Earticle

초록

영어

Glycans, as attached to proteins and lipids on the cell surface, play crucial roles in various physiological phenomena such as cell-cell communication, differentiation and development. Recently, they have been highlighted as stem cell markers used to assess the pluripotency status. However, analysis of cell surface glycans requires difficult and laborious processes for the isolation of plasma membrane from other cellular components. Especially, efficient removal of endoplasmic reticulum (ER) and Golgi membranes are required since the high amount of high-mannose type glycans in ER has hampered the analysis of complex type glycans on the cell surface. In the present study, we employed the simple adhesion-based method for the isolation of plasma membrane to analyze the glycans coating on the cell surface efficiently. First, cells adhered to an adsorbed layer of polylysine on glass plates and then were lysed by hypotonic pressure method using ice-cold distilled water. After the cell lysis and successive washing steps, plasma membrane fractions containing glycopeptides could be collected from the plate by trypsin digestions. The purity of the isolated plasma membranes was evaluated by fluorescence imaging using organelle-specific probes and optimal isolation conditions were established to minimize the contamination of ER and Golgi fractions. Using this method, N-glycans obtained from the plasma membranes of embryonic carcinoma NTERA-2 and Chinese hamster ovary (CHO) cells were analyzed and compared to N-glycan profiles derived from the corresponding total cell fractions. Complex-type glycans capped with terminal sialic acids were observed as major populations in plasma membrane fractions whereas high-mannose type glycans mainly detected in the profiles of total cells. Taken together, easy and rapid method to isolate plasma membrane was successfully employed and optimized for the analysis of the cell surface N-glycans, which will contribute to elucidating the biological implications of cell surface glycan changes.

저자

• Ji-Young Mun [ Systems & Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) ]
• Kyung Jin Lee [ Systems & Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) ]
• Hoon Seo [ Systems & Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) ]
• Ohsuk Kwonand [ Systems & Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) ]
• Doo-Byoung Oh [ Systems & Synthetic Biology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) ]

참고문헌

발행기관

간행물

표지보기 관심저널 등록

• 간행물명

  한국당과학회 학술대회

• 간기

  연간

• 수록기간

  2006~2022

• 십진분류

  KDC 517 DDC 614