Glycoengineering of plant expression systems is a prerequisite for the production of biopharmaceuticals that are compatible with animal-derived glycoproteins. Large amounts of high-mannose glycans such as Man7GlcNAc2, Man8GlcNAc2, and Man9GlcNAc2 (Man7/8/9), which can be favorably modified by chemical conjugation of mannose-6-phosphate, are desirable for lysosomal enzyme targeting. This study proposed a rice cell-based glycoengineering strategy using two different mannosidase inhibitors, kifunensine (KIF) and swainsonine (SWA), to increase Man7/8/9 glycoforms of recombinant human acid α-glucosidase (rhGAA), which is a therapeutic enzyme for Pompe disease. Response surface methodology was used to investigate the effects of the mannosidase inhibitors and to evaluate the synergistic effect of glycoengineering on rhGAA. Both inhibitors suppressed formation of plant-specific complex and paucimannose type N-glycans. SWA increased hybrid type glycans while KIF significantly increased Man7/8/9. Interestingly, the combination of KIF and SWA more effectively enhanced synthesis of Man7/8/9, especially Man9, than KIF alone. These changes show that SWA in combination with KIF more efficiently inhibited ER α-mannosidase II, resulting in a synergistic effect on synthesis of Man7/8/9. In conclusion, combined KIF and SWA treatment in rice cell culture media can be an effective method for the production of rhGAA displaying dominantly Man7/8/9 glycoforms without genetic manipulation of glycosylation.

Ovotransferrin (OT), a multifunctional glycoprotein with defensive and protective activities, accounts for approximately 13% of chicken egg white proteins and is known as a major egg-associated allergen along with ovomucoid (OM). In contrast to the well-characterized N-glycans of OM, the N-glycan structure of OT has not been reported. Here, using HPLC equipped with a fluorescence detector and mass spectrometric analysis in combination with exoglycosidase digestion, we investigated the N-glycan type and branched pattern of OT, and compared them with those of OM. The HPLC peak area was used to calculate the relative quantity (%) of each glycan. Seventeen N-glycans, including 11 glycans (1 core structure and 10 complex-type oligosaccharides), that commonly exist in ovotransferrin and ovomucoid were identified. Six characteristic glycans (2 truncated structures, 1 complex-type, and 3 hybrid-type oligosaccharides) in OT and eight characteristic glycans in OM were classified. OT contains the following branched complex-type structures: mono-(13.2%), bi-(23.9%), tri-(9.0%), tetra-(2.7%), and penta-(2.8%) antennary oligosaccharides. However, OM contained mostly tri-(33.5%) and penta-(31.2%) antennary oligosaccharides. The N-glycan-containing bisecting N-acetylglucosamine comprised 43.4% and 79.8% of the total glycans in OT and OM, respectively. Moreover, using circular dichroism analysis, we observed that the secondary structure of the deglycosylated OT is quite different from that of the intact protein. To our knowledge, this is the first study to analyze N-glycans in OT in comparison with those of OM.

Most known osteoporosis medicines are effective for bone resorption, and so there is an increasing demand for medicines that stimulate bone formation. Watercress (N. officinale R. Br.) is widely used as a salad green and herbal remedy. This study analyzed a watercress extract using ultra-performance liquid chromatography/mass spectrometry, and identified a rutin as one of its major constituents. Osteogenic-related assays were used to compare the effects of watercress containing rutin (WCR) and rutin alone on the proliferation and differentiation of human osteoblast-like MG-63 cells. The reported data are expressed as percentages relative to the control value (medium alone; assigned as 100%). WCR increased cell proliferation to 125.0±4.0% (mean±SD), as assessed using a cell viability assay, and increased the activity of alkaline phosphatase, an early differentiation marker, to 222.3±33.8%. In addition, WCR increased the expression of collagen type I, another early differentiation marker, to 149.2±2.8%, and increased the degree of mineralization, a marker of the late process of differentiation, to 122.9±3.9%. Rutin alone also increased the activity of ALP (to 154.4±12.2%), the expression of collagen type I (to 126.6±6.2%), and the degree of mineralization (to 112.3±5.0%). Daidzein, which is reported to stimulate bone formation, was used as a positive control; the effects of WCR on proliferation and differentiation were significantly greater than those of daidzein. These results indicate that WCR and rutin can both induce bone formation via the differentiation of MG-63 cells. This is the first study demonstrating the effectiveness of either WCR or rutin as an osteoblast stimulant.

is a wild herb found mainly in the Himalayas, growing at altitudes of 1400-4200 m. is widely consumed as a vegetable and herbal medicine in Asia, but its effects on bone health have not been reported previously. This study investigated the effects of a hot-water extract of roots on bone formation. The hot-water extract significantly increased the proliferation of human osteoblast-like MG-63 cells and the stimulatory effects on osteoblast differentiation were noticeably greater for the hot-water extract than for daidzein (a positive control), as reflected by alkaline phosphatase activity, collagen content, and mineral deposition. Expression of the bone-remodeling marker osteocalcin production and bone microstructural parameters were significantly improved in Sprague-Dawley rats after oral treatment with the hot-water extract compared with their control (saline-administered) counterparts. The chemical compounds of the hot-water extract were characterized by liquid chromatography-mass spectrometry, and alliin, sinapic acid, and ferulic acid, which exert beneficial effects on bone health, were identified. These findings indicate that can be used as a natural resource for increasing bone formation. This is the first report of the anabolic effects of extracts on bone formation and.

Turnip (Brassica rapa L.) root ethanol extract (TRE) was prepared, and its chemical constituents were characterized by ultra-performance liquid chromatography and mass spectrometry. Thirteen glucosinolates (GSLs) were identified, comprising eight aliphatic, four indolic, and one aromatic compounds. The effects of these GSLs on bone formation were investigated in vitro by incubating human osteoblast-like MG-63 cells with TRE and then analyzing their viability, alkaline phosphatase (ALP) activity, collagen content, and mineralization and in vivo by administering TRE orally to normal young rats (500 mg/kg/day) and assessing subsequent changes in serum osteocalcin and bone microstructure in these animals. No TRE-related toxicity was found, and the levels of cell viability, ALP activity, collagen synthesis, and mineralization were significantly increased relative to the negative control. In particular, stimulatory effects on the differentiation of MG-63 cells were strongly enhanced as compared with a positive control (daidzein). Serum osteocalcin was also significantly increased, and some important bone microstructural parameters were improved in TRE-administered rats compared with their saline-administered counterparts. GSLs therefore appear to have a stimulatory effect on bone formation in both MG-63 cells and normal young rats. This is the first report on the usefulness of turnip root and its GSL compounds for bone formation.

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