Weisgerber C, Troy F A
Department of Biological Chemistry, University of California School of Medicine, Davis 95616.
J Biol Chem. 1990 Jan 25;265(3):1578-87.
The nature of endogenous acceptor molecules implicated in the membrane-directed synthesis of the polysialic acid (polySia) capsule in Escherichia coli K1 serotypes is not known. The capsule contains at least 200 sialic acid (Sia) residues that are elongated by the addition of new Sia residues to the nonreducing termini of growing nascent chains (Rohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342). Presumably, chain growth starts when activated Sia residues are transferred to acceptors that are not already sialylated. In the present study, we used an acapsular mutant defective in synthesis of CMP-NeuAc to label acceptors with [14C]NeuAc and an anti-polySia-specific antibody (H.46) to identify the molecules to which the polySia was attached. [14C]Sia-labeled acceptors were solubilized with 2% Triton X-100, immunoprecipitated with H.46, and partially depolymerized with poly-alpha-2,8-endo-N-acetylneuraminidase. Approximately 5% of the [14C]Sia incorporated remained attached to endogenous acceptors. Double-labeling experiments were used to show that the non-Sia moiety of the acceptor was labeled in vivo with [14C]leucine and elongated in vitro with CMP-[3H]NeuAc. Concomitant with desialylation of the [3H]polySia-[14C]Leu acceptor was the appearance of a new [14C]Leu-labeled protein at 20 kDa. After strong acid hydrolysis, the 20-kDa labeled protein was shown to contain [14C]Leu. The acceptor molecules were not labeled metabolically with D-[3H]GlcN, 35SO4, or 32PO4, indicating that they do not appear to contain lipopolysaccharide, peptidoglycan, phosphatidic acid, or phospholipid. Based on these results, we conclude that the endogenous acceptor molecule is a membrane protein of about 20 kDa. The nature of attachment of polySia to acceptor is unknown. There are only 400-500 acceptor molecules/cell, which is about 100-fold fewer than the 50,000 polySia chains/cell. This suggests that each acceptor molecule may participate in the shuttling of about 100 polySia chains/cell. We hypothesize that the acceptor protein may function to translocate polySia chains from their site of synthesis on the cytoplasmic surface of the inner membrane to the periplasm.
目前尚不清楚参与大肠杆菌K1血清型中膜导向的聚唾液酸（polySia）荚膜合成的内源性受体分子的性质。该荚膜含有至少200个唾液酸（Sia）残基，这些残基通过在新生链的非还原末端添加新的Sia残基而延长（Rohr，T. E.，和Troy，F. A.（1980）J. Biol. Chem. 255，2332 - 2342）。据推测，当活化的Sia残基转移到尚未被唾液酸化的受体上时，链的生长开始。在本研究中，我们使用了一个在CMP - NeuAc合成方面有缺陷的无荚膜突变体，用[14C]NeuAc标记受体，并使用抗聚唾液酸特异性抗体（H.46）来鉴定聚唾液酸所附着的分子。用2% Triton X - 100溶解[14C]Sia标记的受体，用H.46进行免疫沉淀，并用聚α - 2,8 - 内切 - N - 乙酰神经氨酸酶进行部分解聚。大约5%掺入的[14C]Sia仍附着在内源性受体上。双标记实验表明，受体的非Sia部分在体内用[14C]亮氨酸标记，并在体外用CMP - [3H]NeuAc延长。伴随着[3H]聚唾液酸 - [14C]亮氨酸受体的去唾液酸化，出现了一种新的20 kDa的[14C]亮氨酸标记蛋白。强酸水解后，显示20 kDa的标记蛋白含有[14C]亮氨酸。受体分子未被D - [3H]葡糖胺、35SO4或32PO4代谢标记，这表明它们似乎不含有脂多糖、肽聚糖、磷脂酸或磷脂。基于这些结果，我们得出结论，内源性受体分子是一种约20 kDa的膜蛋白。聚唾液酸与受体的连接性质尚不清楚。每个细胞中有400 - 500个受体分子，这比每个细胞中50,000条聚唾液酸链少约100倍。这表明每个受体分子可能参与每个细胞中约100条聚唾液酸链的穿梭。我们假设受体蛋白可能起到将聚唾液酸链从其在内膜细胞质表面的合成位点转运到周质的作用。
