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돼지 유래의 β-1,4-N-acetylglucosaminyltransferase A (MGAT4A) 유전자의 동정 및 기능 분석
김지윤(Ji-Youn Kim),황환진(Hwan-Jin Hwang),정학재(Hak-Jae Chung),박미령(Mi-Ryung Park),변승준(Sung June Byun),김경운(Kyung-Woon Kim) 한국생명과학회 2016 생명과학회지 Vol.26 No.3
인체치료용 당단백질은 바이오의약품에 있어서 중요한 요소이며 특히, 단백질 말단에 결합되어있는 시알산은 의약품의 체내 활성이나 안정성에 큰 영향을 미친다. 최근 돼지유즙은 바이오의약품을 생산하기 위한 생체반응기로써 주목받고 있다. β-1,4-N-acetylglucosaminyltransferase A (MGAT4A)는 재조합단백질이 가지는 시알산 함량을 늘리기 위한 필수 효소 중 하나이다. 그러나 돼지 MGAT4A는 아직도 그 서열이나 기능이 불분명하다. 따라서 이번 연구에서는 돼지 MGAT4A의 서열 동정 및 기능분석을 하였다. 돼지 MGAT4A는 535개의 아미노산을 코딩하는 1,638개의 염기서열로 이루어져 있으며, 일반적인 당전이효소들의 특징인 type II membrane topology의 특성을 가지고 있다. Real-time PCR분석법을 통하여, 지금까지 알려진 것과는 조금 다르게 MGAT4A 유전자가 간이나 유선에서 많은 발현을 보였으나 소장이나 위, 방광에서는 그 발현량이 적다는 것도 확인하였다. 또한, 효소의 기능 분석을 위하여 PK-15 세포주에서 MGAT4A효소의 과발현을 유도하였다. 과발현이 확인된 세포주로 렉틴을 이용한 면역형광염색법과 ELISA방법으로 MGAT4A효소의 과발현이 β-1,4-N-acetylglucosamine의 함량을 증가시켰음을 확인하였다. 또한 기질반응을 통해 bi-antennary structure에 대한 반응성도 확인하였으며, MGAT4A의 과발현이 유도된 세포에서 약 3배 이상의 높은 기질 반응성을 보였다. 이러한 연구는 생체반응기로써 돼지를 이용하는데 있어서 중요한 기반이 될 것이라고 생각한다. Glycan modification is important in pharmaceutical industry. Especially, sialic acid affects the bioactivity and stability of medicine. Milk of pig has been used as bioreactor to produce various pharmaceutical proteins. Therefore, it is necessary to modify the glycan chain in pig mammary grand. β-1,4-N-Acetylglucosaminyltransferase A (pMGAT4A) is one of the essential enzymes for increase of sialic acid content, but pig MGAT4A is unclear. In this study, the pMGAT4A was identified and characterized. The pMGAT4A has 1638 nucleotides encoding 535 amino acids and type II membrane topology, which is one of the common features in many glycosyltransferases. The gene was strongly expressed in liver and mammary gland, whereas was weakly expressed in small intestine, stomach and bladder. For functional test, HA-tagged MGAT4A was over-expressed in porcine kidney (PK-15) cell line. Forced expression of pMGAT4A gene was identified by qPCR, and we identified that pMGAT4A is located in Golgi complex by co- staining with HA antibody and BODIPY TR ceramide. In addition, we identified the increase of mannose-β-1,4-N-acetylglucosamine structure by ELISA and immunofluorescence using Datura stramonium agglutinin (DSA), which recognizes mannose-β-1,4-Nacetylglucosamine. Through the specific activity analysis, we showed that pMGAT4A modified bi-antennary to tri-antennary. This event affects sialic acid content. Therefore, we thought that over-expression of pMGAT4A will be necessary in pig mammary grand for improved medicine.
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Kim, Cheorl-Ho,Kang, Bong Seok,Kim, Yeon-Jeong,Shim, Jae-Kyoung,Song, Eun-Young,Park, Young-Guk,Lee, Young-Choon,Nam, Kyung-Soo,Kim, June-Ki,Lee, tae-Kyun,Chung, Tae-Wha The Korea Science and Technology Center 1998 BMB Reports Vol.31 No.6
In a previous paper (Kim et al., 1996a), the immediate 5'-flanking region and coding region of the human UDP-N-acetylglucosamine:-D-mannoside-1, 4-N-acetylglucosaminyltransferase III(N-acetylglucosaminyltransferase-III); GnT-III) gene was reported, isolated and analyzed. Herein, we report on amplification of a new 5'-noncoding region of the GnT-III mRNA by single-strand ligation to single-stranded cDNA-PCR (5'-RACE PCR) using poly(A)+ RNA isolated form human fetal liver cells. A cDNA clone was obtained with 5' sequences (96 bp) that diverged seven nucleotides upstream from the ATG(+1) start codon. A concensus splice junction sequence, TCTCCCGCAG, was found immediately 5' to the position where the sequences of the cDNA diverged. The result suggested the presence of an intron in the 5'-noncoding region and that the cDNA was an incompletely reverse-transcribed cDNA product derived from an mRNA containing a new noncoding exon. When mRAN expression of GnT-III in various human tissues and cancer cell lines was examined, Northern blot analysis indicated high expression levels of GnT-III in human fetal kidney and brain tissues, as well as for a number of leukemia and lymphoma cancer cell lines. Promoter activities of the 5'-flanking regions of exon 1 and the new noncoding region were measured in a human hepatoma cell line, HepG2 by luciferase assays. The 5'-flanking region of exon 1 was the most active, whilst that of exon 2 was inactive.
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Kang, Bong Seok,Kim, Yeon-Jeong,Shim, Jae-Kyoung,Song, Eun-Young,Park, Young-Guk,Lee, Young-Choon,Nam, Kyung-Soo,Kim, June-Ki,Lee, Tae-Kyun,Chung, Tae-Wha,Kim, Cheorl-Ho 경북대학교 병원 2001 경북대학교병원의학연구소논문집 Vol.5 No.1
In a previous paper(Kim et al.,1996a),the immediate 5'-flanking region and coding region of human UDP-N-acetylglucosamine: -D-mannoside-1,4-N-acetylglucosaminyltransferaseⅢ(N-acetylglucosaminyltransferase-Ⅲ;GnT-Ⅲ)gene was reported,isolated and analyzed. Herein, we report on amplification of a new 5'-noncoding region of the GnT-Ⅲ mRNA by single-strand ligation to single-standed cDNA-PCR(5'-RACE PCR) using poly(A)+ RNA isolated from human fetal liver cells.A cDNA clone was obstained with 5'sequences(96bp)that diverged seven nucleotides upstream from the ATG(+1) start codon.A concensus splice junction sequence, TCTCCCGCAG,was found immediately 5' to the position where the sequences of the cDNA diverged. The result suggested the presence of an intron in the 5'-noncoding region and that the cDNA was an incompletely reversetranscribed cDNA product derived from an mRNA containing a new noncoding exon. When mRNA expression of GnT-Ⅲ in various human tissues and cancer cell lines was examined, Northern blot analysis indicated high expression levels of GnT-Ⅲ in human fetal kidney and brain tissues,as well as for a number of leukemia and lymphoma cancer cell lines. Promoter activities of the 5'-flanking regions of exon 1 and the new noncoding region were measured in a human hepatoma cell line, HepG2, by luciferase assays. The 5'-flanking region of exon 1 was the most active, whilst that of exon 2 was inactive.
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