• 한국미생물·생명공학회지
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• 제42권2호
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• pp.190-193
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• 2014

CMP-Neu5Ac synthetase는 sialyated 된 glycoconjugates의 전구체로 사용되는 CMP-Neu5Ac를 합성하는데 관여하는 주요 효소이다. Escherichia coli K1에서 유래한 CMP-Neu5Ac synthetase 유전자 (neuA)는 평소 E. coli BL21(DE3)에서 비수용성으로 생성되는데, 이를 수용성 단백질로 생산하고자 여러 가지 샤페론 단백질 동시 발현기술을 이용하였다. 이를 위해, GroEL-ES와 DnaK-DnaJ-GrpE를 암호화하는 pG-KJE8 plasmid와 neuA를 동시 형질전환 시켰고 0.01 mM IPTG와 0.005 mg/ml의 L-arabinose로 유도하여 $20^{\circ}C$에서 발현시켰다. 그 결과, E. coli에서의 수용성 CMP-Neu5Ac Synthetase 생산이 현저하게 증가하였다.

• 한국발생생물학회지:발생과생식
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• 제2권2호
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• pp.149-155
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• 1998

폴리시알린산 (polysialic acid)은 미생물로부터 인간에 이르기까지 다양한 세포의 세포 표면 복합당사슬에 공유 결합하는 당사슬의 일종이다. 최근 성게 난자의 젤리층과 성게 난자의 표면에서 폴리시알린산이 연결된 당단백질이 발견됨으로써 폴리시알린산의 생합성과 그 기능에 대한 의문이 제기되었다. 둥근성게의 난자와 배에서 추출한 효소액과 CMP-[$^{14}$ C]Neu5Ac를 기질로 사용하여 Neu5Ac를 endogenous acceptor에 전달하는 CMP-Neu5Ac:poly-$\alpha$2, 8 sialosyl sialyltransferase (polyST)를 발견하였다. polyST는 20mM MOPS (pH 7.0), 2$0^{\circ}C$에서 최적 활성을 나타냈다. 10 mM $Mg^2$$^{+}$를 효소 반응액에 첨가하면 polyST의 황성은 2.7배 증가하였다. poiyST는 또한 포유류의 gang1ioside인 GD3에 폴리시알린산을 합성할 수 있었다. 이와 같은 폴리시알린산이 존재하는 ganglioside가 현재까지 자연계에서 알려진 바가 없다는 사실은 하나의 polyST가 endogenous acceptor와 ganglioside에 폴리시알린산을 합성했다고 결론지을 수 있다. 과량의 GD3를 exogenous acceptor로 사용하여 둥근성게에 존재하는 polyST의 활성을 조사한 결과 mesenchyme 포배기 때부터 그 활성이 급격히 증가하고 낭배기 때에 최고에 도달했다. 성게의 배발생 때에 polyST의 활성이 조절된다는 것은 낭배기 때에 일어나는 세포나 조직 사이에 일어나는 변화와 초기 단계 spicule 형성에 어떤 중요한 기능을 담당하고 있을 가능성이 있다.

• BMB Reports
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• 제41권1호
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• pp.48-54
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• 2008

PM0188 is a newly identified sialyltransferase from P. multocida which transfers sialic acid from cytidine 5'-monophosphonuraminic acid (CMP-NeuAc) to an acceptor sugar. Although sialyltransferases are involved in important biological functions like cell-cell recognition, cell differentiation and receptor-ligand interactions, little is known about their catalytic mechanism. Here, we report the X-ray crystal structures of PM0188 in the presence of an acceptor sugar and a donor sugar analogue, revealing the precise mechanism of sialic acid transfer. Site-directed mutagenesis, kinetic assays, and structural analysis show that Asp141, His311, Glu338, Ser355 and Ser356 are important catalytic residues; Asp141 is especially crucial as it acts as a general base. These complex structures provide insights into the mechanism of sialyltransferases and the structure-based design of specific inhibitors.

• Molecules and Cells
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• 제23권2호
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• pp.138-144
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• 2007

Previously we showed that the human GM3 synthase gene was expressed during the induction of megakaryocytic differentiation in human leukemia K562 cells by phorbol 12-myristate 13-acetate (PMA). In this study we found that treatment of PMA-induced K562 cells with $G{\ddot{o}}6976$, a specific inhibitor of PKC, and U0126, an inhibitor of the extracellular signal-regulated kinase (ERK) reduced expression of GM3 synthase, whereas wortmannin, an inhibitor of phosphoinositide 3-kinase (PI3K) did not. Moreover, activation of ERK and cAMP response element binding protein (CREB) was prevented by pretreatment with $G{\ddot{o}}6976$ and U0126. PMA stimulated the promoter activity of the 5'-flanking region from -177 to -83 region of the GM3 synthase gene, and mutation or deletion of a CREB site located around -143 of the promoter reduced PMA-stimulated promoter activity, as did the inhibitors $G{\ddot{o}}6976$ and U0126. Our results demonstrate that induction of GM3 synthase during megakaryocytic differentiation in PMA-stimulated human leukemia K562 cells depends upon the PKC/ERK/CREB pathway.

• Archives of Pharmacal Research
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• 제23권5호
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• pp.525-530
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• 2000

mST3GaIV synthesizes ganglioside GM3, the precursor for simple and complex a- and b- series gangliosides, and the expression and regulation of mST3GaIV (CMP-NeuAc: lactosylceramide $\alpha$2,3-sialyltransferase) activity is central to the production of almost all gangliosides, a class of glycosphingolipids implicated in variety of cellular processes such as transmembrane signaling, synaptic transmission, specialized membrane domain formation and cell-cell interactions. To understand the developmental expression of mST3GaIV in mice, we investigated the spatial and temporal expression of mST3GaIV mRNA during the mouse embryogenesis [embryonic (E) days; 19, E11, E13, E15] by in situ hybridization with digoxigenin-labeled RNA probes. All tissues from 19 and E11 were positive for mST3GaIV mRNA. On E13, mST3GaIV mRNA was expressed in various neural and non-neural tissues. In contrast to these, on E15, the telencephalon and liver produced a strong expression of mST3GaIV which was a quite similar to that of E13. In this stage, mST3GaIV mRNA was also expressed in some non-neural tissues. These data indicate that mST3GaIV is differently expressed at developmental stages of embryo, and this may be importantly related with regulation of organogenesis in mice.