STRUCTURE, BIOSYNTHESIS AND FUNCTION OF GLYCOPROTEINS

摘要：本研究计划的目的是获得磷酸甘氨酰基靶向系统的分子理解，该系统的功能是将新合成的酸水解酶传递给溶酶体。这种细胞内蛋白质运输途径的缺陷导致严重的溶酶体贮积病。具体目标包括：(1)表征dna酶I上的蛋白质识别结构域，这是与磷酸转移酶相互作用所必需的，磷酸转移酶催化了Man-6-P识别标记物产生的第一步。我们将使用位点定向诱变来鉴定dna酶I表面决定与磷酸转移酶结合的残基，并分析这些残基如何在选定的糖基化位点指导磷酸化。(2)磷酸二酯α - glcnacase细胞质尾部氨基酸的鉴定，这些氨基酸作为其在反式高尔基网络（TGN）和质膜之间运输的信号。这种酶从酸水解酶寡糖中去除覆盖的GlcNAc，暴露介导与Man-6-P受体结合的Man-6-P残基。具有这些氨基酸的GST肽将用于分离识别这些基序的蛋白质。针对细胞质尾部的抗体将用于免疫分离TGN以表征其蛋白质组成。(3)分析磷酸化/去磷酸化事件如何调节GGA蛋白与Man-6-P受体和外壳蛋白AP-1的相互作用。最近的证据表明，gga结合高尔基体中的Man-6-P受体，并将其呈递给AP-1，包装成网格蛋白包被的囊泡。我们将(a)确定与AP-1 γ亚基耳结构域结合的gga铰链片段中的氨基酸。(b)确定AP-1相关酪蛋白激酶2对GGAs 1,3的磷酸化如何改变GGA蛋白构象，导致与AP-1分离并释放结合的Man-&P受体。(c)鉴定作用于GGAs 1,3的磷酸酶。一个候选是已知与MPR细胞质尾部结合的蛋白磷酸酶2a。(d)进行体外实验，以确定在没有AP-1的情况下，gga是否能成核网格蛋白包被的囊泡。这解决了gga除了作为AP-1囊泡组装的辅助蛋白外，是否形成自己的运输囊泡的问题。(4)继续研究AP-1募集到脂质体所需的胞质蛋白。(1)我们工作的主要目标是进一步了解GGA蛋白与mpr和AP-1的相互作用。迄今为止，我们的研究结果表明，GGAs 1和3的磷酸化/去磷酸化调节了这些过程。我们发现ap - 1含有紧密结合的酪蛋白激酶2e (CK-2)，使GGAs磷酸化1/3。我们将确定四聚体AP-1的哪个亚基结合CK-2b？用胰蛋白酶消化分子，选择性地切割s61和？？并确定CK-2是否被释放或留在AP-1的主干部分。在此结果的指导下，我们将使用AP-1的适当结构域进行gst下拉实验，以下拉可溶性CK-2。接下来，我们将确定使GGAs113去磷酸化的磷酸酶，从而使这些分子与mpr和AP-1结合。一个候选是蛋白磷酸酶2a (PP2a)，已知它与包括MPR在内的几种高尔基蛋白结合。有了CK-2和磷酸酶，我们将通过凝胶过滤、蔗糖密度梯度和蛋白酶敏感性来研究磷酸化对GGAs 1/3构象的影响。这些试剂也将使我们能够证实我们的初步数据，即1/3的GGAs磷酸化会损害与AP-1的结合。亚基，从而为这种复合物的解离提供了一种机制。为了更好地理解GGA- ap -1相互作用，我们将使用诱变技术来鉴定与ap -1相互作用的GGA铰链结构域的氨基酸。耳朵。我们还将确定GGA铰链是否与AP-1的同一表面结合？耳朵是？？协同蛋白和rabaptin 5，两种已知与？？耳朵。这些实验将以最近发表的？？耳朵的结构。与Hans Geuze合作，我们将使用免疫E/M分析丝氨酸355突变为丙氨酸（防止磷酸化）或天冬氨酸（模拟磷酸化）的GGAl分子的亚细胞分布。我们假设这两个突变体都将被招募到TGN上，但后者将无法与mpr或AP-1结合，因此其定位到含有AP-1的网格蛋白包被芽将受到损害。这些研究将有助于将我们的体外研究结果与GGAs在完整细胞中的行为联系起来。最后，我们将研究纯化的GGAs在高尔基膜和脂质体上的募集，以确定GGAs是否可以成核ccv。虽然已知gga与网格蛋白结合，但没有证据表明它们能使ccv成核。关于gga是否独立于与AP-1的相互作用而形成ccv，这是一个重要的问题。(2)我们将继续定义DNase I上允许与磷酸转移酶结合的蛋白质识别结构域。这种相互作用是理解酸水解酶如何选择性磷酸化的核心。到目前为止，我们的发现表明赖氨酸残基只是结合表面的一个组成部分。通过在细菌中表达的非糖基化DNase I突变体来抑制磷酸转移酶对完整酸水解酶的磷酸化，我们希望确定参与结合的其他氨基酸。重组磷酸转移酶（由Novazyme的William Canfield博士提供）的可用性将使我们首次能够进行该酶与其蛋白质底物之间的直接结合研究。我们将扩展我们的研究！？发现酶! ?通过识别UCE细胞质尾部允许从TGN退出的氨基酸残基来进行转运。一旦这些被定义，我们将使用含有这些残基的gst融合蛋白在下拉试验中寻找相互作用的蛋白。使用类似的方法，我们将尝试鉴定一种与含有486- y基序结合的蛋白质，该基序将UCE保留在核内体中以返回TGN。这些蛋白质的鉴定将促进我们对UCE贩运如何受到监管的理解。我们已经制备了针对UCE细胞质尾部的高亲和力抗体，用于TGN（大部分UCE定位的地方）的免疫分离。如果成功，这将是特异性高尔基腔室的首次免疫分离。我们将通过二维凝胶分析，FABOMass光谱分析和使用候选蛋白质抗体的western blotting来鉴定蛋白质成分。新的蛋白将被克隆，并通过免疫荧光确认其在TGN中的定位。这些实验可能会发现TGN的新功能。(4)我们将继续纯化AP-1募集到脂质体所需的胞质蛋白。如果成功，我们将获得氨基酸序列，以确定它是否是已知的蛋白质。如果它是新的，我们将克隆它以获得它的序列，并鉴定可能为其功能提供线索的蛋白质结构域。然后我们将研究蛋白质如何促进AP-1的募集。

Abstract The objective of the research proposal is to obtain a molecular understanding of the phosphomannosyl targeting system which functions to deliver newly synthesized acid hydrolases to lysosomes. Defects in this intracellular protein transport pathway give rise to severe lysosomal storage diseases. The specific aims include: (1) Characterization of the protein recognition domain on DNase I that is necessary for interaction with phosphotransferase which catalyzes the first step in the generation of the Man-6-P recognition marker. We will use site-directed mutagenesis to identify residues on the surface of DNase I that determine binding to phosphotransferase and analyze how these residues direct phosphorylation at selected glycosylation sites. (2) Identification of amino acids in the cytoplasmic tail of phosphodiester alpha-GlcNAcase that serve as signals for its