Cahill James, Ahuja Shivani, Whorton Matthew R
Vollum Institute, Oregon Health and Science University, 3181 SW Sam Jackson Park Rd., Portland, OR 97239, USA.
Bio Protoc. 2020 Mar 20;10(6):e3551. doi: 10.21769/BioProtoc.3551.
Nucleotide-sugar transporters (NSTs) facilitate eukaryotic cellular glycosylation by transporting nucleotide-sugar conjugates into the Golgi lumen and endoplasmic reticulum for use by glycosyltransferases, while also transferring nucleotide monophosphate byproducts to the cytoplasm. Mutations in this family of proteins can cause a number of significant cellular pathologies, and wild type members can act as virulence factors for many parasites and fungi. Here, we describe an assay to measure the transport activity of the CMP-sialic acid transporter (CST), one of seven NSTs found in mammals. While transport assays have been previously described for CST, these studies failed to account for the fact that 1) commercially available stocks of CMP-sialic acid (CMP-Sia) are composed of ~10% of the higher-affinity CMP and 2) CMP-Sia is hydrolyzed into CMP and sialic acid in aqueous solutions. Herein we describe a method for treating CMP-Sia with a nonselective phosphatase, Antarctic phosphatase, to convert all free CMP to cytidine. This allows us to accurately measure substrate affinities and transport kinetics for purified CST reconstituted into proteoliposomes.
核苷酸糖转运蛋白（NSTs）通过将核苷酸糖共轭物转运到高尔基体腔和内质网中供糖基转移酶使用，促进真核细胞的糖基化，同时还将核苷酸单磷酸副产物转移到细胞质中。该蛋白家族中的突变可导致许多严重的细胞病变，野生型成员可作为许多寄生虫和真菌的毒力因子。在此，我们描述了一种测定CMP-唾液酸转运蛋白（CST）转运活性的方法，CST是哺乳动物中发现的七种NSTs之一。虽然之前已经描述了CST的转运测定方法，但这些研究没有考虑到以下事实：1）市售的CMP-唾液酸（CMP-Sia）库存中约10%是高亲和力的CMP；2）CMP-Sia在水溶液中会水解成CMP和唾液酸。在此，我们描述了一种用非选择性磷酸酶南极磷酸酶处理CMP-Sia的方法，将所有游离CMP转化为胞苷。这使我们能够准确测量重组到蛋白脂质体中的纯化CST的底物亲和力和转运动力学。
