Hohmann H P, Gerisch G, Lee R W, Huttner W B
J Biol Chem. 1985 Nov 5;260(25):13869-78.
An 80-kDa glycoprotein of Dictyostelium discoideum, designated contact site A, has been implicated in EDTA-stable cell adhesion. This protein is known to be the major sulfated protein of aggregation-competent cells and has been shown to contain two types of carbohydrate, sulfated type 1 and unsulfated type 2 carbohydrate moieties. Here we investigate the cell-free sulfation of this protein. In the homogenate of developing cells, [35S]sulfate was transferred by endogenous sulfotransferase from [35S]3'-phosphoadenosine-5'-phosphosulfate to the contact site A glycoprotein and to various other endogenous proteins. The sulfate was transferred to carbohydrate rather than to tyrosine residues. After differential centrifugation of the homogenate, the capacity for sulfation of the contact site A glycoprotein was barely detected in the plasma membrane-enriched 10,000 X g pellet fraction which contained the bulk of this glycoprotein, but was largely recovered in the 100,000 X g pellet fraction which contained only a small portion of this glycoprotein. After sucrose gradient centrifugation, the membranes containing the sulfation capacity were found to have a density characteristic for Golgi membranes. In immunoblots, monoclonal antibodies raised against the contact site A glycoprotein recognized not only this 80-kDa protein, but also a sulfatable 68-kDa protein found in the 100,000 X g pellet fraction. The 68-kDa protein did not react with monoclonal antibodies against type 2 carbohydrate but was converted by endoglycosidases F and H into a 53-kDa protein, indicating that it was a partially glycosylated form of the 80-kDa glycoprotein containing only type 1 carbohydrate. Isoelectric focusing showed that a substantial portion of the 68-kDa glycoprotein was unsulfated, even after cell-free sulfation. The 68-kDa glycoprotein was not found in the plasma membrane-enriched 10,000 X g pellet fraction and did not accumulate in parallel with the 80-kDa contact site A glycoprotein during cell development. We conclude that the 68-kDa glycoprotein is a precursor that is converted by attachment of type 2 carbohydrate and sulfation of type 1 carbohydrate into the mature 80-kDa glycoprotein. The precursor nature of the 68-kDa glycoprotein was supported by results obtained with mutant HL220 which is defective in glycosylation (Murray, B. A., Wheeler, S., Jongens, T., and Loomis, W. F. (1984) Mol. Cell. Biol. 4, 514-519). This mutant specifically lacks type 2 carbohydrate and produces a 68-Kda glycoprotein instead of the 80-kDa contact site A glycoprotein (Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984) EMBO J. 3, 2663-2670).(ABSTRACT TRUNCATED AT 400 WORDS)
盘基网柄菌的一种80 kDa糖蛋白，称为接触位点A，与EDTA稳定的细胞黏附有关。已知该蛋白是具有聚集能力的细胞的主要硫酸化蛋白，并且已显示含有两种类型的碳水化合物，硫酸化的1型和未硫酸化的2型碳水化合物部分。在此，我们研究了该蛋白的无细胞硫酸化作用。在发育中细胞的匀浆中，[35S]硫酸盐被内源性硫酸转移酶从[35S]3'-磷酸腺苷-5'-磷酸硫酸转移到接触位点A糖蛋白和各种其他内源性蛋白上。硫酸盐被转移到碳水化合物而非酪氨酸残基上。对匀浆进行差速离心后，在富含质膜的10,000×g沉淀组分中几乎检测不到接触位点A糖蛋白的硫酸化能力，该组分含有大部分这种糖蛋白，但在100,000×g沉淀组分中大量恢复，该组分仅含有一小部分这种糖蛋白。经过蔗糖梯度离心后，发现具有硫酸化能力的膜具有高尔基体膜的密度特征。在免疫印迹中，针对接触位点A糖蛋白产生的单克隆抗体不仅识别这种80 kDa蛋白，还识别在100,000×g沉淀组分中发现的一种可硫酸化的68 kDa蛋白。68 kDa蛋白不与抗2型碳水化合物的单克隆抗体反应，但被内切糖苷酶F和H转化为53 kDa蛋白，表明它是仅含有1型碳水化合物的80 kDa糖蛋白的部分糖基化形式。等电聚焦显示，即使经过无细胞硫酸化，68 kDa糖蛋白的很大一部分仍未硫酸化。在富含质膜的10,000×g沉淀组分中未发现68 kDa糖蛋白，并且在细胞发育过程中它不会与80 kDa接触位点A糖蛋白平行积累。我们得出结论，68 kDa糖蛋白是一种前体，通过附着2型碳水化合物和1型碳水化合物的硫酸化转化为成熟的80 kDa糖蛋白。糖基化缺陷的突变体HL220的实验结果支持了68 kDa糖蛋白的前体性质（默里，B.A.，惠勒，S.，琼根斯，T.，和卢米斯，W.F.（1984年）《分子与细胞生物学》4，514 - 519）。该突变体特异性地缺乏2型碳水化合物，并产生一种68 kDa糖蛋白而非80 kDa接触位点A糖蛋白（吉田，M.，施塔德勒，J.，贝特霍尔德，G.，和杰里施，G.（1984年）《欧洲分子生物学组织杂志》3，2663 - 2670）。（摘要截断于400字）