trafficking between the trans-Golgi network (TGN) and the plasma membrane. This enzyme removes the covering GlcNAc from acid hydrolase oligosaccharides to expose Man-6-P residues that mediate binding to Man-6-P receptors. GST peptides with these amino acids will be used to isolate proteins that recognize these motifs. Antibodies to the cytoplasmic tail will used to immunoisolate the TGN for characterization of its protein composition. (3) Analysis of how phosphorylation/dephosphorylation events regulate the interaction of the GGA proteins with the Man-6-P receptors and the coat protein AP-1. Recent evidence shows that the GGAs bind Man-6-P receptors in the Golgi and present then to AP-1 for packaging into clathrin-coated vesicles. We will (a) define the amino acids in the hinge segments of the GGAs that bind to the ear domain of the gamma subunit of AP-1. (b) determine how phosphorylation of GGAs 1, 3 by AP-1 associated casein kinase 2 alters GGA protein conformation resulting in dissociation from AP-1 and the release of bound Man-&P receptors. (c) identify the phosphatase that acts on GGAs 1,3. A candidate is protein phosphatase 2a that is known to bind to the MPR cytoplasmic tail. (d) perform in vitro assays to determine whether the GGAs can nucleate clathrin coated vesicles in the absence of AP-1. This addresses the issue of whether GGAs form their own transport vesicles in addition to serving as accessory proteins for AP-1 vesicle assembly. (4) Studies to identify the cytosolic proteins required for AP-1 recruitment onto liposomes will be continued. Research Plan for the Extension (1) A major goal of our work will be to further our understanding of the interactions of the GGA proteins with the MPRs and AP-1. Our findings to date indicate that phosphorylation / dephosphorylation of GGAs 1 and 3 regulate these processes. We have found that AP-I contains tightly bound casein kinas2e (CK-2) that phosphorylates GGAs 1/3. We will identify which subunit of the tetrameric AP-1 binds CK-2b?? digesting the molecule with trypsin to selectively cleave the hinge region of the s61 and ?? subunits and determining if the CK-2 is released or remains with the trunk portion of the AP-1. Guided by this result we will perform GST-pull down experiments using appropriate domains of AP-1 to pull down soluble CK-2. Next we will identify the phosphatase that dephosphorylates GGAs113, thereby allowing these molecules to bind to the MPRs and AP-1. A candidate is protein phosphatase 2a (PP2a) that is known to bind to several Golgi proteins including the MPR. With CK-2 and the phosphatase in hand, the effect of phosphorylation on the conformation of GGAs 1/3 will be studied by gel filtration, sucrose density gradients and protease sensitivity. These reagents will also allow us to confirm our preliminary data that phosphorylation of GGAs 1/3 impairs binding to the AP-1 ?? subunit, thereby providing a mechanism for the dissociation of this complex. To better understand the GGA-AP-1 interaction,w e will use mutagenesis to identify the amino acids of the GGA hinge domain that interact with the AP-I ?? ear. We will also determine whether or not the GGA hinge binds to the same surface of the AP-1 ?? ear as ?? synergin and rabaptin 