Engineering PH20 Hyaluronidase via Rational Design for Enhanced Catalytic Activity and Thermostability
- 주제(키워드) PH20 , Hyaluroidase , Enzyme engineering , protein engineering , thermostability , catalytic activity , rational design
- 주제(DDC) 547
- 발행기관 아주대학교 일반대학원
- 지도교수 Yong-Sung Kim
- 발행년도 2025
- 학위수여년월 2025. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000035241
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Hyaluronan (HA), a major glycosaminoglycan in the extracellular matrix (ECM), is frequently overproduced in solid tumors, where it forms a dense, hydrated barrier that elevates interstitial fluid pressure and impedes therapeutic penetration. Recombinant human PH20 (rHuPH20), an HA-specific endo-β-N-acetylglucosaminidase, has been explored to overcome this barrier by enzymatically degrading ECM HA and enhancing drug distribution. However, wild-type rHuPH20 suffers from low thermal stability and diminished enzymatic activity under near-neutral pH, limiting its efficacy in both solid tumor environments and subcutaneous (SC) co-formulation settings. To address these limitations, we established a structure- and sequence-guided protein engineering strategy aimed at improving both catalytic activity and thermostability of rHuPH20. Mutation-permissive second-shell residues were identified through conservation and structural filtering, followed by substrate-informed and consensus-directed mutagenesis. Initial screening yielded eight activity-enhancing mutants, and their combinations led to AC12, the most active intermediate clone. Stability-focused mutations were then predicted using four complementary computational tools, and validated substitutions (L200M, N240E) were integrated into the AC12 background, resulting in dual-optimized variants (ATC1–ATC6). Among them, ATC5 retained ~4.4-fold higher relative activity than WT and exhibited a +7.6 °C increase in melting temperature (Tₘ). Further optimization targeted a flexible loop near the catalytic cleft, where additional mutations such as M70L were introduced to generate ATC7–ATC12. The final variant, ATC11, showed superior catalytic efficacy (3.6-fold increase in kcat/Km over WT) along with the greatest thermal improvements (+8.0 °C in Tₘ and +5.2 °C in T₅₀). Benchmarking against the clinical standard HM261 confirmed the enhanced functional performance of ATC11. These findings highlight the potential of engineered rHuPH20 variants in improving therapeutic accessibility and durability for tumor ECM remodeling and long-acting subcutaneous formulations.
more목차
1. Introduction 1
1.1 Hyaluronic acid(HA) 1
1.2 Human Hyaluronidase PH20 (HuPH20) 2
1.3 Therapeutic Applications and Engineering Challenges of Recombinant Human PH20 3
2. Materials and methods 4
2.1 Expression plasmids Construction 4
2.2 Expression and purification of rHuPH20 and variants in mammalian cell 4
2.3 Modeling of AlphaFold2 5
2.4 Molecular Docking 5
2.5 Sequence Conservation and Mutational Tolerance Analysis 5
2.6 Computational Prediction of Thermostabilizing Mutations 6
2.7 B-factor Analysis 7
2.8 Turbidimetric assay 7
2.9 Morgan-elson assay 8
2.10 Operational Thermostability (T₅₀) Assay 8
2.11 Thermal Stability Assay 8
2.12 Structure-Based Interaction and Visualization Analysis 9
3. Results 10
3.1 Structure-Guided Identification and Refinement of Mutation-Permissive Hotspots 10
3.1.1. Conservation of the Catalytic Architecture Across Mammalian PH20 Homologs 10
3.1.2. Structural Definition of Second-Shell Mutation-Permissive Residues 11
3.2 Consensus- and Substrate-Informed Substitution Design at Refined Hotspot Sites 14
3.2.1 Conservation-Guided Refinement of Mutation-Permissive Residues 14
3.2.2 Structural Definition of Second-Shell Mutation-Permissive Residues 16
3.3 Rational Design and Functional Validation of Single-Point Mutants 17
3.4 Structural Dissection of Activity-Enhancing Mutants Using Docking-Based Enzyme–Substrate Complexes 19
3.5 Development of rHuPH20 Combination Mutants Derived from Single-Point Mutations 22
3.6 Computational Design Strategies for Improving Thermal Stability of rHuPH 25
3.7 Validation of Enhanced Thermostability in rHuPH20 Single-Point Mutants 26
3.8 Design of Dual-Optimized rHuPH20 Variants with Enhanced Activity and Thermal Stability 31
3.9 Further Enhancement of Catalytic Activity and Thermal Stability of rHuPH20 via Advanced Combinatorial Design 34
4. Discussion 38
CONCLUSION 40
REFERENCES 41
ABSTRACT IN KOREAN 48
