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: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711311
• 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，对脊椎动物细胞周围基质的结构和功能至关重要。平均每个成年人体内有15克HA，其中1/3每天通过组织更替被替换。此外，HA在细胞增殖和迁移时增加，在细胞成熟时通过调节HA周转而减少。已经确定了三种广泛表达的降解透明质酸的透明质酸酶（HYALs）和三种结合和内化透明质酸的受体，但它们如何促进透明质酸的降解却知之甚少。