A new member of the UDP-N-acetylglucosamine: bgalactose b1,3-N-acetylglucosaminyltransferase (b3Gn-T) family having the b3-glycosyltransferase motifs was identified using an in silico method. This novel b3Gn-T was cloned from a human colon cancer cell line and named b3Gn-T8 based on its position in a phylogenetic tree and enzymatic activity. b3Gn-T8 transfers GlcNAc to the non-reducing terminus of the Galb1-4GlcNAc of tetraantennary N-glycan in vitro. HCT15 cells transfected with b3Gn-T8 cDNA showed an increase in reactivity to both LEA and PHA-L4 in a flow cytometric analysis. These results indicated that b3Gn-T8 is involved in the biosynthesis of poly-Nacetyllactosamine chains on tetraantennary (b1,6-branched) N-glycan. In most of the colorectal cancer tissues examined, the level of b3Gn-T8 transcript was significantly higher than in normal tissue. b3Gn-T8 could be an enzyme involved in the synthesis of poly-N-acetyllactosamine on b1-6 branched N-glycans in colon cancer.

No abstract

Chondroitin sulfate E (CSE) markedly enhanced plasminogen activation by tissue plasminogen activators (t-PAs) and urinary plasminogen activator (u-PA) in vitro; in the presence of 10 microg/ml of CSE, the potentiation factors of single chain of t-PA, two chain of t-PA and u-PA were 400, 140 and 130, respectively. Though the potentiation activity of CSE gradually decreased when it was depolymerized by chondroitinase ABC, the specific disaccharide from CSE still showed significant activity. Glycosaminoglycan (GAG) from sea cucumber, which possesses a very similar core structure to CSE, but has additional sulfated fucose branches exhibit very weak activity. These results suggested that the minimal structural requirement in CSE to enhance plasminogen activation by plasminogen activators is GlcUAbeta1-3GalNAc(4S,6S) and that additional branching sugars abolish the activity.

Glucagon-like peptide-1 (GLP-1) is an incretin peptide that plays a crucial role in lowering blood glucose levels and holds promise for treating type II diabetes. In this study, we synthesized GLP-1 derivatives that were conjugated with glycosaminoglycans (GAGs), i.e., chondroitin (CH) or heparosan (HPN), to address the major limitation in their clinical use of GLP-1, which is its short half-life in the body. After exploring a variety of CHs with different molecular sizes and heterobifunctional linkers having different alkyl chains, we obtained CH-conjugated GLP-1 derivatives that stayed in blood circulation much longer (T1/2 elim > 25 h) than unconjugated GLP-1 and showed blood glucose-lowering efficacy up to 120 h after subcutaneous injection in mice. By using the same optimized linker design, we eventually obtained a HPN-conjugated GLP-1 derivative with efficacy lasting 144 h. These results demonstrate that conjugation with GAG is a promising strategy for improving the duration of peptide drugs.

A novel, long-acting insulin conjugate was developed by modification with a glycosaminoglycan (GAG; i.e., chondroitin [CH] or heparosan [HPN]) at different positions (GlyA1, LysB29, or both) via different linkers with varying arm lengths. The in vitro glucose uptake-enhancing activity of conjugates inversely correlated with the circulatory in vivo half-life in mice and was affected primarily by the conjugation position rather than linker arm length. Conjugation at GlyA1, which provided the best balanced profile of in vitro activity and circulation period in mice, also provided the strongest glucose-lowering efficacy, exhibiting 12 h durability, which was superior to that of insulin glargine and comparable to that of insulin degludec in streptozotocin (STZ)-treated mice. The use of different GAGs did not significantly affect blood circulation or efficacy in mice; however, blood levels of CH-conjugated insulin and efficacy were lower in rats, indicating species differences in the performance of CH. Finally, optimal activity was obtained by conjugation with HPN at GlyA1 using a C3 linker. These results demonstrate the applicability of GAG modification for prolonging the efficacy of peptide drugs, similar to our previous application to glucagon-like peptide 1.

No abstract

Amyloid precursor protein (APP) and amyloid precursor-like proteins 1 and 2 (APLP1 and APLP2) are members of a large gene family. Although APP is known to be the source of the beta-amyloid peptides involved in the development of Alzheimer's disease, the normal functions of APP, APLP1 and APLP2 in cells are poorly understood. In this study, we carried out gene silencing analysis by means of RNA interference with synthetic small interfering RNA duplexes targeting the App, Aplp1 and Aplp2 genes in Neuro2a (N2a) cells, a mouse neuroblastoma cell line. The results demonstrated that cell viability and neurite outgrowth of N2a cells undergoing knockdown of Aplp1 were significantly reduced, compared with N2a cells undergoing knockdown of either App or Aplp2.

Sea cucumber glycosaminoglycan (SC-GAG) was isolated from the body wall of the sea cucumber Stichopus japonicus. The SC-GAG consists of a chondroitin sulfate E-type core polymer with sulfated fucose branches attaching glycosidically to almost every disaccharide unit of the core polymer at the C-3 position of the GlcA or at C-4 and/or C-6 position(s) of GalNAc. SC-GAG was subjected to mild acid-hydrolysis, which cleaved selectively the glycosidic linkages between the core polymer and the fucose branches, resulting in two types of partially defucosylated SC-GAG derivatives. One type (type A), obtained by 3 h-hydrolysis, contained 33% of the fucose branches and the other type (type B), obtained by 6-h hydrolysis, contained 10% of the fucose branches. The molecular masses of types A and B were determined to be 8 and 4 kDa, respectively, by gel permeation HPLC. A chondroitinase ABC (Chase ABC)-digestion demonstrated that types A and B contained 46 and 66% of digestable disaccharide units, respectively, and both types contained 29% of E-type unsaturated disaccharide units bearing no fucose branches. Intact SC-GAG and types A and B were compared for t-PA-mediated plasminogen activation by an in vitro assay system. Although intact SC-GAG and type B exhibited rather weak activity at 6.25 microg/ml, type A exhibited 5 to 10-fold higher activity than intact SC-GAG and type B at the same concentration. The activity of type A was almost one-third that of purified chondroitin sulfate E (127 kDa containing 64.5% E-type disaccharide units) from squid cartilage at 6.25 microg/ml concentration. These results suggest that t-PA-mediated plasminogen activation requires the presence of E-type disaccharide units bearing no fucose branches and a molecular mass larger than 7.5 kDa in terms of the chondroitin sulfate E structure with or without fucose branching.