We have reported two brain-specific agalactobiantennary N-linked sugar chains with the bisecting GlcNAc and α1-6Fuc residues, (GlcNAcβ1-2)_{0 or 1}Manα1-3(GlcNAcβ1-2Manα1-6)(GlcNAcβ1-4)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Here, the reason of the absence of Gal on the sugar chains was analyzed through the detection of the other complex type sugar chains in brain. Sia-Gal or Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains was not found. We therefore have investigated the substrate specificity of galactosyltransferase activities in brain by using as acceptor substrates pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and α1-6Fuc residues. While the β1-4galactosyltransferases in liver and kidney could utilize all nine oligosaccharides as substrates, the β1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both the bisecting GlcNAc and Fuc residues, but could utilize the other three acceptors. Similar results were obtained using glycopeptides with the agalactobiantennary sugar chains with or without the bisecting GlcNAc and α1-6Fuc residues as substrates. The β1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain specific sugar chains and different from β1-4galactosyltransferase-I.

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Liquid chromatography/mass spectrometry equipped with a graphitized carbon column is useful for the simultaneous analysis of oligosaccharides. By using capillary column and nanoelectrospray ion source, the method can be used for the oligosaccharide profiling of sub microgram quantities of glycoproteins. This oligosaccharide profiling is expected to be a powerful tool for the glycome analysis. We demonstrate a potential application of oligosaccharide profiling in glycomics with two examples, the structural analysis of N-linked oligosaccharides from a gel-separated glycoprotein, and the differential analysis of N-linked oligosaccharides in cells.

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“Functional glycomics”is a coming area of glycoscience and glycotechnology, which aims at elucidating biological roles of protein glycosylation from a holistic viewpoint, in a particular context of post-genomics and post-proteomics. However, analysis of glycans, i.e., the third bio-informative chain, should meet various difficulties due to many factors which other bio-macromolecules lack, e.g., branching and linkage isomerism. Recently, unique methods, which enable a large scale of identification methods of glycoproteins have been developed independently in two groups. In this chapter, advanced methods for glycoproteomics enabling identification of both core proteins and glycosylation sites are described as well as subsequent glycan profiling by means of lectin-affinity technologies.

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Elucidation of the biological role of glycan is one of the most important subjects to be resolved following the genome project. Glycosylation of proteins and lipids is performed in the Golgi apparatus by glycosyltransferases, which are responsible for synthesizing the huge diversity of complex oligosaccharides attached to glycoproteins and glycolipids. Our molecular evolutionary study showed that a prototype of each glycosyltransferase was conserved in Drosophila, suggesting common roles of glycans in humans and Drosophila.
RNA interference (RNAi) was first reported in 1998 as a biological response of C. elegans to exogeneous double-strand RNA (dsRNA), which induces sequence-specific gene silencing. It is a multi-step process including the generation of active small interfering RNA (siRNA) by reaction with an RNase III endonuclease, Dicer. The resulting 21- to 23-nt siRNA mediates degeneration of the complementary homologous RNA. RNAi has recently emerged as a powerful reverse genetics tool for studying gene function in many model organisms, including Drosophila.
To analyze the basic physiological functions of glycans, we established the Drosophila RNAi knock down system of glycosyltransferases and verified the system using the Drosophila proteoglycan UDP-galactose: β-xylose β1, 4galactosyltransferase I (dβ4GalTI). The expression of the target gene was disrupted specifically and the degree of interference was correlated with the phenotype. This study was the first to use reverse genetics, RNA interference, to study Drosophila glycosyltransferase systematically. The inducible glycosyltransferase RNAi knock down fly obtained using the GAL4-UAS system will open a new way for the analysis of the biological role of glycans.

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In-gel competitive reassociation (IGCR) technique is one of the DNA subtraction methods with a high capacity. We have improved the IGCR technique and overcame several disadvantages such as time-consuming and complex process with poor reproducibility. The improved IGCR method was applied to the analysis of human gastric adenocarcinoma genomic DNA. The genomic DNA library, which was constructed after the subtraction by IGCR of a normal gastric tissue genomic DNA from an adenocarcinoma genomic DNA in the same patient, contained 13.9% clones of Epstein-Barr virus (EBV) genomic DNA fragment, which is known to be a cause of Burkitt lymphoma. The quantification of EBV genomic DNA revealed that the detection of these EBV fragments originated in the gastric adenocarcinoma genomic DNA was due to the condensation from a few copies to about 100 thousand copies by the improved IGCR technique. This suggests that our improved IGCR technique can efficiently enrich differences between two genomic DNAs with high simplicity.

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Alkaline phosphatase (ALP) in serum of Korean native goat is composed of isoenzymes derived from intestine, bone and liver. From the band patterns of polyacrylamide gel electrophoresis (PAGE), the ALP organ type was classified into five patterns. The F type band detected by starch gel electrophoresis (SGE) originated from liver ALP. As the ALP type of goat serum has been classified into F type and 0/0 type by SGE, the F and 0/0 types could be classified respectively into F/F or F/0 types and F/0 or 0/0 types by PAGE (AlkPhor SYSTEM). The ALP types changed by aging. The 0/0 type individual, which was composed of only bone ALP, was undetectable in individuals over three years old. This suggests that skeletogeny is completed by the age of three in Korean native goat. As 0/0 type individuals died before three years old, this may indicate that liver ALP is necessary for their survival beyond three years of age. The Korean native goat population was characteristically divided into a high (over 500IU/l) and low (under 500IU/l) group by ALP activity value.

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A mechanism of oxidative modification of apolipoproteins (apo) in human very-low-density lipoprotein (VLDL) was investigated in vitro. Lipid peroxidation was promoted by cupric ion in VLDL. Modification of apoE and apoB-100 was observed in the VLDL oxidation. N^ε-(Hexanonyl)lysine, one of the lipid hydroperoxide-modified lysine residue, was detected in VLDL oxidized for 18 hours by immunoblot analysis and enzyme-linked immunosorbent assay. The results indicate that lysine residues of apoE and apoB-100 were modified by lipid hydroperoxides. The heparin-binding activity of apoE and apoB-100 which seems to reflect their low-density lipoprotein receptor (LDLr)-binding activity decreased in the VLDL oxidation. This demonstrates that the heparin-binding site of apoE and apoB-100 which includes lysine residues was modified in the VLDL oxidation. Our data suggest that lysine residues of the LDLr-binding site of apoE and apoB-100 might be damaged by lipid hydroperoxides produced in the VLDL oxidation.

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Cattle milk proteins are composed of casein components (Cn) such as α, β and κ-Cn, and whey components such as β-lactoglobulin (Lg). These milk protein components were separated by two-dimensional electrophoresis and compared with the separation patterns of yak, goat, sheep, deer, camel, llama, pig, horse and human milk. Based on our results, cattle and yak, goat and sheep, and camel and llama had very similar patterns, respectively.
As for components like β-Lg in deer, were separated at the same location as that of cattle, components like α- and β-Cn resembled the patterns of goat and sheep. Also, the separation patterns of κ-Cn were divided into two groups: animals (cattle, yak, goat, sheep and llama) whose milk separated near pI6.3, and animals (deer, horse and human) whose milk separated near pI6.9.

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