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N-Linked Glycosylation of Ribosomal Protein S3 (rpS3) Is Required for Secretion through ER-Golgi Dependent Pathway

  • 이민선 (Lee, Min Seon, 대학원 생명과학과 생화학전공)

초록/요약

RpS3 is a component of the 40S ribosomal small subunit and consists of 243 amino acids. RpS3 has a role in translation as well as extra-ribosomal functions such as apoptosis and DNA repair. Previous data have been revealed that the functions of rpS3 are regulated by post-translational modifications including phosphorylation, methylation and ubiquitination. Here, it was found that rpS3 is glycosylated at Asn165 residue through mass spectrometry analysis. And the secretion of rpS3 was inhibited by both brefeldin A which is the ER-Golgi transport inhibitor, and tunicamycin which is a N-linked glycosylation inhibitor. It was demonstrated that the N–linked glycosylation of rpS3 is necessary for its secretion into the culture media via the ER-Golgi dependent pathway. Furthermore, it was found that rpS3 was bound to Concanavalin A, a lectin protein having carbohydrate binding ability, and PNGase F treatment shifted down the secreted rpS3 into lower molecular weight band. In addition, it was shown that N165G mutant of rpS3 reduces its secretion level compared to wild-type. Through in vitro binding assay, rpS3 was identified to form homodimers via N-terminal region (rpS3:1-85) and a middle region (rpS3:95-158). These results suggested that Asn 165 residue of rpS3 is a critical site for N–linked glycosylation and the ER - Golgi secretion pathway.

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초록/요약

rpS3는 진핵생물의 40S 리보솜을 구성하는 단백질 중의 하나이며 243개의 아미노산으로 이루어져 있다. rpS3는 번역의 역할 뿐만 아니라 손상된 DNA의 복구와 세포사멸에도 기능을 한다. rpS3의 기능은 인산화, 메틸화 및 유비퀴틴 등의 번역 후 변형에 의해 조절된다. 소포체에서 골지체로의 수송을 막는 Brefeldin A와 N-연결당화를 막는 Tunicamycin을 처리했을 때 RpS3의 분비가 감소했다. 이를 통해 rpS3의 N-연결당화가 소포체와 골지체를 통한 분비에 중요한 역할을 하는 것을 확인했다. 또한 분비된 RpS3에 PNGase F를 처리하였을 때 RpS3의 사이즈가 줄었고, 당에 결합하는 단백질인 Concanavalin A렉틴에 결합하였다. 질량 분석기를 통해 rpS3의 165번째 asparagine에 당화가 되는 것을 확인했다. 또한 HT1080세포에서 rpS3의 N165G돌연변이가 야생형에 비해 분비가 감소했다. In vitro binding을 통해 rpS3가 N-말단부분(rpS3:1-85)과 중간부분(rpS3:96-159)을 통해 동종이량체를 형성하는 것을 확인했다. 이러한 결과는 rpS3의 아스파라긴 165번째 잔기가 N-연결사슬과 소포체와 골지체를 통한 분비에 중요한 역할을 하는 것을 제안한다.

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목차

List of Tables ---------------------------------------------------------------------------------ⅰ
List of Figures ------------------------------------------------------------------------------ ⅱ
Abstract ------------------------------------------------------------------------------------- ⅲ
Introduction ---------------------------------------------------------------------------------- 1
Materials and methods --------------------------------------------------------------------- 7
Results ---------------------------------------------------------------------------------------- 14
Secretion of rpS3 is inhibited by both BFA(Brefeldin A) and Tunicamycin --------------------------------------------------------------------------------------------- 14
The secreted rpS3 is N-glycosylated. --------------------------------------------- 17
Enrichment of secreted rpS3 for identification of N-linked glycosylation sites. ------------------------------------------------------------------------------------- 20
Identification of N-linked glycosylation sites in rpS3 by mass spectrometry analysis. -------------------------------------------------------------------------------- 22
The effect of N-linked glycosylation on rpS3 secretion. ---------------------- 28
Homodimerization of rpS3 protein. ---------------------------------------------- 32
Discussion ------------------------------------------------------------------------------------ 35
References ----------------------------------------------------------------------------------- 38
Abstract in Korean------------------------------------------------------------------------- 42
Acknowledgement-------------------------------------------------------------------------- 43

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