a GWAS and results from three in vitro experiments. We show that one predicted reaction is enzymatically feasible and that one rejected reaction does not occur in vitro. Moreover, in contrast to previous knowledge, enzymes involved in our predictions colocalize in the Golgi of two cell lines, further confirming the in silico predictions.IgG glycosylation is an important factor in immune function, yet the molecular details of protein glycosylation remain poorly understood. The data-driven approach presented here uses large-scale plasma IgG mass spectrometry measurements to infer new biochemical reactions in the glycosylation pathway.

HumanN-acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite.

Significance Cell-surface and secreted glycoproteins are initially synthesized and glycosylated in the endoplasmic reticulum. Glycan structures are trimmed and remodeled as they transit the secretory pathway, resulting in multi-branched complex-type structures. The enzymes that remodel these structures have precise linkage and branch specificities, with the product of one reaction being specifically recognized as the substrate for the following reaction. These reactions include N-acetylglucosaminyltransferase II (MGAT2), an enzyme that initiates complex branch extension by precise recognition of its glycan substrate. The structural basis for MGAT2 substrate recognition is the subject of the present study. Structures of MGAT2-substrate complexes reveal both modular and convergent mechanisms for selective substrate recognition and catalysis and provide a generalized model for template-based synthesis of glycan structures by glycosyltransferases. Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by N-acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcβ1,2- branch from the trimannosyl glycan core using UDP-GlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn2+-UDP donor analog complex and as a GlcNAcMan3GlcNAc2-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GT-A Rossmann-like fold that employs conserved divalent cation-dependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate “recognition arm.” Recognition arm interactions are similar to the enzyme–substrate interactions for Golgi α-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. These data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells.

Structural Characterization of a Heparan Sulfate Pentamer Interacting with LAR-Ig1-2

Leukocyte common antigen-related (LAR) protein is one of the type IIa receptor protein tyrosine phosphatases (RPTPs) that are important for signal transduction in biological processes, including axon growth and regeneration. Glycosaminoglycan chains, including heparan sulfate (HS) and chondroitin sulfate (CS), act as ligands that regulate LAR signaling. Here, we report the structural characterization of the first two immunoglobulin domains (Ig1-2) of LAR interacting with an HS pentasaccharide (GlcNS6S-GlcA-GlcNS3,6S-IdoA2S-GlcNS6S-OME, fondaparinux) using multiple solution-based NMR methods. In the course of the study, we extended an assignment strategy useful for sparsely labeled proteins expressed in mammalian cell culture supplemented with a single type of isotopically enriched amino acid ([15N]-Lys in this case) by including paramagnetic perturbations to NMR resonances. The folded two-domain structure for LAR-Ig1-2 seen in previous crystal structures has been validated in solution using residual dipolar coupling data, and a combination of chemical shift perturbation on titration of LAR-Ig1-2 with fondaparinux, saturation transfer difference (STD) spectra, and transferred nuclear Overhauser effects (trNOEs) have been employed in the docking program HADDOCK to generate models for the LAR-fondaparinux complex. These models are further analyzed by postprocessing energetic analysis to identify key binding interactions. In addition to providing insight into the ligand interaction mechanisms of type IIa RPTPs and the origin of opposing effects of CS and HS ligands, these results may assist in future design of therapeutic compounds for nervous system repair.

Association mapping, transcriptomics, and transient expression identify candidate genes mediating plant–pathogen interactions in a tree

