Characterization of HYAL2 and non-HYAL2 dependent pathways of hyaluronan degradation

透明质酸降解的 HYAL2 和非 HYAL2 依赖性途径的表征

• 批准号: RGPIN-2017-04953
• 负责人: TriggsRaine, Barbara
• 金额: $ 2.91万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=667142
• 关键词: Characterization; HYAL2; non; dependent; pathways

**Hyaluronan (HA) is a polysaccharide that is essential to the structure and function of the matrix surrounding vertebrate cells. There is 15 g of HA present in an average adult human, 1/3 of which is replaced each day through constitutive turnover. In addition HA is increased when cells multiply and migrate and decreased when cells mature through regulated HA turnover. Three widely expressed hyaluronidase enzymes (HYALs) that degrade HA and three receptors that bind and internalize HA have been identified, but how they contribute to HA degradation is poorly understood. ***OBJECTIVES****The long term objective of my laboratory is to determine how three HYAL enzymes (HYAL1, HYAL2, HYAL3) work with HA receptors to mediate HA degradation. In the current model for HA degradation, HYAL2 is proposed to cleave extracellular HA to fragments that are internalized for degradation in the lysosome. Consistent with this, mice lacking HYAL2 (HYAL2 KO) accumulate extracellular HA. However, HYAL2 activity is not reproducibly detected. Further, cells from HYAL2 KO mice internalize and degrade HA. Based on these findings, we hypothesize that HYAL2 is an HA-degrading enzyme whose activity is regulated, and that non-HYAL2 dependent pathways of HA degradation exist. My immediate goals (next 5 years) to address this hypothesis are:****1. To determine if HYAL2 functions as a HA-degrading enzyme and/or receptor. The size of HA and whether it is internalized in the presence of wild type or mutant forms of HYAL2 expressed in HYAL2 KO cells will be examined. Increased HA size in the presence of mutant HYAL2 will suggest HYAL2 is an enzyme. If activity for HYAL2 is demonstrated, we will develop novel assays for HYAL2 activity by mass spectrometry and/or using HA protein complexes to replace exogenous HA.****2. To identify HYAL2 binding proteins that can modulate its activity. Interacting partners for HYAL2 will be identified from developing mouse hearts using immunoprecipitation followed by mass spectrometry. Confirmed interacting partners will be overexpressed in wild type and HYAL2 KO cells and HA size, HYAL2 activity, and HA internalization, will be examined.****3. To identify and differentiate pathways of HA degradation. Uptake of fluorescent HA by wild type and Hyal2 KO cells will be monitored in the presence and absence of inhibitors of internalization pathways, and/or blocking antibodies for known HA receptors. Pathways identified in vitro will be verified in vivo by monitoring the uptake of fluorescently labelled HA by optical imaging.****SIGNIFICANCE****With my HQP, we will advance the understanding of HA degradation while giving HQP valuable skills in glycoscience that are needed by industries using HA in cosmetic, veterinary and tissue engineering applications. HYAL2, or its activators, are likely to be identified as valuable targets by these industries for the development of inhibitors that could increase the half life of exogenous HA products. **

**透明质酸 (HA) 是一种多糖，对于脊椎动物细胞周围基质的结构和功能至关重要。一个普通成年人体内含有 15 克 HA，其中 1/3 每天通过结构更新而被替换。此外，当细胞增殖和迁移时，HA 会增加，而当细胞成熟时，HA 会通过调节 HA 周转而减少。三种广泛表达的可降解 HA 的透明质酸酶 (HYAL) 和三种结合并内化 HA 的受体已被鉴定，但人们对它们如何促进 HA 降解知之甚少。 ***目标****我实验室的长期目标是确定三种 HYAL 酶（HYAL1、HYAL2、HYAL3）如何与 HA 受体一起介导 HA 降解。在当前的 HA 降解模型中，HYAL2 被认为可以将细胞外的 HA 裂解成片段，然后内化到溶酶体中进行降解。与此一致的是，缺乏 HYAL2 (HYAL2 KO) 的小鼠会积累细胞外 HA。然而，HYAL2 活性并未被重复检测到。此外，来自 HYAL2 KO 小鼠的细胞内化并降解 HA。基于这些发现，我们假设 HYAL2 是一种 HA 降解酶，其活性受到调节，并且存在非 HYAL2 依赖的 HA 降解途径。我解决这一假设的近期目标（未来 5 年）是：****1。确定 HYAL2 是否起到 HA 降解酶和/或受体的作用。将检查 HA 的大小以及它是否在 HYAL2 KO 细胞中表达的 HYAL2 野生型或突变形式存在的情况下内化。在突变体 HYAL2 存在的情况下，HA 大小增加表明 HYAL2 是一种酶。如果 HYAL2 的活性得到证实，我们将通过质谱法和/或使用 HA 蛋白复合物替代外源 HA 来开发新的 HYAL2 活性检测方法。****2。鉴定可调节其活性的 HYAL2 结合蛋白。 HYAL2 的相互作用伙伴将通过免疫沉淀和质谱分析从发育中的小鼠心脏中鉴定出来。已确认的相互作用伙伴将在野生型和 HYAL2 KO 细胞中过表达，并且将检查 HA 大小、HYAL2 活性和 HA 内化。****3。识别和区分 HA 降解的途径。在存在或不存在内化途径抑制剂和/或已知HA受体的阻断抗体的情况下，将监测野生型和Hyal2 KO细胞对荧光HA的摄取。通过光学成像监测荧光标记的 HA 的摄取，体外确定的途径将在体内得到验证。****意义****通过我的 HQP，我们将增进对 HA 降解的理解，同时为 HQP 提供糖科学方面的宝贵技能，化妆品、兽医和组织工程应用中使用 HA 的行业需要这些产品。 HYAL2 或其激活剂可能被这些行业确定为有价值的目标，用于开发可以延长外源 HA 产品半衰期的抑制剂。 **