• BMB Reports
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• v.41 no.1
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• pp.72-78
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• 2008

Human NeuNAc-9-P synthase is a two-domain protein with ability to synthesize both NeuNAc-9-P and KDN-9-P. Its mouse counterpart differs by only 20 out of 359 amino acids but does not produce KDN-9-P. By replacing the AFL domain of the human NeuNAc-9-P synthase which accommodates 12 of these differences, with the mouse AFL domain we examined its importance for the secondary KDN-9-P synthetic activity. The chimeric protein retained almost half of the ability of the human enzyme for KDN-9-P synthesis while the NeuNAc-9-P production was reduced to less than 10%. Data from the homology modeling and the effect of divalent ions and temperature on the enzyme activities suggest conformational differences between the human and mouse AFL domains that alter the shape of the cavity accommodating the substrates. Therefore, although the AFL domain itself does not define the ability of the human enzyme for KDN-9-P synthesis, it is important for both activities by aiding optimal positioning of the substrates.

• 한국전자현미경학회:학술대회논문집
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• 2000.05a
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• pp.33-33
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• 2000

• Applied Microscopy
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• v.30 no.1
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• pp.101-111
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• 2000

In this study, the distribution of lectin receptors in Paragonimus westermani tissue was explored using colloidal gold label complexed with lectin WGA purified from wheat germ (Triticum vulgare). The lectin WGA gold complex, shown to recognize GlcNAc (N-acetylgalactosamine) and NeuNAc (N-acetylneuraminic acid) regions, was applied to detect binding sites in Lowicryl HM 20 sections viewed under electron microscope. Labeled sections of the metacercaria revealed gold particles specifically distributed on the tegumental syncytium and lamella of the excretory canal. Labeling of young adult tissue was then quantified and compared to that of adult worm tissue. Adult worm tissue sections resulted in specific gold particle distribution on the lamella of caecal epithelium and excretory canal. These results indicate that lectin WGA receptors are located in the tegumental syncytium and lamella of the excretory canal of the metacercariae, and in the lamella of the caecum and excretory canal of the young adult and adult. Therefore, the GlcNAc and NeuNAc regions in the tegumental syncytium appear to be functionally associated with cell-recognition and protection from the immune system of the host, and linked with membrane transport and absorption of nutrients in the lamella of the excretaory canal and caecal epithelia.

• Applied Microscopy
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• v.31 no.1
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• pp.49-57
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• 2001

In this study, the distribution of lectin receptors in culutured fibroblast was explored using colloidal gold label complexed with lectin WGA purified from wheat germ (Triticum vulgare). The lectin WGA gold complex, shown to recognize GlcNAc (N-acetylgalactosamine) and NeuNAc (N-acetylneuraminic acid) regions, was applied to detect binding sites in Lowicryl HM 20 sections viewed under electron microscope Labeled sections of the culutured fibroblast revealed gold particles specifically distributed on the cytoplasm and cell surface of the fibroblast. Labeling of 24 hours culutured fibroblast was then quantified and compared to that of 72 hours culutured fibroblast. 24 hours culutured fibroblast sections resulted in specific gold particle distribution on the cytoplasmic vesicle of the culutured fibroblast. These results indicate that lectin WGA receptors are located in the cytoplasmic vesicle and cell surface of the 24 hours culutured fibroblast, and on the cell surface of the 72 hours culutured fibroblast. Therefore, the GlcNAc and NeuNAc regions on the cell surface appear to be functionally associated with cell-recognition and protection from other cell of the tissue, and linked with secretion and exocytosis of the fibroblast cytoplasm.

• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.142-148
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• 2004

Erythropoietin is a main regulator of human erythropoiesis. Recombinant human erythropoietin (rhEPO) is one of the glycoproteins produced in animal cells, and it has oligo saccharides chains which comprise about 40％ of its molecular mass. Because the content of sialic acid can extend circulatory lifetime, the high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the sialylation of rhEPO produced by chinese hamster ovary cell culture was maximized by supplementing the culture medium with N-acetylm-annosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en), a sialidase inhibitor. Feeding of 20 mM ManNAc/0.5 mM NeuAc2en into culture medium increased the sialic acid content by nearly tenfold compared with unsupplemented medium. This effect was achieved without affecting the cell growth or product yield. Six erythropoietin fractions differing in sialic acid content, ranging from 11∼15％ of EPO, were identified from chinese hamster ovary cell-derived rhEPO by mono Q column chromatography. It was found that, at 20 mM ManNAc/0.5 mM NeuAc2en feeding, productivity of hyper-glycosylated EPO increased up to 50％, compared with the unsupplemented medium.

• Applied Microscopy
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• v.32 no.2
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• pp.163-170
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• 2002

Experimatal maxillary sinusitis was induced in New Zealand white rabbits by blocking the maxillary sinus ostium. The distribution of lectin receptors was explored in the mucosa with induced maxillary sinusitis using colloidal gold label complex with lectin WGA purified from wheat germ (Triticum vulgaris). The lectin WGA gold complex, shown to recognize GlcNac (N-acetylglucosamine) and NeuNAc (N-acetylneuraminic acid) regions, was applied to detect binding sites in Lowicryl HM 20 sections and viewed under the electron microscope. An increased height of the cylindric cells, ciliary loss and hyperplasia of the secretory cells were observed. Examination of normal sinus mucosa labeled with gold-labeled lectins showed the distribution of sialoglycoconjugates to be mainly in the ciliary layer and the granules in the secretory cells. Inflamed mucosa had increased labeling intensity of gold-labeled WGA in the cilia and the secretory granules. These results indicate that lectin WGA receptors are located in the cilia and secretory granules. Specific changes in the lectin binding pattern were apparent in the inflamed mucosa in the experimentally induced acute sinusitis, in comparison with normal mucosa, conceivably as a part of host defense reactions.