Faculty of Veterinary Medicine, Institute of Physiological Chemistry, Ludwig-Maximilians-University Munich, Munich, Germany.
Membrane Biology and Axonal Repair Laboratory, Hospital Nacional de Parapléjicos (SESCAM), Toledo, Spain.
Methods Mol Biol. 2022;2442:247-288. doi: 10.1007/978-1-0716-2055-7_15.
Mammalian galectins have no signal peptide, and it is not known what would happen if a galectin is directed to take the classical export route. The corresponding engineering of galectin-specific cDNA will answer questions on the fate of a signal peptide-bearing protein variant after its entry into the endoplasmic reticulum (ER). Affinity chromatography and mass-spectrometric analysis of occupancy of potential N-glycosylation sites for the galectin, binding and functional assays with cells as well as subcellular fractionation by density gradient ultracentrifugation and immunocytochemical colocalization with ER/Golgi markers report on aspects of the consequences of letting a galectin enter new territory. Applying these methods will help to clarify why galectins are leaderless and thus produced by free ribosomes.
哺乳动物半乳糖凝集素没有信号肽，如果将半乳糖凝集素引导到经典的输出途径，会发生什么尚不清楚。对半乳糖凝集素特异性 cDNA 的相应工程改造将回答内质网 (ER) 中进入信号肽携带蛋白变异体的命运问题。半乳糖凝集素潜在 N-糖基化位点的亲和层析和质谱分析、与细胞的结合和功能测定以及通过密度梯度超速离心的亚细胞分级分离和与 ER/Golgi 标记物的免疫细胞化学共定位报告了让半乳糖凝集素进入新领域的后果的各个方面。应用这些方法将有助于阐明为什么半乳糖凝集素没有信号肽，因此是由游离核糖体产生的。
