Heparosan-glucuronate 5-epimerase: Molecular cloning and characterization of a novel enzyme.

Iduronic acid (IdoA) is a critical component of heparan sulfate in its interaction with functional proteins. Heparosan-N-sulfate-glucuronate 5-epimerase (HNSG-5epi) converts d-glucuronic acid (GlcA) residues in N-sulfated heparosan (NS-heparosan), as an intermediate in heparan sulfate biosynthesis, to IdoA. In the present study, the authors discovered a different 5-epimerase, designated HG-5epi (heparosan-glucuronate 5-epimerase), that is involved in acharan sulfate biosynthesis and possesses novel substrate specificity. A candidate cDNA of HG-5epi was cloned from the cDNA library of Achatina fulica. The cloned cDNA contained a whole coding region that predicts a type II transmembrane protein composed of 601 amino acid residues. The amino acid sequence of HG-5epi is homologous to that of HNSG-5epi. Recombinant HG-5epi was expressed in insect cells and its enzymatic properties characterized. As expected, HG-5epi epimerizes GlcA residues in heparosan, but not in NS-heparosan. Conversion of IdoA to GlcA was also catalyzed by HG-5epi when completely desulfated N-acetylated heparin was used as the substrate, indicating a reversible reaction mechanism. At equilibrium of the epimerization, the proportion of IdoA in the reaction product reached up to 30% of total hexuronic acid. To our knowledge, this is the first report to describe an enzyme that catalyzes the epimerization of non-sulfated heparosan. This new enzyme may be applied to the study of synthetic heparan sulfate-related polysaccharides having certain biological and pharmacological activities. In addition, a new method using anion-exchange HPLC connected to a post-column fluorescent labeling system was developed for analyzing hexuronic acid isomers.

Glucuronyltransferase activity of KfiC from Escherichia coli strain K5 requires association of KfiA: KfiC and KfiA are essential enzymes for production of K5 polysaccharide, N-acetylheparosan.

Heparan sulfate is a ubiquitous glycosaminoglycan in the extracellular matrix of most animals. It interacts with various molecules and exhibits important biological functions. K5 antigen produced by Escherichia coli strain K5 is a linear polysaccharide N-acetylheparosan consisting of GlcUA beta1-4 and GlcNAc alpha1-4 repeating disaccharide, which forms the backbone of heparan sulfate. Region 2, located in the center of the K5-specific gene cluster, encodes four proteins, KfiA, KfiB, KfiC, and KfiD, for the biosynthesis of the K5 polysaccharide. Here, we expressed and purified the recombinant KfiA and KfiC proteins and then characterized these enzymes. Whereas the recombinant KfiC alone exhibited no GlcUA transferase activity, it did exhibit GlcUA transferase and polymerization activities in the presence of KfiA. In contrast, KfiA had GlcNAc transferase activity itself, which was unaffected by the presence of KfiC. The GlcNAc and GlcUA transferase activities were analyzed with various truncated and point mutants of KfiA and KfiC. The point mutants replacing aspartic acid of a DXD motif and lysine and glutamic acid of an ionic amino acid cluster, and the truncated mutants deleting the C-terminal and N-terminal sites, revealed the essential regions for GlcNAc and GlcUA transferase activity of KfiC and KfiA, respectively. The interaction of KfiC with KfiA is necessary for the GlcUA transferase activity of KfiC but not for the enzyme activity of KfiA. Together, these results indicate that the complex of KfiA and KfiC has polymerase activity to synthesize N-acetylheparosan, providing a useful tool toward bioengineering of defined heparan sulfate chains.

Purification, characterization, and molecular cloning of a novel keratan sulfate hydrolase, endo-beta-N-acetylglucosaminidase, from Bacillus circulans.

Keratan sulfate (KS) is degraded by various enzymes including endo-beta-galactosidase, keratanase, and keratanase II, which are used for the structural analysis of KS. We purified a novel KS hydrolase, endo-beta-N-acetylglucosaminidase, from the cell pellet and conditioned medium of Bacillus circulans, by sequential chromatography using DE52 and phenyl-Sepharose columns with approximately 63- and 180-fold purity and 58 and 12.5% recovery, respectively. Like keratanase II of Bacillus sp. Ks36, the enzyme, designated Bc keratanase II, hydrolyzed KS between the 4GlcNAcbeta1-3Gal1 structure (endo-beta-N-acetylglucosaminidase), but not hyaluronan, heparan sulfate, heparin, and chondroitin sulfate C, demonstrating a strict specificity to KS. The enzyme digested shark cartilage KS to disaccharides and tetrasaccharides and bovine cornea KS to hexasaccharide, indicating that it prefers highly sulfated KS. Distinct from keratanase II of strain Ks36, the enzyme digested shark cartilage KS at an optimal temperature of 55 degrees C. Based on partial peptide sequencing of the enzyme, we molecularly cloned the gene, which encodes a protein with a predicted molecular mass of approximately 200 kDa. From the deduced protein sequence, Bc keratanase II contained a domain at the C terminus, homologous to the S-layer-like domain of pullulanase from Thermoanaerobacterium thermosulfurigenes and endoxylanase from Thermoanaerobacterium saccharolyticum, and a carbohydrate-binding domain, which may serve to specifically recognize KS chains. A full-length recombinant enzyme showed keratanase II activity. These results may prove useful for the structural analysis of KS toward achieving an understanding of its function.

A synthetic ceramide analog ameliorates spatial cognition deficit and stimulates biosynthesis of brain gangliosides in rats with cerebral ischemia.

A synthetic ceramide analog, L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (L-PDMP) upregulates ganglioside biosynthesis in several cell lines. In cultured cortical neurons, neurotrophic effects of L-PDMP on neurite outgrowth and synaptic activity were demonstrated. In addition, it was found that L-PDMP could ameliorate the spatial cognition deficit in rats with ischemia. To elucidate this effect, we evaluated the effect of L-PDMP on brain ganglioside biosynthesis and its therapeutic efficacy against spatial cognition deficit in rats made ischemic. Rats were trained for 2 weeks, using an 8-arm radial maze task, and then forebrain ischemia was induced. L-PDMP was injected i.p. at 40 mg/kg twice a day starting from day 1 or 3 after ischemia induction for 6 or 4 days, respectively. The first study showed significantly reduced spatial cognition deficit at 12 h after the final drug administration, and L-PDMP tended to attenuate apoptosis in hippocampal CA1. To examine the effect of L-PDMP on brain ganglioside biosynthesis, N-[3H]acetyl-D-mannosamine was infused into the lateral ventricle via an injection cannula at 12 h after the final drug administration. After 4 h, the brain gangliosides were purified and analyzed. Upregulation of ganglioside biosynthesis by L-PDMP was observed on days 3 and 5 after ischemia. These results are an indication that L-PDMP may ameliorate spatial cognition deficit by upregulating ganglioside biosynthesis in ischemic brain.