Sarah R. Hanson

Glycoproteomic probes for fluorescent imaging of fucosylated glycans in vivo.

Glycomics is emerging as a new field for the biology of complex glycoproteins and glycoconjugates. The lack of versatile glycan-labeling methods has presented a major obstacle to visualizing at the cellular level and studying glycoconjugates. To address this issue, we developed a fluorescent labeling technique based on the Cu(I)-catalyzed [3 + 2] cycloaddition, or click chemistry, which allows rapid, versatile, and specific covalent labeling of cellular glycans bearing azide groups. The method entails generating a fluorescent probe from a nonfluorescent precursor, 4-ethynyl-N-ethyl-1,8-naphthalimide, by clicking the fluorescent trigger, the alkyne at the 4 position, with an azido-modified sugar. Using this click-activated fluorescent probe, we demonstrate incorporation of an azido-containing fucose analog into glycoproteins via the fucose salvage pathway. Distinct fluorescent signals were observed by flow cytometry when cells treated with 6-azidofucose were labeled with the click-activated fluorogenic probe or biotinylated alkyne. The intracellular localization of fucosylated glycoconjugates was visualized by using fluorescence microscopy. This technique will allow dynamic imaging of cellular fucosylation and facilitate studies of fucosylated glycoproteins and glycolipids.

Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells

Developing tools for investigating the cellular activity of glycans will help to delineate the molecular basis for aberrant glycosylation in pathological processes such as cancer. Metabolic oligosaccharide engineering, which inserts sugar-reporting groups into cellular glycoconjugates, represents a powerful method for imaging the localization, trafficking, and dynamics of glycans and isolating them for glyco-proteomic analysis. Herein, we show that the alkyne-reporting group can be incorporated into cellular glycans. The alkyne group is a small, inert, bio-orthogonal handle that can be chemoselectively labeled by using the Cu(I) catalyzed [3 + 2] azide-alkyne cycloaddition, or click chemistry. Alkynyl sugar monomers, based on fucose (Fuc) and N-acetylmannosamine (ManNAc), were incorporated into fucosylated and sialylated glycans in several cancer cell lines, allowing for cell surface and intracellular visualization of glycoconjugates, as well as, observation of alkyne-bearing glycoproteins. Similarly to our previous results with an azido Fuc/alkynyl probe system, we demonstrated that click-activated fluorogenic probes are practical tools for efficiently and selectively labeling alkynyl-modified glycans. Because Fuc and sialic acid are terminal glycan residues with a notably increased presence in many tumors, we hope that our method will provide useful information about their roles in cancer and ultimately can be used for diagnostic and therapeutic purposes.

The core trisaccharide of an N-linked glycoprotein intrinsically accelerates folding and enhances stability

The folding energetics of the mono-N-glycosylated adhesion domain of the human immune cell receptor cluster of differentiation 2 (hCD2ad) were studied systematically to understand the influence of the N-glycan on the folding energy landscape. Fully elaborated N-glycan structures accelerate folding by 4-fold and stabilize the β-sandwich structure by 3.1 kcal/mol, relative to the nonglycosylated protein. The N-glycans first saccharide unit accounts for the entire acceleration of folding and for 2/3 of the native state stabilization. The remaining third of the stabilization is derived from the next 2 saccharide units. Thus, the conserved N-linked triose core, ManGlcNAc2, improves both the kinetics and the thermodynamics of protein folding. The native state stabilization and decreased activation barrier for folding conferred by N-glycosylation provide a powerful and potentially general mechanism for enhancing folding in the secretory pathway.

