Ling V, Luxenberg D, Wang J, Nickbarg E, Leenen P J, Neben S, Kobayashi M
Genetics Institute, Cambridge, MA 02140, USA.
Eur J Immunol. 1997 Feb;27(2):509-14. doi: 10.1002/eji.1830270223.
The monoclonal antibody ER-MP12 was recently described to recognize an antigen present on cell subpopulations of adult mouse bone marrow, including pluripotent hematopoietic stem cells. In an effort to understand the function of ER-MP12 antigen in hematopoiesis, we used biochemical and physical methods to determine its identity. ER-MP12 antigen was isolated by immunoprecipitation from FDCP-1 cell membrane proteins, yielding a glycosylated 113-kDa band upon analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Thirteen peptides derived from trypsinized ER-MP12 antigen were analyzed by electrospray ionization mass spectrometry and compared to a protein sequence database. The search revealed the identity of the ER-MP12 antigen as platelet endothelial cell adhesion molecule-1, CD31 (PECAM-1). This result was subsequently confirmed by Edman sequencing of a single ER-MP12 peptide fragment followed by comparison with PECAM-1 sequence. In addition, flow cytometric analysis of bone marrow and embryonic stem cells revealed highly similar profiles between ER-MP12 and CD31 (MEC 13.3 antibody)-stained cells. The presence of PECAM-1 on primitive hematopoietic stem cells supports the theory for the interaction of hematopoietic progenitor and stem cells with bone marrow stroma and transendothelial migration.
单克隆抗体ER-MP12最近被描述为可识别成年小鼠骨髓细胞亚群上存在的一种抗原，包括多能造血干细胞。为了了解ER-MP12抗原在造血过程中的功能，我们使用生化和物理方法来确定其身份。通过免疫沉淀从FDCP-1细胞膜蛋白中分离出ER-MP12抗原，在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳分析时，得到一条糖基化的113-kDa条带。对经胰蛋白酶消化的ER-MP12抗原衍生的13个肽段进行电喷雾电离质谱分析，并与蛋白质序列数据库进行比较。搜索结果显示ER-MP12抗原的身份为血小板内皮细胞黏附分子-1，即CD31（PECAM-1）。随后通过对单个ER-MP12肽段进行埃德曼测序，然后与PECAM-1序列进行比较，证实了这一结果。此外，对骨髓和胚胎干细胞的流式细胞术分析显示，ER-MP12和CD31（MEC 13.3抗体）染色的细胞之间具有高度相似的图谱。原始造血干细胞上存在PECAM-1支持了造血祖细胞和干细胞与骨髓基质相互作用及跨内皮迁移的理论。
