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

**透明质酸(HA)是一种对脊椎动物细胞周围基质的结构和功能至关重要的多糖。平均每个成年人体内含有15克HA，其中1/3是通过组织周转每天被替换的。此外，当细胞增殖和迁移时，HA增加，当细胞成熟时，HA通过调节HA的周转而降低。三种广泛表达的降解HA的透明质酸酶(HYALs)和三种结合和内化HA的受体已被确定，但它们如何促进HA降解尚不清楚。*目标*我的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的降解。在目前的HA降解模型中，Hyal2被提议将细胞外的HA裂解成片断，这些片断被内化以供在溶酶体中降解。与此一致的是，缺乏透明质酸2(Hyal2 KO)的小鼠会积聚细胞外HA。但是，不能重复检测到Hyal2活性。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，其活性是受调节的，并且存在非依赖于Hyal2的HA降解途径。我的近期目标(未来5年)是：*1.确定Hyal2是否作为HA降解酶和/或受体发挥作用。将检测HA的大小以及在Hyal2 KO细胞中表达的野生型或突变形式的Hyal2是否内化。在突变的Hyal2存在的情况下，HA大小增加将表明Hyal2是一种酶。如果证实了Hyal2的活性，我们将通过质谱仪和/或使用HA蛋白复合体取代外源HA来开发新的Hyal2活性分析方法。*2.鉴定能够调节其活性的Hyal2结合蛋白。将使用免疫沉淀和质谱分析从发育中的小鼠心脏中确定与Hyal2相互作用的伙伴。确认的相互作用伙伴将在野生型和Hyal2 KO细胞中过表达，并将检测HA大小、Hyal2活性和HA内化。*3.鉴定和区分HA降解的途径。野生型和Hyal2 KO细胞对荧光HA的摄取将在存在和不存在内化途径的抑制剂和/或阻断已知HA受体的抗体的情况下进行监测。体外确定的途径将通过光学成像监测荧光标记的HA的摄取在体内得到验证。*意义*通过My HQP，我们将促进对HA降解的理解，同时为HQP提供在糖科学方面的宝贵技能，这些技能是使用HA的行业在化妆品、兽医和组织工程应用中所需的。Hyal2或其激活剂很可能被这些行业确定为有价值的靶标，用于开发可以延长外源性HA产品的半衰期的抑制剂。**

**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. **