Fibroblast growth factor-based signaling through synthetic heparan sulfate blocks copolymers studied using high cell density three-dimensional cell printing.

Four well-defined heparan sulfate (HS) block copolymers containing S-domains (high sulfo group content) placed adjacent to N-domains (low sulfo group content) were chemoenzymatically synthesized and characterized. The domain lengths in these HS block co-polymers were ~40 saccharide units. Microtiter 96-well and three-dimensional cell-based microarray assays utilizing murine immortalized bone marrow (BaF3) cells were developed to evaluate the activity of these HS block co-polymers. Each recombinant BaF3 cell line expresses only a single type of fibroblast growth factor receptor (FGFR) but produces neither HS nor fibroblast growth factors (FGFs). In the presence of different FGFs, BaF3 cell proliferation showed clear differences for the four HS block co-polymers examined. These data were used to examine the two proposed signaling models, the symmetric FGF2-HS2-FGFR2 ternary complex model and the asymmetric FGF2-HS1-FGFR2 ternary complex model. In the symmetric FGF2-HS2-FGFR2 model, two acidic HS chains bind in a basic canyon located on the top face of the FGF2-FGFR2 protein complex. In this model the S-domains at the non-reducing ends of the two HS proteoglycan chains are proposed to interact with the FGF2-FGFR2 protein complex. In contrast, in the asymmetric FGF2-HS1-FGFR2 model, a single HS chain interacts with the FGF2-FGFR2 protein complex through a single S-domain that can be located at any position within an HS chain. Our data comparing a series of synthetically prepared HS block copolymers support a preference for the symmetric FGF2-HS2-FGFR2 ternary complex model.

Structure/function analysis of Pasteurella multocida heparosan synthases: toward defining enzyme specificity and engineering novel catalysts.

The Pasteurella multocida heparosan synthases, PmHS1 and PmHS2, are homologous (∼65% identical) bifunctional glycosyltransferase proteins found in Type D Pasteurella. These unique enzymes are able to generate the glycosaminoglycan heparosan by polymerizing sugars to form repeating disaccharide units from the donor molecules UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc). Although these isozymes both generate heparosan, the catalytic phenotypes of these isozymes are quite different. Specifically, during in vitro synthesis, PmHS2 is better able to generate polysaccharide in the absence of exogenous acceptor (de novo synthesis) than PmHS1. Additionally, each of these enzymes is able to generate polysaccharide using unnatural sugar analogs in vitro, but they exhibit differences in the substitution patterns of the analogs they will employ. A series of chimeric enzymes has been generated consisting of various portions of both of the Pasteurella heparosan synthases in a single polypeptide chain. In vitro radiochemical sugar incorporation assays using these purified chimeric enzymes have shown that most of the constructs are enzymatically active, and some possess novel characteristics including the ability to produce nearly monodisperse polysaccharides with an expanded range of sugar analogs. Comparison of the kinetic properties and the sequences of the wild-type enzymes with the chimeric enzymes has enabled us to identify regions that may be responsible for some aspects of both donor binding specificity and acceptor usage. In combination with previous work, these approaches have enabled us to better understand the structure/function relationship of this unique family of glycosyltransferases.

Comamonas testosteronan synthase, a bifunctional glycosyltransferase that produces a unique heparosan polysaccharide analog.

Glycosaminoglycans (GAGs) are linear hexosamine-containing polysaccharides. These polysaccharides are synthesized by some pathogenic bacteria to form an extracellular coating or capsule. This strategy forms the basis of molecular camouflage since vertebrates possess naturally occurring GAGs that are essential for life. A recent sequence database search identified a putative protein from the opportunistic pathogen Comamonas testosteroni that exhibits similarity with the Pasteurella multocida GAG synthase PmHS1, which is responsible for the synthesis of a heparosan polysaccharide capsule. Initial supportive evidence included glucuronic acid (GlcUA)-containing polysaccharides extracted from C. testosteroni KF-1. We describe here the cloning and analysis of a novel Comamonas GAG synthase, CtTS. The GAG produced by CtTS in vitro consists of the sugars d-GlcUA and N-acetyl-D-glucosamine, but is insensitive to digestion by GAG digesting enzymes, thus has distinct glycosidic linkages from vertebrate GAGs. The backbone structure of the polysaccharide product [-4-D-GlcUA-α1,4-D-GlcNAc-α1-](n) was confirmed by nuclear magnetic resonance. Therefore, this novel GAG, testosteronan, consists of the same sugars as the biomedically relevant GAGs heparosan (N-acetyl-heparosan) and hyaluronan but may have distinct properties useful for future medical applications.

