Glycosaminoglycans promote osteogenesis from human induced pluripotent stem cells via neural crest induction.

The stepwise development of bone is rigidly controlled from mesenchymal cells through osteoblasts. Dysregulation of this process causes various bone diseases, such as osteoporosis and osteogenesis imperfecta. Recently, it has been noted that the decrease in bone density due to aging occurs not only in the axial skeleton but also in the facial bone. To address this issue, we focused on neural crest-derived osteoblasts that form craniofacial bone, and evaluated several functional ingredients that have been reported to activate osteoblast function using mineralization ability as an index. Glucosamine is a major component of glycosaminoglycans, is highly expressed in connective and cartilage tissues, and is known as a health food that improves joint function. Recent studies suggest that glucosamine promotes osteoblast activation; however, the underlying mechanism of this phenomenon remains unclear. This study is the first to elucidate the effects of glucosamine on neural crest-derived osteoblast differentiation using human induced pluripotent stem cells. We confirmed that glucosamine promotes osteogenesis of neural crest-derived mesenchymal stromal cells and osteoblasts. Furthermore, glucosamine increased the gene expression as well as the protein levels of osteopontin (OPN) and screlostin (SOST) which are involved in the following two processes: (1) conversion of mesenchymal stromal cells into osteoblasts, and (2) maturation of osteoblasts. These findings suggest that glucosamine plays a role in promoting osteogenesis and contributes to maintaining a healthy bone condition.

Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling.

To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.

A mutant phospholipase D with enhanced thermostability from Streptomyces sp.

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329-441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.

Study on thermostability of phospholipase D from Streptomyces sp.

Four phospholipases D (PLDs) in the culture supernatants from Streptomyces strains were purified to conduct a comparative study of their thermostabilities. Among the four purified PLDs, the enzyme from Streptomyces halstedii K1 lost its activity at 45 degrees C. PLD from Streptomyces septatus TH-2 was stable at the same temperature. We determined the nucleotide sequence encoding the PLD gene from S. halstedii K1 (K1PLD). The deduced amino acid sequence showed high homology to that of the PLD gene from S. septatus TH-2 (TH-2PLD). By comparison of the optimum temperature and the thermostability among recombinant PLDs, K1PLD, TH-2PLD and T/KPLD that possessed the N-terminus of TH-2PLD and the C-terminus of K1PLD, T/KPLD showed the properties midway between those of K1PLD and TH-2PLD. It was suggested that the 176 amino acids at C-terminus of Streptomyces PLD were important for its thermostability.

Inhibition of Streptomyces chromofuscus phospholipase D activity by dichloro-(2,2':6',2''-terpyridine)-platinum (II) dihydrate.

To determine the catalytic site of Streptomyces chromofuscus phospholipase D (PLD), which lacks an HKD motif, we examined the effects of inhibitors on the hydrolytic activity of the PLD by comparing it with cabbage and Streptomyces PLDs, which have two HKD motifs. We showed that dichloro-(2,2':6',2''-terpyridine)-platinum (II) dihydrate, a His- and Cys-directed chemical modifier, had inhibitory effects on the activities of all types of PLD examined. On the other hand, N-ethylmaleimide, a thiol-directed modifier had no such effects on PLD activity. These results suggest that the His residue plays an important role in the activity of Streptomyces chromofuscus PLD.