• YAKHAK HOEJI
• /
• v.44 no.6
• /
• pp.588-594
• /
• 2000

To investigate the effects of iota-carrageenan (CAR) and/or alum on the adjuvancity as well as the structural difference of oligosaccharide on the IgG2b in the adjuvant effect, C57BL/6 mice were immunized twice with fetuin as a model antigen. CAR alone showed no significant effect on induction of antibody except IgG1. In contrast, Alum-CAR (after mixing of antigen-Alum, CAR adjuvant was prepared) and CAR-Alum (after formulation of antigen-CAR, Alum adjuvant was prepared) enhanced production of antibody, especially, IgG2b. After separation of IgG2b, changes of glycosylation were investigated using enzymelinked lectin assay. High affinity of IgG2b to N-acetylneuraminic acid, galactose and mannose-specific lectin were induced by CAR-Alum adjuvant, however, the affinity of IgG2b induced by CAR-Alum to GlcNAc and GalNAc-specific lectin were much less than that induced by Alum-CAR.

• YAKHAK HOEJI
• /
• v.44 no.2
• /
• pp.197-203
• /
• 2000

The enzymatic properties of $\beta$-galactosidases from Aspergillus oryzae, bovine liver and Saccharomyces pragilis have been studied using enzyme-linked lectin assay based on the RC $A_{120}$ and BS-II lectins which specifically bind to terminal galactose and GlcNAc residue, respectively. Asialofetuin, a monomeric glycoprotein with approximately 48 kDa in molecular weight, was used as a substrate. This glycoprotein contains three N-linked triantennary complex type carbohydrate chains with each of which terminating in Ga1$\beta$P1 longrightarrow4G1cNAc (74%). Their optimal pHs were 3.5 and 6.5 (A. oryzae), and 3.5~5.5 (bovine liver and S. fragilis) at 37$^{\circ}C$ during 24 hrs, and the effective concentrations were 0.9, 2.9, and 1.7 mg/ml, respectively The enzyme from A oryzae requires 100 mM N $a^{+}$ or $K^{+}$, while the enzyme from bovine liver requires $Ba^{2+}$ for activity. However all of the three $\beta$-galactosidases were inactivated by SDS and C $u^{2+}$. These results indicate that the hydrolysis of glycoprotein such as asialofetuin depends on the reaction conditions of $\beta$-galactosidases and some metal ions. ions.

• Asian-Australasian Journal of Animal Sciences
• /
• v.30 no.8
• /
• pp.1086-1092
• /
• 2017

Objective: O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzes the addition of O-GlcNAc and GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling and transcription. Pig OGT is located near the region of chromosome X that affects follicle stimulating hormone level and testes size. The objective of this study was to find the variations of OGT between European and Chinese pigs. Methods: Pigs were tested initially for polymorphism in OGT among European and Chinese pigs by polymerase chain reaction and sequencing at the U.S. Meat Animal Research Center (USMARC). The polymorphism was also determined in an independent population of pigs including European and Chinese Meishan (ME) breeds at the National Institute of Animal Science (NIAS, RDA, Korea). Results: The intron 20 of OGT from European and Chinese pigs was 514 and 233 bp, respectively, in the pigs tested initially. They included 1 White composite (WC) boar and 7 sows ($2Minzu{\times}WC$, $2Duroc\;[DU]{\times}WC$, $2ME{\times}WC$, $1Fengzing{\times}WC$) at USMARC. The 281-bp difference was due to an inserted 276-bp element and GACTT in European pigs. When additional WC and ME boars, the grandparents that were used to generate the $1/2ME{\times}1/2WC$ parents, and the 84 boars of 16 litters from mating of $1/2ME{\times}1/2WC$ parents were analyzed, the breeds of origin of X chromosome quantitative trait locus (QTL) were confirmed. The polymorphism was determined in an independent population of pigs including DU, Landrace, Yorkshire, and ME breeds at NIAS. OGT was placed at position 67 cM on the chromosome X of the USMARC swine linkage map. Conclusion: There was complete concordance with the insertion in European pigs at USMARC and NIAS. This polymorphism could be a useful marker to identify the breed of origin of X chromosome QTL in pigs produced by crossbreeding Chinese and European pigs.

• Korean Journal of Veterinary Research
• /
• v.43 no.1
• /
• pp.139-144
• /
• 2003

A chitinase cDNA named CHT1 was cloned from the hard tick, Haemaphysalis longicornis, and the enzymatic properties of its recombinant proteins were characterized. The CHT1 cDNA encodes 930 amino-acid (aa) residues including a 22 aa putative signal peptide, with the calculated molecular mass of the putative mature protein 104 kDa. The E coli-expressed rCHT1 exhibited weak chitinolytic activity against $4MU-(GlcNAc)_3$. The rCHT1 protein with higher activity was obtained using recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), which expresses rCHT1 under polyhedrin promoter. These findings suggest that the rCHT1 expressed in baculovirus-mediated Sf 9 cells has a high activity than E coli-expressed rCHT1.

