ENAMEL MATRIX SERINE PROTEINASE 1

釉质基质丝氨酸蛋白酶1（搪瓷基质丝氨酸蛋白酶1，EMSP1）在分泌阶段加工搪瓷蛋白，在成釉发育早期降解搪瓷蛋白。EMSP1被认为对牙釉质的形成至关重要，而人类EMSP1基因的缺陷被怀疑会导致无牙釉质发育不全。我们验证了两个假设：1 . EMSP1功能在牙釉质形成分泌阶段对牙釉质基质蛋白的正确加工至关重要，功能缺失可导致牙釉质发育不全。2. 在牙釉质形成的过渡/早期成熟阶段，EMSP1功能对牙釉质基质蛋白的降解至关重要，功能丧失可导致牙釉质低聚合。以下4具体。我们的目的是验证这些假设：1 .确定分泌过程中EMSP1的时空表达。过渡。牙釉质生物矿化的成熟阶段。对小鼠上颌和下颌骨进行原位杂交和免疫组化。从新生儿、产后（PN）第2、4、6、8、14天及成人开始对第一、第二磨牙和门牙进行分析。2. 表征EMSP1对淀粉原蛋白的酶活性。用重组猪EMSP1体外消化重组猪淀粉原蛋白。消化产物将通过Edman降解和质谱法进行表征，并与体内发生的淀粉原蛋白裂解位点进行比较。3. 在小鼠缺乏EMSP1表达的情况下表征牙釉质形成。生成EMSP1基因敲除小鼠并进行表征。4. 确定人EMSP1基因与牙釉质形成遗传性缺陷的遗传联系。与3个实验室合作，这些实验室收集了大量患有淀粉性发育不全症（AI）的种类，将使用一种候选基因方法来建立人类EMSP1基因与AI之间的联系。完成这些目标将对EMSP1在牙釉质生物矿化过程中牙釉质基质蛋白的组织、加工、转换和降解中的作用有重要的见解。

Enamel matrix serine proteinase 1 (EMSP1) is proposed to process enamel proteins during the secretory stage, and degrade enamel proteins during the early maturation stage of amelogenesis. EMSP1 is believed to be critical for proper dental enamel formation and defects in the human EMSP1 gene are suspected of causing amelogenesis imperfecta. Two hypotheses are tested: l. EMSP1 function is essential for the correct processing of enamel matrix proteins during the secretory stage of enamel formation, and that a loss of function can result in hypoplastic enamel. 2. EMSP1 function is essential for the degradation of enamel matrix proteins during the transition/early maturation stages of enamel formation, and that a loss of function can result in hypommeralized enamel. The following 4 Specific.Aims are proposed to test these hypotheses: l. To determine the temporal and spatial expression of EMSP1 during the secretory. transition. and maturation stages of enamel biomineralization. In situ hybridization and immunohistochemistry will be performed on mouse maxillae and mandibles. The first and second molars and incisors will be analyzed from newborn, postnatal (PN) days 2, 4, 6, 8 and 14 and adult. 2. To characterize the enzymatic activity of EMSP1 on amelogenin. Recombinant pig amelogenin will be digested by recombinant pig EMSP1 in vitro. The digestion products will be characterized by Edman degradation and mass spectrometry and compared to amelogenin cleavage sites that occur in vivo. 3. To characterize enamel formation in the absence of mouse EMSP1 expression. EMSP1 knock-out mice will be generated and characterized. 4. To determine the genetic linkage of the human EMSP1 gene with inherited defects of dental enamel formation. In collaboration with 3 laboratories that have assembled large numbers of kindreds suffering for amelogenesis imperfecta (AI), a candidate gene approach will be used to establish linkage between the human EMSP1 gene and AI . Accomplishing these aims will gain important insights into the action of EMSP1 in the organization, processing, turnover, and degradation of enamel matrix proteins during enamel biomineralization.