Daniel R. Studelska

INHIBITION OR ACTIVATION OF APERT SYNDROME FGFR2 (S252W) SIGNALING BY SPECIFIC GLYCOSAMINOGLYCANS

Most Apert syndrome patients harbor a single amino acid mutation (S252W) in fibroblast growth factor (FGF) receptor 2 (FGFR2), which leads to abnormal FGF/FGFR2 signaling. Here we show that specific combinations of FGFs and glycosaminoglycans activate both alternative splice forms of the mutant but not of the wild-type FGF receptors. More importantly, 2-O- and N-sulfated heparan sulfate, prepared by a combined chemical and enzymatic synthesis, antagonized the over-activated FGFR2b (S252W) to basal levels at nanomolar concentrations. These studies demonstrated that specific glycosaminoglycans could be useful in treating ligand-dependent FGFR signaling-related diseases, such as Apert syndrome and cancer.

Glycosaminoglycans in Human and Bovine serum: Detection of Twenty-Four Heparan sulfate and chondroitin sulfate Motifs Including a Novel Sialic Acid-modified Chondroitin Sulfate Linkage Hexasaccharide

Heterogeneous heparan sulfate and chondroitin sulfate glycosaminoglycan (GAG) polysaccharides are important components of blood circulation. Changes in GAG quantity and structure in blood have been indicated in cancers and other human diseases. However, GAG quantities and structures have not been fully characterized due to lack of robust and sensitive analytical tools. To develop such tools, we isolated GAGs from serum and plasma. We employed liquid chromatography (LC) for GAG quantification and LC/mass spectrometry (MS) for GAG structural analysis. Twenty-four heparan and chondroitin sulfate motifs were identified, including linkage hexasaccharides, repeating disaccharide compositions, reducing, and non-reducing end mono-, di-, tri-, and tetrasaccharide structures. Disaccharides were detectable at picomolar level without radiolabeling or derivitization, so only a few ml of human and fetal bovine serum was required for this study. The detection of different reducing end structures distinct from GAG linkage hexasaccharides revealed that free GAG chains generated by GAG degradation enzymes co-existed with proteoglycans in serum. In addition, a novel sialic acid-modified linkage hexasaccharide was found conjugated to bikunin, the most abundant serum proteoglycan.

Rotational-echo double-resonance NMR-restrained model of the ternary complex of 5-enolpyruvylshikimate-3-phosphate synthase.

The 46-kD enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase catalyzes the condensation of shikimate-3-phosphate (S3P) and phosphoenolpyruvate to form EPSP. The reaction is inhibited by N-(phosphonomethyl)-glycine (Glp), which, in the presence of S3P, binds to EPSP synthase to form a stable ternary complex. We have used solid-state NMR and molecular modeling to characterize the EPSP synthase–S3P–Glp ternary complex. Modeling began with the crystal coordinates of the unliganded protein, published distance restraints, and information from the chemical modification and mutagenesis literature on EPSP synthase. New inter-ligand and ligand-protein distances were obtained. These measurements utilized the native 31P in S3P and Glp, biosynthetically 13C-labeled S3P, specifically 13C and 15N labeled Glp, and a variety of protein-15N labels. Several models were investigated and tested for accuracy using the results of both new and previously published rotational-echo double resonance (REDOR) NMR experiments. The REDOR model is compared with the recently published X-ray crystal structure of the ternary complex, PDB code 1G6S. There is general agreement between the REDOR model and the crystal structure with respect to the global folding of the two domains of EPSP synthase and the relative positioning of S3P and Glp in the binding pocket. However, some of the REDOR data are in disagreement with predictions based on the coordinates of 1G6S, particularly those of the five arginines lining the binding site. We attribute these discrepancies to substantive differences in sample preparation for REDOR and X-ray crystallography. We applied the REDOR restraints to the 1G6S coordinates and created a REDOR-refined xray structure that agrees with the NMR results.

High affinity glycosaminoglycan and autoantigen interaction explains joint specificity in a mouse model of rheumatoid arthritis

In the K/BxN mouse model of rheumatoid arthritis, autoantibodies specific for glucose-6-phosphate isomerase (GPI) can transfer joint-specific inflammation to most strains of normal mice. Binding of GPI and autoantibody to the joint surface is a prerequisite for joint-specific inflammation. However, how GPI localizes to the joint remains unclear. We show that glycosaminoglycans (GAGs) are the high affinity (83 nm) joint receptors for GPI. The binding affinity and structural differences between mouse paw/ankle GAGs and elbows/knee GAGs correlated with the distal to proximal disease severity in these joints. We found that cartilage surface GPI binding was greatly reduced by either chondroitinase ABC or β-glucuronidase treatment. We also identified several inhibitors that inhibit both GPI/GAG interaction and GPI enzymatic activities, which suggests that the GPI GAG-binding domain overlaps with the active site of GPI enzyme. Our studies raise the possibility that GAGs are the receptors for other autoantigens involved in joint-specific inflammatory responses.

Glycosaminoglycans and Activated Contact System in Cancer Patient Plasmas

Oncogenic mutations create cancer cells. Cancer cells require thrombin for growth, angiogenesis, and metastasis. All cancer patients display a hypercoagulable state, which includes platelet activation, blood coagulation, complement activation, vasodilatation, and inflammation. This often results in thrombosis, the second leading cause of death in cancer patients. It is established that chemically oversulfated glycosaminoglycans (GAGs) induce thrombin generation through contact system activation in human plasma. Thrombin is responsible for thrombosis. In this chapter, we show that plasmas from lung cancer patients contain activated contact systems apparent by the absence of high molecular weight kininogen and processed C1inh, by abnormal kallikrein and thrombin activities, and by increased glucosamine, galactosamine, and GAG levels. Activated contact systems were also evident in plasmas from breast, colon, and pancreatic cancer patients. These data suggest that GAGs or other molecules produced by tumors induce abnormal thrombin generation through contact system activation. Therefore, the contact system and glycans represent new targets for cancer diagnosis, prevention, and treatment.

Distance between phosphine-sulfide sidechains of a disubstituted peptide by DRAMA 31P NMR.

Dipolar restoration at the magic angle (DRAMA) has been used to measure the 31P-31P internuclear distance between phosphine-sulfide substituted sidechanins on the fourth and eighth residues of a 12-residue helical peptide. The 7.4 A distance is the same for the peptide lyophilized in bulk or isolated in a cryo- and lyoprotected matrix of poly(ethylene glycol) and sucrose. However, the 31P linewidth for the undiluted peptide is an order of magnitude greater than for the matrix-isolated peptide indicating charge and hydration heterogeneity in the bulk state.