• 한국당과학회:학술대회논문집
• /
• 2006.11a
• /
• pp.40-40
• /
• 2006

• 한국당과학회:학술대회논문집
• /
• 2006.11a
• /
• pp.42-43
• /
• 2006

• The Korean Journal of Zoology
• /
• v.36 no.1
• /
• pp.123-132
• /
• 1993

곤충의 탈피작용에서 일어나는 키틴 분해대사를 구명하기 위한 목적으로 배추흰나비 (Pieris rupae L.)에서 3종의 서N-acetylglucosaminldase(El, Ell, Elll)를 분리, 정제하였다. Polvacrylamide gel 상에서 이들 효소의 활성은 기질과 triphenvltetrazolium chloride와의 반응 결과 생기는 발색정도로 확인하였다. 각각의 분자량은 76,000, 55,000, 35,000 da, pl 값은 모두 5.8이었으며 후p$\beta$GlcNAc와 각p$\beta$GaINAc에 대해서 다같이 기질특이성을 나타내었다. 또한 El과 Elll는 탈피 시기와 일치하는 전용 말기에 최대 활성을 나타내었으며 Ell는 용화 직후에 최대 활성을 나타내었다.

• BMB Reports
• /
• v.51 no.12
• /
• pp.609-610
• /
• 2018

Glycosylation is one form of protein modification and plays a key role in protein stability, function, signaling regulation and even cancer. NleB and SseK are bacterial effector proteins and possess glycosyltransferase activity, even though they have different substrate preferences. NleB/SseKs transfer the GlcNAc sugar to an arginine residue of host proteins, leading to reduced $NF-{\kappa}B-dependent$ responses. By combining X-ray crystallography, NMR, molecular dynamics, enzyme kinetic assays and in vivo experiments, we demonstrated that a conserved HEN (His-Glu-Asn) motif in the active site plays a key role in enzyme catalysis and virulence. The lid-domain regulates the opening and closing of the active site and the HLH domain determines the substrate specificity. Our findings provide evidence for the enzymatic mechanism by which arginine can be glycosylated by SseK/NleB enzymes.

• Journal of Life Science
• /
• v.28 no.4
• /
• pp.389-397
• /
• 2018

The structure of glycan residues attached to glycoproteins can influence the biological activity, stability, and safety of pharmaceutical proteins delivered from transgenic pig milk. The production of therapeutic glycoprotein in transgenic livestock animals is limited, as the glycosylation of mammary gland cells and the production of glycoproteins with the desired homogeneous glycoform remain a challenge. The ${\beta}$-1,3-N-acetylglucosaminylatransferase1 (B3GNT1) gene is an important enzyme that attaches N-acetylglucosamine (GlcNAc) to galactose (Gal) residues for protein glycosylation; however, there is limited information about pig glycosyltransferases. Therefore, we cloned the pig B3GNT1 (pB3GNT1) and investigated its functional properties that could attach N-acetylglucosamine to galactose residue. Using several different primers, a partial pB3GNT1 mRNA sequence containing the full open reading frame (ORF) was isolated from liver tissue. The ORF of pB3GNT1 contained 1,248 nucleotides and encoded 415 amino acid residues. Organ-dependent expression of the pB3GNT1 gene was confirmed in various organs from adult and juvenile pigs. The pB3GNT1 mRNA expression level was high in the muscles of the heart and small intestine but was lower in the lungs. For functional characterization of pB3GNT1, we established a stable expression of the pB3GNT1 gene in the porcine kidney cell line (PK-15). As a result, it was suggested that the glycosylation pattern of pB3GNT1 expression in PK-15 cells did not affect the total sialic acid level but increased the poly N-acetyllactosamine level. The results of this study can be used to produce glycoproteins with improved properties and therapeutic potential for the generation of desired glycosylation using transgenic pigs as bioreactors.

• Journal of The Korean Society of Inherited Metabolic disease
• /
• v.5 no.1
• /
• pp.65-75
• /
• 2005

Purpose: To understand genetic differences and similarities between mucolipidosis and control. Methods: Using the fibroblast of the mucolipidosis II and control, forward and reverse subtracted libraries were constructed. Among these clones, we investigated mutations in the GNPTA (MGC4170) gene, which codes for the ${\alpha}/{\beta}$ subunits of phosphotransferase, and in the GNPTAG gene, which codes for the ${\gamma}$ subunits in 5 Korean patients with mucolipidosis type II or IIIA. Result: Several differentially expressed cDNAs were cloned and their sequences were determined. Mutation analysis of the interested gene, GNPTA was performed and we identified 7 mutations in the GNPTA gene, but none in the GNPTAG gene. The mutations in type II patients included p.Q104X(c.310C>T), p.R1189X(c.3565C>T), p.S1058X(c.3173C>G), p.W894X(c.2681G>A) and p.H1158fsX15(c.3474_3475delTA), all of which are non-sense or frame shift mutations. However, a splicing site mutation, IVS13+1G>A (c.2715+1G>A) was detected along with a non-sense or a frame shift mutation (p.R1189X or p.E858fsX3(c.2574_2575delGA)) in two mucolipidosis type IIIA patients. Conclusion: This report shows that mutations in the GNPTA gene coding for the ${\alpha}{\beta}$subunits of phosphotransferase, and not mutations in the GNPTAG gene, account for most of mutations found in Korean patients with mucolipidosis type II or IIIA.