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: $ 5.83万
• 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: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=744197
• 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. OBJECTIVESThe 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.SIGNIFICANCEWith 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，对脊椎动物细胞的基质的结构和功能至关重要。一个普通的成年人中存在15克HA，每天通过构成营业额替换1/3。另外，当细胞通过调节的HA周转成熟时，细胞繁殖并迁移并减少时，HA会增加。已经确定了三种降解HA和三个受体的透明酶酶（透明质酸酶），但已经鉴定出结合和内在化的HA，但是对HA降解的贡献却很少了解。我实验室的长期目标是确定三种透明酶（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，提出了透明2将细胞外HA裂解至溶酶体内部降解的片段。与此相一致，缺乏透明的小鼠（透明2 KO）会积累细胞外HA。但是，未检测到透明活性。此外，来自Hyal2 KO小鼠的细胞内在化和降解HA。基于这些发现，我们假设透明是一种降解的酶，其活性受到调节，并且存在非氢化依赖的HA降解途径。我的直接目标（接下来的5年）解决了这一假设：1。确定Hyal2是否充当降解酶和/或受体。 HA的大小以及在透明2 KO细胞中表达的野生型或突变形式的存在中的内部化。在存在突变透明的情况下，HA大小的增加将表明透明2是一种酶。如果证明了透明度的活性，我们将通过质谱法和/或使用HA蛋白复合物替代外源性HA.2的HA.2。识别可以调节其活性的透明结合蛋白。使用免疫沉淀，随后进行质谱法，将通过开发小鼠心脏来确定透视2的相互作用伴侣。确认的相互作用伴侣将在野生型和透明2 KO细胞中过表达，HA大小，透明活性和HA内在化将被检查。3。识别和区分HA降解途径。在存在和不存在内部化途径抑制剂的情况下，将监测野生型和透明2 KO细胞对荧光HA的吸收，并且/或阻断已知HA受体的抗体。通过光学成像监测荧光标记的HA的摄取，可以在体内验证，通过我的HQP，我们将提高对HA降解的理解，同时为HQP提供高度的糖基含量技能，而该行业在质量，兽医，兽医，兽医和组织中都需要使用HA，而这些技能是在糖节上的宝贵技能。 这些行业可以将Hyal2或其激活剂确定为有价值的目标，以开发抑制剂，从而增加外源性HA产品的半衰期。