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Active Expression of Human Hyaluronidase PH20 and Characterization of Its Hydrolysis Pattern.

ABSTRACT: Hyaluronidases are a group of glycosidases catalyzing the degradation of hyaluronic acid (HA). Because of the advantages of effectively hydrolyzing the HA-rich matrix and low immunogenicity, human hyaluronidase PH20 (hPH20) is widely used in the medical field. Here, we realized the active expression of recombinant hPH20 by Pichia pastoris under a methanol-induced promoter P_AOX1. By optimizing the composition of the C-terminal domain and fusing protein tags, we constructed a fusion mutant AP₂-△491C with the extracellular hyaluronidase activity of 258.1 U·L^-1 in a 3-L bioreactor, the highest expression level of recombinant hPH20 produced by microbes. Furthermore, we found recombinant hPH20 hydrolyzed the β-1,4 glycosidic bonds sequentially from the reducing end of o-HAs, with HA₆ ^NA as the smallest substrate. The result will provide important theoretical guidance for the directed evolution of the enzyme to prepare multifunctional o-HAs with specific molecular weights.

SUBMITTER: Pang B

PROVIDER: S-EPMC9136067 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Active Expression of Human Hyaluronidase PH20 and Characterization of Its Hydrolysis Pattern.

Pang Bo B He Jing J Zhang Weijiao W Huang Hao H Wang Yang Y Wang Miao M Du Guocheng G Kang Zhen Z

Frontiers in bioengineering and biotechnology 20220513

Hyaluronidases are a group of glycosidases catalyzing the degradation of hyaluronic acid (HA). Because of the advantages of effectively hydrolyzing the HA-rich matrix and low immunogenicity, human hyaluronidase PH20 (hPH20) is widely used in the medical field. Here, we realized the active expression of recombinant hPH20 by <i>Pichia pastoris</i> under a methanol-induced promoter P<sub>AOX1</sub>. By optimizing the composition of the C-terminal domain and fusing protein tags, we constructed a fus ...[more]

PMID: 35646856

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Project description:INTRODUCTION:There is currently no consensus in the literature concerning the impact of aging on the properties of hyaluronan (HA) in the subcutaneous (SC) space. Recombinant human hyaluronidase PH20 (rHuPH20) facilitates SC administration of injected therapeutics by depolymerizing SC HA, facilitating bulk fluid flow, dispersion and absorption. This study assessed the impact of intrinsic aging on HA in the SC space and thus the ability of rHuPH20 to enhance delivery of co-administered therapeutics. METHODS:Histologic evaluations of HA levels and degradation were performed on human skin samples from six age groups, aged from 20 to 100 years. HA levels were evaluated by HA staining and degradation by staining samples for HA following incubation with rHuPH20. HA was extracted from samples and HA size determined by gel electrophoresis. Dermal reconstitution was assessed in young (aged 1.5 months) and elderly (aged?>?16 months) mice. Baseline dye dispersion was measured at 5 and 20 min post-intradermal dye injection. Following treatment with rHuPH20, dye dispersion was measured again at 2, 24, 48, 72 and 96 h. RESULTS:Distribution of HA was confined to the interstitial space between adipocytes, with similar pericellular presence and levels of HA found across all age groups. Substantial levels of high-molecular-weight HA were observed in all age groups at baseline. Incubation with a clinically relevant dose of rHuPH20 resulted in degradation of all SC HA and similar degradation profiles independent of age. No difference in dye dispersion time was observed between young and elderly mice across the range of time points assessed, with dye dispersion returning to baseline levels by 24 h after rHuPH20 treatment. CONCLUSIONS:Subcutaneous delivery of approved therapeutics facilitated by co-administration with rHuPH20 should not be impacted by intrinsic aging, with this study providing no evidence for an effect of aging on HA distribution, structure or a loss of rHuPH20 efficacy.

Dataset Information

Active Expression of Human Hyaluronidase PH20 and Characterization of Its Hydrolysis Pattern.

Publications

Active Expression of Human Hyaluronidase PH20 and Characterization of Its Hydrolysis Pattern.

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