Protein Native-State Stabilization by Placing Aromatic Side Chains in N-Glycosylated Reverse Turns

Protein reverse turns that interact with a phenlyalanine group allow stable introduction of glycan groups at asparagine residues. N-glycosylation of eukaryotic proteins helps them fold and traverse the cellular secretory pathway and can increase their stability, although the molecular basis for stabilization is poorly understood. Glycosylation of proteins at naïve sites (ones that normally are not glycosylated) could be useful for therapeutic and research applications but currently results in unpredictable changes to protein stability. We show that placing a phenylalanine residue two or three positions before a glycosylated asparagine in distinct reverse turns facilitates stabilizing interactions between the aromatic side chain and the first N-acetylglucosamine of the glycan. Glycosylating this portable structural module, an enhanced aromatic sequon, in three different proteins stabilizes their native states by –0.7 to –2.0 kilocalories per mole and increases cellular glycosylation efficiency.

Extracellular sulfatases support cartilage homeostasis by regulating BMP and FGF signaling pathways

The balance between anabolic and catabolic signaling pathways is critical in maintaining cartilage homeostasis and its disturbance contributes to joint diseases such as osteoarthritis (OA). A unique mechanism that modulates the activity of cell signaling pathways is controlled by extracellular heparan endosulfatases Sulf-1 and Sulf-2 (Sulfs) that are overexpressed in OA cartilage. This study addressed the role of Sulfs in cartilage homeostasis and in regulating bone morphogenetic protein (BMP)/Smad and fibroblast growth factor (FGF)/Erk signaling in articular cartilage. Spontaneous cartilage degeneration and surgically induced OA were significantly more severe in Sulf-1−/− and Sulf-2−/− mice compared with wild-type mice. MMP-13, ADAMTS-5, and the BMP antagonist noggin were elevated whereas col2a1 and aggrecan were reduced in cartilage and chondrocytes from Sulf−/− mice. Articular cartilage and cultured chondrocytes from Sulf−/− mice showed reduced Smad1 protein expression and Smad1/5 phosphorylation, whereas Erk1/2 phosphorylation was increased. In human chondrocytes, Sulfs siRNA reduced Smad phosphorylation but enhanced FGF-2-induced Erk1/2 signaling. These findings suggest that Sulfs simultaneously enhance BMP but inhibit FGF signaling in chondrocytes and maintain cartilage homeostasis. Approaches to correct abnormal Sulf expression have the potential to protect against cartilage degradation and promote cartilage repair in OA.

N-Glycosylation of Enhanced Aromatic Sequons to Increase Glycoprotein Stability

N‐glycosylation can increase the rate of protein folding, enhance thermodynamic stability, and slow protein unfolding; however, the molecular basis for these effects is incompletely understood. Without clear engineering guidelines, attempts to use N‐glycosylation as an approach for stabilizing proteins have resulted in unpredictable energetic consequences. Here, we review the recent development of three “enhanced aromatic sequons,” which appear to facilitate stabilizing native‐state interactions between Phe, Asn‐GlcNAc and Thr when placed in an appropriate reverse turn context. It has proven to be straightforward to engineer a stabilizing enhanced aromatic sequon into glycosylation‐naïve proteins that have not evolved to optimize specific protein–carbohydrate interactions. Incorporating these enhanced aromatic sequons into appropriate reverse turn types within proteins should enhance the well‐known pharmacokinetic benefits of N‐glycosylation‐based stabilization by lowering the population of protease‐susceptible unfolded and aggregation‐prone misfolded states, thereby making such proteins more useful in research and pharmaceutical applications.

Glucosamine-6-sulfamate analogues of heparan sulfate as inhibitors of endosulfatases.

Keeping Sulfate. The extracellular endosulfatases, which modulate signalling pathways by removing sulfate groups from heparan, can be inhibited by replacing the 6-sulfate destined for cleavage with an inhibitory sulfamate motif, as demonstrated by simple glucosamine-6-sulfamate analogs of heparan sulfate.

