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. **

** Hybryonan（HA）是一种多糖，对脊椎动物细胞周围基质的结构和功能至关重要。平均成年人体内存在15 g HA，其中1/3每天通过组成性周转被替换。此外，HA在细胞增殖和迁移时增加，在细胞成熟时通过调节的HA周转减少。已经鉴定了三种广泛表达的降解HA的透明质酸酶（HYAL）和三种结合和内化HA的受体，但是对它们如何促进HA降解知之甚少。本实验室的长期目标是确定三种HYAL酶（HYAL 1、HYAL 2、HYAL 3）如何与HA受体一起介导HA降解。在目前的HA降解模型中，HYAL 2被提出将细胞外HA切割成片段，这些片段被内化以在溶酶体中降解。与此一致，缺乏HYAL 2（HYAL 2 KO）的小鼠积累细胞外HA。然而，HYAL 2活性不能重复检测。此外，来自HYAL 2 KO小鼠的细胞内化并降解HA。基于这些发现，我们假设HYAL 2是一种活性受到调节的HA降解酶，并且存在非HYAL 2依赖性的HA降解途径。我的近期目标（未来5年），以解决这个假设是：*1。确定HYAL 2是否作为HA降解酶和/或受体发挥功能。将检查HA的大小以及在HYAL 2 KO细胞中表达的HYAL 2的野生型或突变形式存在下HA是否被内化。在突变体HYAL 2存在下HA大小增加将表明HYAL 2是一种酶。如果HYAL 2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性HA来开发新的HYAL 2活性测定法。2.鉴定能够调节HYAL 2活性的结合蛋白。HYAL 2的相互作用伴侣将使用免疫沉淀法随后通过质谱法从发育中的小鼠心脏中鉴定。确认的相互作用配偶体将在野生型和HYAL 2 KO细胞中过表达，并将检查HA大小、HYAL 2活性和HA内化。* 3.鉴定和区分HA降解途径。在存在和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，监测野生型和Hyal 2 KO细胞对荧光HA的摄取。通过光学成像监测荧光标记HA的摄取，在体内验证体外鉴定的途径。*重要性 * 通过我的HQP，我们将促进对HA降解的理解，同时为HQP提供糖科学方面的宝贵技能，这些技能是在化妆品、兽医和组织工程应用中使用HA的行业所需要的。 HYAL 2或其激活剂可能被这些工业鉴定为用于开发可增加外源HA产物的半衰期的抑制剂的有价值的靶标。**