Doctor of Philosophy

dissertationGlycosaminoglycans are carboyhydrate side chains of proteoglycans that have a myriad of biological functions. In the brain, these molecules are implicated in everything from development to plasticity to disease. Two of the main types of glycosaminoglycans (GAGs), heparan sulfate and chondroitin sulfate, have been implicated in both plasticity and learning; however, the exact role they play has remained unclear. One of the more interesting sensorimotor systems in the brain involves the learning and production of vocalizations. The goal of this work was to investigate the role GAGs play in two different aspects of this complex behavior, the neural control of vocal ontogeny and superfast muscle involvement in song production of zebra finch. In order to fully understand the role GAGs play in complex biological behaviors, such as vocalizations, it is imperative that the proper tools be synthesized, characterized, and produced for the study of these carbohydrates. Enzymes, specifically sulfated polymers and oligosaccharides, and small molecules provide unique opportunities to examine the role of GAGs. The use of enzymes in the song-specific nucleus, HVC, allowed the validation of the functionality of these enzymes in the model system of interest. Changes in stereotyped song were observed showing that GAG modulation could lead to alteration of a learned behavior. After this confirmation that GAGs were present and involved in song, small molecules called xylosides were used to examine the role of chondroitin sulfate iv biosynthesis during vocal ontogeny. Infusion of xyloside into RA (robust nucleus of the arcopallium), a nucleus important for vocal ontogeny, led to a change in the development of song. This implies that regulated biosynthesis of chondroitin sulfate during the critical period for vocal ontogeny is important. Lastly, the role of superfast syringeal muscles in song production was examined. Heparan sulfate degradation in these muscles alters the ability of the syrinx to modulate airflow. This change in muscle kinetics was correlated with significant, but temporary, differences in acoustic structure and frequency modulation while long-term differences showed aberrant syllable production

Identification of a critical sulfation in chondroitin that inhibits axonal regeneration.

The failure of mammalian CNS neurons to regenerate their axons derives from a combination of intrinsic deficits and extrinsic factors. Following injury, chondroitin sulfate proteoglycans (CSPGs) within the glial scar inhibit axonal regeneration, an action mediated by the sulfated glycosaminoglycan (GAG) chains of CSPGs, especially those with 4-sulfated (4S) sugars. Arylsulfatase B (ARSB) selectively cleaves 4S groups from the non-reducing ends of GAG chains without disrupting other, growth-permissive motifs. We demonstrate that ARSB is effective in reducing the inhibitory actions of CSPGs both in in vitro models of the glial scar and after optic nerve crush (ONC) in adult mice. ARSB is clinically approved for replacement therapy in patients with mucopolysaccharidosis VI and therefore represents an attractive candidate for translation to the human CNS

Correction: A novel cytoskeletal action of xylosides.

[This corrects the article DOI: 10.1371/journal.pone.0269972.]

Sulfation Patterns Determine Cellular Internalization of Heparin-Like Polysaccharides

Heparin is a highly sulfated polysaccharide that serves biologically relevant roles as an anticoagulant and anticancer agent. While it is well-known that modification of heparin’s sulfation pattern can drastically influence its ability to bind growth factors and other extracellular molecules, very little is known about the cellular uptake of heparin and the role sulfation patterns serve in affecting its internalization. In this study, we chemically synthesized several fluorescently labeled heparins consisting of a variety of sulfation patterns. These polysaccharides were thoroughly characterized using anion exchange chromatography and size exclusion chromatography. Subsequently, we utilized flow cytometry and confocal imaging to show that sulfation patterns differentially affect the amount of heparin uptake in multiple cell types. This study provides the first comprehensive analysis of the effect of sulfation pattern on the cellular internalization of heparin or heparan sulfate like polysaccharides. The results of this study expand current knowledge regarding heparin internalization and provide insights into developing more effective heparin-based drug conjugates for applications in intracellular drug delivery

A Nanosensor for Ultrasensitive Detection of Oversulfated Chondroitin Sulfate Contaminant in Heparin

Heparin has been extensively used as an anticoagulant for the last eight decades. Recently, the administration of a contaminated batch of heparin caused 149 deaths in several countries including USA, Germany, and Japan. The contaminant responsible for the adverse effects was identified as oversulfated chondroitin sulfate (OSCS). Here, we report a rapid, ultrasensitive method of detecting OSCS in heparin using a nanometal surface energy transfer (NSET) based gold-heparin-dye nanosensor. The sensor is an excellent substrate for heparitinase enzyme, as evidenced by ∼70% recovery of fluorescence from the dye upon heparitinase treatment. However, the presence of OSCS results in diminished fluorescence recovery from the nanosensor upon heparitinase treatment, as the enzyme is inhibited by the contaminant. The newly designed nanosensor can detect as low as 1 × 10^–9 % (w/w) OSCS making it the most sensitive tool to date for the detection of trace amounts of OSCS in pharmaceutical heparins

Chemoenzymatically Prepared Heparan Sulfate Containing Rare 2‑O-Sulfonated Glucuronic Acid Residues

The structural diversity of natural sulfated glycosaminoglycans (GAGs) presents major promise for discovery of chemical biology tools or therapeutic agents. Yet, few GAGs have been identified so far to exhibit this promise. We reasoned that a simple approach to identify such GAGs is to explore sequences containing rare residues, for example, 2-O-sulfonated glucuronic acid (GlcA<i>p</i>2S). Genetic algorithm-based computational docking and filtering suggested that GlcA<i>p</i>2S containing heparan sulfate (HS) may exhibit highly selective recognition of antithrombin, a key plasma clot regulator. HS containing only GlcA<i>p</i>2S and 2-N-sulfonated glucosamine residues, labeled as HS_2S2S, was chemoenzymatically synthesized in just two steps and was found to preferentially bind antithrombin over heparin cofactor II, a closely related serpin. Likewise, HS_2S2S directly inhibited thrombin but not factor Xa, a closely related protease. The results show that a HS containing rare GlcA<i>p</i>2S residues exhibits the unusual property of selective antithrombin activation and direct thrombin inhibition. More importantly, HS_2S2S is also the first molecule to activate antithrombin nearly as well as the heparin pentasaccharide although being completely devoid of the critical 3-<i>O</i>-sulfonate group. Thus, this work shows that novel functions and mechanisms may be uncovered by studying rare GAG residues/sequences