Evaluation of Sulfatase-Directed Quinone Methide Traps for Proteomics

Sulfatases hydrolytically cleave sulfate esters through a unique catalytic aldehyde, which is introduced by a posttranslational oxidation. To profile active sulfatases in health and disease, activity-based proteomic tools are needed. Herein, quinone methide (QM) traps directed against sulfatases are evaluated as activity-based proteomic probes (ABPPs). Starting from a p-fluoromethylphenyl sulfate scaffold, enzymatically generated QM-traps can inactivate bacterial aryl sulfatases from Pseudomonas aeruginosa and Klebsiella pneumoniae, and human steroid sulfatase. However, multiple enzyme-generated QMs form, diffuse, and non-specifically label purified enzyme. In complex proteomes, QM labeling is sulfatase-dependent but also non-specific. Thus, fluoromethylphenyl sulfates are poor ABPPs for sulfatases.

Sulfotransferases and sulfatases: Sulfate modification of carbohydrates

Class I HAS belongs to the GT2 family of glycosyltransferases, which include other β-glycosyltransferases such as cellulose and chitin synthases (Griffiths et al., 1998), and it has been proposed that metazoan HAS evolved through the addition of β-1-3 glyco-syltransferase activity to a pre-existing β-1-4 glycosyltransferase enzyme (cellulose or chitin synthase) based on sequence homology and residual activity of some HAS enzymes (Lee and Spicer, 2000). The subgroup of the GT2 family that includes streptococcal and ver-tebrate HAS also includes rhizobial oligochitin synthases, a group of putative archaic glycyl transferases as well as some orphan genes (Blank et al., 2008). The finding of HAS genes on a Bacillus anthracis plasmid and in a viral genome indicates that HAS can be mobilized (Blank et al., 2008) and HAS was identified by the Human Genome Project as one of 223 candidates for lateral gene transfer from bac-teria to humans (Lander et al., 2001). Vertebrate HAS, however, is no more closely related to streptococcal HAS than to rhizobial oligochi-tin synthases or the putative archaic glycosyltranferases (Salzberg et al., 2001). Thus, streptococcal HAS may have evolved from cellu-lose or chitin synthase in a similar manner to metazoan HAS, but in a separate event. 16.3  Streptococcal Hyaluronic Acid ProductionHA for commercial applications is produced by extraction from animal tissues (e.g., rooster comb, bovine eyes, and umbilical cord) or through microbial fermentation. Concerns regarding the potential contamination of animal tissues with adventitious agents have seen the market shift toward microbial production.HA is produced as an exopolysaccharide coat by group A Streptococcus pyogenes and several group C streptococci, including Streptococcus equi, Streptococcus uberis, and Streptococcus equisimilis (Fig. 16.2). The HA capsule aids these (opportunistic) pathogens evade the host immune system and mutant microbes without HA capsules are less virulent than parental strains (Chung et al., 2001). The HA capsule may also aid migration of the bacterium through epi-thelial layers into tissue (Cywes and Wessels, 2001).The two HA precursors, UDP-GlcUA and UDP-GlcNAc, are synthesized from glucose-6-phosphate and fructose-6-phosphate, respectively (Fig. 16.3). The same pathways are used in the biosynthesis of cell wall constituents: cell wall polysaccharides, teichoic acid, and peptidoglycan. In streptococci, the HAS is expressed from a polycistronic operon containing one or more of the enzymes responsible for precursor biosynthesis. Unlike Bacillus subtilis, streptococci do not produce teichuronic acid and the has operons characterized to date all included hasB responsible for the final step in UDP-GlcUA biosynthesis. This minimal two-gene operon is found in S. uberis (Ward et al., 2001). The has operon in S. pyogenes includes an additional gene, hasC, obtained through duplication of galU (Dougherty and van de Rijn 1994). The operons in S. equi subspecies also contain hasC obtained through duplication of galU (Blank et al., 2008); sequence analysis indicates that this duplication event is distinct from that in S. pyogenes, but occurred prior to subspeciation into S. equi subsp. equi (S. equi) and S. equi subsp. zooepidemicus (S. zooepidemicus). The S. zooepidemicus operon includes a duplicated glmU gene (hasD) and is located immediately upstream of pgi, which is transcribed both from the has operon promoter and its own promoter (Blank et al., 2008).