Cytosolic Glycosylation

描述(申请人提供)：这项研究的长期目标是确定蛋白质是如何以及为什么被复杂的0-连接的多糖修饰的 真核细胞质。我们现在的关注点是一种五糖，与一种 多蛋白SCF E3泛素连接酶的亚基Skp1上的羟脯氨酸， 在Dictvostelial.Skp1被一组全新的酶修饰 与那些在分泌途径中的人不同。几乎所有的Skp1通常都是糖基化的。 但突变形式的修饰效果很差，而且是异质性的。非糖基化的 形态未能集中在细胞核内，这一影响可能是 通过突变的或药理上的糖基化干扰来概括。 基于这一点和其他关于Skp 1-酶的生化证据 相互作用，我们假设Skp1修饰途径具有 促进和监控折叠的监护人/质量控制活动 Skp1用于进入SCF复合体并最终进入细胞核。这款车有 与N-连接的葡聚糖依赖的伴侣/质量控制相似 承租人的保留制度。在接下来的项目期间，我们将继续 对4种Skp1修饰酶的研究，最终目的是利用 新发现的信息来测试对伴侣/质量的某些预测 控制假说。网柄苔藓含有三种4-脯氨酰羟基酶样酶 预计存在于细胞质中的基因。我们假设其中一个 这些修饰了Skp1，而突变的Skp1在体内的羟化程度很低 因为与RER中一样，Pro羟基酶具有类似伴侣的活性。 GnT5 1与GlcNAcTase活性结合，似乎与 高尔基体的粘蛋白型多肽aGalNActase。我们假设GnT5 修饰Skp1，但在体内对突变体Skp1的修饰很差，因为它形成了一个 具有长寿命、不依赖催化作用的络合物。Bl、3GalTase和 A1，2FucTase活性驻留在同一蛋白质中，我们将研究 假设它有一个过程动作，以确保快速扩展 在GlcNAc添加承诺步骤之后的Skp1葡聚糖。我们假设一个 部分纯化的aGalTase活性增加了一个外部末端a-Gal Skp1上的残基，它与低半乳糖化的 主池Skp1的前身，直到Skp1准备退出，并且 突变体Skp1将表现出其末端Gal的过度回收。这些研究 预计将确定修饰Skp1的酶(可能除外 对于aGalTase之一)，并测试关于 监护人/质量控制模式。此外，预计还会有酶序列。 用于鉴定同源基因，在细胞质或 高尔基，随着它们的基因组序列的完成，它们的基因组序列也变得更加复杂。

DESCRIPTION (provided by applicant): The long-term goal of the study is to determine how and why proteins are modified by complex 0-linked glycans in the eukaryotic cytoplasm. Our immediate focus is on a pentasaccharide attached to a hydroxyproline on Skp1, a subunit of the multiprotein SCF E3 ubiquitin ligase, in Dictvostelium. Skp1 is modified by an entirely novel set of enzymes separate from those in the secretory pathway. Nearly all Skp1 is normally glycosylated but mutant forms are poorly and heterogeneously modified. The non-glycosylated forms failed to concentrate in the nucleus, an effect that could be recapitulated by disturbing glycosylation mutationally or pharmacologically. Based on this and additional biochemical evidence for Skp 1-enzyme interactions, we hypothesize that the Skp1 modification pathway has chaperone/quality control activity that facilitates and monitors folding of Skp1 for entry into the SCF complex and ultimately the nucleus. This model has parallels with the N-linked glycan-dependent chaperone/quality control retention system of the rER. in the coming project period, we will continue the study of 4 of the Skp1 modification enzymes with the ultimate goal of using the new-found information to test certain predictions of the chaperone/quality control hypothesis. Dictyostelium contains three 4-prolyl hydroxylase-like genes that are predicted to reside in the cytoplasm. We hypothesize that one of these modifies Skp1 and that a mutant Skp1 is poorly hydroxylated in vivo because of chaperone-like activity of the prolyl hydroxylase as in the rER. GnT5 1 copurifies with the GlcNAcTase activity and appears to be homologous to the mucin-type polypeptide aGalNAcTases of the Golgi. We hypothesize that GnT5l modifies Skp1, but poorly modifies mutant Skp1 in vivo because it forms a long-lived, catalysis-independent complex with it. The Bl,3GalTase and the al,2FucTase activities reside in the same protein and we will examine the hypothesis that it has a processive action to ensure rapid extension of the Skp1 glycan after the GlcNAc addition commitment step. We hypothesize that a partially-purified aGalTase activity adds one of the outer terminal a-Gal residues on Skp1, that it physically associates with an undergalactosylated precursor of the main pool of Skp1 until the Skp1 is ready to exit, and that mutant Skp1 will exhibit excessive recycling of its terminal Gal. These studies are expected to firmly identify the enzymes that modify Skp1 (except possibly for one of the aGalTases) and test specific hypotheses with regard to the chaperone/quality control model. In addition, the enzyme sequences are expected to be useful for the identification of homologous genes, in the cytoplasm or Golgi, of other 'lower' organisms as their genome sequences are completed.