ROLE OF PROTEIN GLYCOSYLATION IN DEVELOPMENT

盘状网柄菌细胞黏菌中modB基因突变的研究 产生蛋白质连接的多糖，这种多糖似乎缺乏其原始的大部分 质量。在该基因座突变等位基因纯合的二倍体菌株中， 还观察到1)假定的接触部位A糖蛋白(Gp80) 降至其正常水平的约10%，2)解离的重新解离 聚集期细胞明显减少，3)子实体变小 都形成了。另外两种正常携带的糖蛋白的表达 缺失的碳水化合物表位似乎没有在这个水平上受到影响 免疫荧光和SDS凝胶分析的分辨率。基于这些 发现，我们假设细胞表面gp80表达减少 导致解离后细胞重新结合减少，并导致减少 结实的身体大小。我们将在以下背景下研究这一假设 更大的问题是多聚糖在积累和定位中的作用 发育调节的糖蛋白。我们将首先调查 Gp80水平下降的基础，使用合成和周转研究 以及EM定位方法。然后我们将产生新的突变， 改变modB依赖的糖链，根据以下条件检查这些修饰 单糖组成、多糖大小分布和互补 分析并检验协同效应对gp80表达、细胞 再结合和子实体大小(孢子数)。我们推测到 修复丢失的部分多糖，或者只移除一部分， 通过突变手段，我们应该能够将modB的这些影响分离出来 关于gp80的积累和细胞重新联合 结构性基础。如果我们不能将这些影响与甚至 碳水化合物结构的细微变化，我们就会推断它们在 由于共同的机制(采用共同的确认过程)而产生的事实 基于公共结构)，这阻止了gp80到达细胞 表面(或在它到达时将其移除)。最后，糖链参数为 另外两个携带碳水化合物表位的糖蛋白将是 对比这些对于gp80的不同之处寻求解释 ModB对其积累的影响。 我们将比较依赖modB的糖链与另一种糖链的作用， 在孢子壳蛋白上表达，该蛋白携带一个由 另一组单抗。使用为 依赖于modB的多糖，我们将研究这种不同的多糖的作用 在将孢子壳蛋白分布到前孢子囊中(其 胞吐作用是发育调节的)，在分泌之后， 将孢子壳蛋白定位于孢子壳、粘液鞘和 茎管。

The modB mutation in the cellular slime mold Dictyostelium discoideum produces protein-linked glycans which appear to lack most of their original mass. In diploid strains homozygous for mutant alleles at this locus, it is also observed that 1) the putative contact sites A glycoprotein (gp80) is reduced to ca. 10% of its normal level, 2) reassociation of dissociated aggregation stage cells is markedly reduced, and 3) smaller fruiting bodies are formed. The expression of two other glycoproteins which normally bear the missing carbohydrate epitope do not appear to be affected at the level of resolution of immunofluorescence and SDS gel analysis. Based on these findings, we hypothesize that reduced gp80 expression on the cell surface results in reduced cell reassociation following dissociation and in reduced fruiting body size. We will investigate this hypothesis in the context of the larger question of glycan function in the accumulation and localization of developmentally-regulated glycoproteins. We will first investigate the basis for the decreased level of gp80, using synthesis and turnover studies and EM localization methods. We will then generate new mutations which alter the modB-dependent glycan, examine these modifications in terms of monosaccharide composition, glycan size distribution, and complementation analysis and then test for coordinate effects on gp80 expression, cell reassociation and fruiting body size (spore number). We speculate that by restoring a part of the missing glycan, or by removing only a part of it, by mutational means, we should be able to dissociate these effects of modB on gp80 accumulation and cell reassociation if they have a different structural basis. If we are unable to to dissociate the effects with even subtle changes in carbohydrate structure, we will infer that they are in fact due to a common mechanism (employing a common recognition process based on common structure) which prevents gp80 from arriving at the cell surface (or removes it once it arrives). Finally, glycan parameters for the other two glycoproteins carrying the carbohydrate epitope will be compared with these for gp80 to seek an explanation for the different effects of modB on their accumulation. We will compare the role of the modB-dependent glycan with another glycan, expressed on spore coat proteins, which carries an epitope recognized by another group of monoclonal antibodies. Using methods worked out for the modB-dependent glycan, we will investigate the role of this distinct glycan in distributing the spore coat proteins into prespore vesicles (whose exocytosis is developmentally regulated) and, following secretion, localizing the spore coat proteins into the spore coat, slime sheath and stalk tube.