5, two proteins known to bind to the ?? ear. These experiments will be guided by the recently published ?? ear structure. In collaboration with Hans Geuze, we will use immuno E/M to analyze the subcellular distributions of GGAl molecules that have serine 355 mutated to alanine (to prevent phosphorylation)o r to aspartate (to mimic phosphorylation). We postulate that both mutants will be recruited onto the TGN, but the latter will be incapable of binding to MPRs or AP-1 and therefore its localization into AP-I containing clathrin-coated buds will be impaired. These studies will help to correlate our in vitro findings with the behavior of the GGAs in intact cells. Finally, we will study the recruitment of purified GGAs onto Golgi membranes and liposomes to determine whether GGAs can nucleate CCVs. While GGAs are known to bind clathrin, there is no evidence that they nucleate CCVs. This is an important issue in regards to whether the GGAs form CCVs independent of their interaction with AP-1. (2) We will continue to define the protein recognition domain on DNase I that allows binding to phosphotransferase. This interaction is central to understanding how acid hydrolases are selectively phosphorylated. Our findings to date indicate that lysine residues are only one component of the binding surface. By using non-glycosylated DNase I mutants expressed in bacteria to inhibit phosphorylation of intact acid hydrolases by phosphotransferase, we hope to identify the other amino acids that are involved in binding. The availability of mg amounts of recombinant phosphotransferase (provided by Dr. William Canfield at Novazyme) will allow us for the first time to perform direct binding studies between this enzyme and its protein substrates. (3) We will extend our studies of !?Uncovering Enzyme!? trafficking by identifying the amino acid residues in the cytoplasmic tail of UCE that allow exit from the TGN. Once these are defined, we will use a GST-fusion protein containing these residues in pull-down assays to search for an interacting protein(s). Using a similar approach we will try to identify a protein(s) that binds to the 486-Ycontaining motif that retains UCE in the endosome for return to the TGN. Identification of such proteins will advance our understanding of how the trafficking of UCE is regulated. We have prepared high affinity antibodies to the cytoplasmic tail of UCE for use in the immuno isolation of the TGN (where the bulk of UCE is localized). If successful this would be the first immunoisolation of a specific Golgi compartment. We will identify the protein components by 2-D gel analysis, FABOMass Spectrometry and western blotting using antibodies to candidate proteins. New proteins will be cloned and their localization in the TGN confirmed by immuno fluorescence. These experiments may identify novel functions of the TGN. (4) We will pursue the purification of the cytosolic protein required for AP-1 recruitment onto liposomes. If successful, we will obtain amino acid sequence to determine if it is a known protein. If it is novel, we will clone it to obtain its sequence and identify protein domains that may give clues as to its function. We will then study how the protein facilitates AP-1 recruitment.