Konrad R J, Tolar J F, Hale J E, Knierman M D, Becker G W, Kudlow J E
Department of Diagnostic and Experimental Medicine, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, IN 46285, USA.
Biochem Biophys Res Commun. 2001 Nov 16;288(5):1136-40. doi: 10.1006/bbrc.2001.5902.
We have previously shown that rat pancreatic islets contain a predominant 135 kDa O-glycosylated protein (p135) that is recognized by immunoprecipitation and Western blotting with anti-O-GlcNAc antibody. In this paper, we show that p135 is also detectable in other rat tissues including brain, heart, liver, spleen, and lung, but not kidney. To identify p135, the protein was purified from rat brain using a multistep procedure including selective absorption with anti-O-GlcNAc antibody. After electrophoresis, and Coomassie staining, the protein was excised from the gel for tryptic digestion. Next, O-methylisourea was used to convert lysine residues to homoarginine to increase the sequence coverage, and MALDI-TOF mass spectrometry detection was performed. MALDI-TOF identified p135 as rat O-GlcNAc transferase (OGT), an identity confirmed by LC/MS of individual peptides. The identification of p135 as OGT is consistent with previous reports of the tissue distribution of OGT, as well as reports that OGT is itself O-glycosylated.
我们之前已经表明，大鼠胰岛含有一种主要的135 kDa O-糖基化蛋白（p135），该蛋白可通过用抗O-GlcNAc抗体进行免疫沉淀和蛋白质印迹法检测到。在本文中，我们表明p135在大鼠的其他组织中也可检测到，包括脑、心脏、肝脏、脾脏和肺，但在肾脏中未检测到。为了鉴定p135，使用包括用抗O-GlcNAc抗体进行选择性吸附在内的多步程序从大鼠脑中纯化该蛋白。电泳和考马斯亮蓝染色后，从凝胶中切下该蛋白进行胰蛋白酶消化。接下来，使用O-甲基异脲将赖氨酸残基转化为高精氨酸以增加序列覆盖率，并进行基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）检测。MALDI-TOF将p135鉴定为大鼠O-GlcNAc转移酶（OGT），单个肽段的液相色谱/质谱（LC/MS）证实了这一身份。将p135鉴定为OGT与之前关于OGT组织分布的报道以及OGT本身是O-糖基化的报道一致。