Chemoenzymatic synthesis with distinct Pasteurella heparosan synthases: monodisperse polymers and unnatural structures.

Heparosan (-GlcUA-beta1,4-GlcNAc-alpha1,4-)(n) is a member of the glycosaminoglycan polysaccharide family found in the capsule of certain pathogenic bacteria as well as the precursor for the vertebrate polymers, heparin and heparan sulfate. The two heparosan synthases from the Gram-negative bacteria Pasteurella multocida, PmHS1 and PmHS2, were efficiently expressed and purified using maltose-binding protein fusion constructs. These relatively homologous synthases displayed distinct catalytic characteristics. PmHS1, but not PmHS2, was able to produce large molecular mass (100-800 kDa) monodisperse polymers in synchronized, stoichiometrically controlled reactions in vitro. PmHS2, but not PmHS1, was able to utilize many unnatural UDP-sugar analogs (including substrates with acetamido-containing uronic acids or longer acyl chain hexosamine derivatives) in vitro. Overall these findings reveal potential differences in the active sites of these two Pasteurella enzymes. In the future, these catalysts should allow the creation of a variety of heparosan and heparinoids with utility for medical applications.

Social defeat, a paradigm of depression in rats that elicits 22-kHz vocalizations, preferentially activates the cholinergic signaling pathway in the periaqueductal gray.

Gene expression profiles in the periaqueductal gray (PAG) of adult Long-Evans rats as a function of a stressful social defeat in inter-male fighting encounters were examined. This social subordination model mimics prototypical behavioral changes that parallel aspects of clinical depression, has been postulated to simulate early changes in the onset of depression in the losers, and has been successfully utilized for the evaluation of antidepressant activity. The 22-kHz ultrasonic vocalizations (USVs) have been shown to reflect negative emotional states akin to anxiety and depression. Social defeat is the most robust and reliable method of eliciting these calls. The PAG has been shown to be a key brain region for the generation of 22-kHz ultrasonic vocalizations, and 22-kHz USVs have been shown to be controlled by the mesolimbic cholinergic system. In this present study, we examined gene expression changes in the PAG of social subordinate rats compared to dominant rats that do not Exhibit 22-kHz USVs. We found that social defeat significantly altered the genes associated with cholinergic synaptic transmission in the PAG. The most robust of these were the increased expression of the beta2 subunit of the nicotinic acetylcholine receptor (CHRNB2) and the T subunit of acetylcholinesterase (ACHE) in the subordinate animals. These changes were corroborated by quantitative real-time polymerase chain reaction (qRT-PCR) and found to be exclusive to the PAG compared to seven other brain regions examined. These data suggest that cholinergic transmission in the PAG is involved in the generation of 22-kHz USVs and provide potential therapeutic targets for the treatment of affective disorders.

Distinct patterns of gene expression in the left and right hippocampal formation of developing rats.

A central problem in neurobiology is the elucidation of the mechanisms that underlie left-right asymmetries in brain structure and function. Using a transcriptome screening approach, we found asymmetric gene expression patterns in the right when compared with the left hippocampal formation at postnatal days (P) 6, 9, and 60 in the rat. Of those genes that were differentially expressed, most were predominantly expressed in the right hippocampus at P6, whereas most were predominantly expressed in the left at P9 and P60. Real-time PCR analysis of genes associated with synaptic vesicle trafficking confirmed this pattern. At P6, 9 of 13 such genes were more robustly expressed in the right hippocampus, while only 1 gene was predominantly expressed in the left. Conversely, at P9, 5 of the 13 genes were more highly expressed in the left hippocampus and only 1 gene was predominantly expressed in the right. This pattern persisted at P60: eight genes were more robustly expressed in the left hippocampus, and the remaining five showed no hemispheric preference. These data demonstrate a pattern of early lateralized gene expression that is likely to underlie the establishment of functional asymmetry in the adult hippocampus.