Significance International trade has resulted in the introduction of plant diseases into natural ecosystems around the world. These introductions have potentially catastrophic impacts on ecosystem structure and function. Leveraging genomic tools, natural variation within a tree species, and a high-throughput phenotyping platform, we present a framework that can be broadly applied to rapidly identify candidate genes associated with resistance and susceptibility to introduced plant diseases. The unprecedented speed and accuracy with which the candidate genes can be identified in woody trees demonstrates the potential of genomics to mitigate the impacts of invasive diseases on forest health. Invasive microbes causing diseases such as sudden oak death negatively affect ecosystems and economies around the world. The deployment of resistant genotypes for combating introduced diseases typically relies on breeding programs that can take decades to complete. To demonstrate how this process can be accelerated, we employed a genome-wide association mapping of ca. 1,000 resequenced Populus trichocarpa trees individually challenged with Sphaerulina musiva, an invasive fungal pathogen. Among significant associations, three loci associated with resistance were identified and predicted to encode one putative membrane-bound L-type receptor-like kinase and two receptor-like proteins. A susceptibility-associated locus was predicted to encode a putative G-type D-mannose–binding receptor-like kinase. Multiple lines of evidence, including allele analysis, transcriptomics, binding assays, and overexpression, support the hypothesized function of these candidate genes in the P. trichocarpa response to S. musiva.

Identification of Key Enzymes for Pectin Synthesis in Seed Mucilage

Mutations in two glycosyltransferase-encoding genes severely impair the elongation of pectic rhamnogalacturonan I, resulting in hydrophobic seeds that do not release mucilage polymers. Pectin is a vital component of the plant cell wall and provides the molecular glue that maintains cell-cell adhesion, among other functions. As the most complex wall polysaccharide, pectin is composed of several covalently linked domains, such as homogalacturonan (HG) and rhamnogalacturonan I (RG I). Pectin has widespread uses in the food industry and has emerging biomedical applications, but its synthesis remains poorly understood. For instance, the enzymes that catalyze RG I elongation remain unknown. Recently, a coexpression- and sequence-based MUCILAGE-RELATED (MUCI) reverse genetic screen uncovered hemicellulose biosynthetic enzymes in the Arabidopsis (Arabidopsis thaliana) seed coat. Here, we use an extension of this strategy to identify MUCI70 as the founding member of a glycosyltransferase family essential for the accumulation of seed mucilage, a gelatinous wall rich in unbranched RG I. Detailed biochemical and histological characterization of two muci70 mutants and two galacturonosyltransferase11 (gaut11) mutants identified MUCI70 and GAUT11 as required for two distinct RG I domains in seed mucilage. We demonstrate that, unlike MUCI70, GAUT11 catalyzes HG elongation in vitro and, thus, likely is required for the synthesis of an HG region important for RG I elongation. Analysis of a muci70 gaut11 double mutant confirmed that MUCI70 and GAUT11 are indispensable for the production and release of the bulk of mucilage RG I and for shaping the surface morphology of seeds. In addition, we uncover relationships between pectin and hemicelluloses and show that xylan is essential for the elongation of at least one RG I domain.

Publisher Correction: Network inference from glycoproteomics data reveals new reactions in the IgG glycosylation pathway

Correction to:Nature Communications (2017) 8:1231. doi:10.1038/s41467-017-01525-0

Paramagnetic Tag for Glycosylation Sites in Glycoproteins: Structural Constraints on Heparan Sulfate Binding to Robo1

An enzyme- and click chemistry-mediated methodology for the site-specific nitroxide spin labeling of glycoproteins has been developed and applied. The procedure relies on the presence of single N-glycosylation sites that are present natively in proteins or that can be engineered into glycoproteins by mutational elimination of all but one glycosylation site. Recombinantly expressing glycoproteins in HEK293S (GnT1-) cells results in N-glycans with high-mannose structures that can be processed to leave a single GlcNAc residue. This can in turn be modified by enzymatic addition of a GalNAz residue that is subject to reaction with an alkyne-carrying TEMPO moiety using copper(I)-catalyzed click chemistry. To illustrate the procedure, we have made an application to a two-domain construct of Robo1, a protein that carries a single N-glycosylation site in its N-terminal domains. The construct has also been labeled with 15N at amide nitrogens of lysine residues to provide a set of sites that are used to derive an effective location of the paramagnetic nitroxide moiety of the TEMPO group. This, in turn, allowed measurements of paramagnetic perturbations to the spectra of a new high affinity heparan sulfate ligand. Calculation of distance constraints from these data facilitated determination of an atomic level model for the docked complex.