Praetorius J, Backlund P, Yergey A L, Spring K R
Transport Physiology Section, Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, The National Institutes of Health, Bethesda, MD 20892-1603, USA.
J Membr Biol. 2001 Dec 1;184(3):273-81. doi: 10.1007/s00232-001-0093-2. Epub 2002 Feb 5.
Although lectins have previously been used to identify specific cell types in the kidney and various other tissues, the proteins labeled were not identified. We hypothesized that fluorescently labeled lectins could provide a useful tool for direct labeling of membrane-associated glycoproteins. Protein fractions from Madin-Darby canine kidney (MDCK) cells were exposed to a panel of 16 fluorescently labeled lectins to identify suitable lectin-protein pairs. Peanut agglutinin (PNA) selectively bound a 220-240 kDa O-linked glycoprotein with a slightly acidic isoelectric point, while Sambucus nigra agglutinin (SNA) labeled a 130 kDa glycoprotein with a highly acidic isoelectric point. Both proteins were readily labeled by lectins applied to the apical surface of living confluent cells. The proteins were isolated by lectin affinity columns and identified by mass spectrometry. Peptides from the PNA-binding protein shared molecular weight and amino acid composition with fibronectin. Fragments of the SNA-binding protein showed amino-acid identity with peptides from beta1 integrin. The identities of these proteins were validated by Western blotting. Binding of PNA to a 220 kDa protein was inhibited by an anti-fibronectin antibody, and binding of a 130 kDa protein by SNA was diminished by an anti-beta1 integrin antibody. We conclude that PNA and SNA can be used as specific markers for fibronectin and beta1 integrin, respectively, in MDCK cells.
尽管凝集素此前已被用于识别肾脏及其他多种组织中的特定细胞类型，但所标记的蛋白质尚未得到鉴定。我们推测，荧光标记的凝集素可为膜相关糖蛋白的直接标记提供一种有用的工具。将来自犬肾上皮细胞（MDCK）的蛋白质组分与一组16种荧光标记的凝集素进行孵育，以确定合适的凝集素 - 蛋白质对。花生凝集素（PNA）选择性结合一种220 - 240 kDa的O - 连接糖蛋白，其等电点略呈酸性，而黑接骨木凝集素（SNA）标记一种130 kDa的糖蛋白，其等电点呈强酸性。将凝集素应用于汇合的活细胞顶表面时，这两种蛋白质均能被轻易标记。通过凝集素亲和柱分离这些蛋白质，并通过质谱进行鉴定。来自PNA结合蛋白的肽段与纤连蛋白的分子量和氨基酸组成相同。SNA结合蛋白的片段与β1整合素的肽段具有氨基酸同一性。通过蛋白质免疫印迹法验证了这些蛋白质的身份。抗纤连蛋白抗体可抑制PNA与220 kDa蛋白质的结合，抗β1整合素抗体可减少SNA与130 kDa蛋白质的结合。我们得出结论，在MDCK细胞中，PNA和SNA可分别用作纤连蛋白和β1整合素的特异性标记物。
