Fig 1.
Charge-charge interactions involving the catalytic Glu131 in HYAL1 and sequence and structure comparison.
(A) Schematic diagram highlighting induction of proximal negative charges by acidic residues to catalytic Glu131 in HYAL1, resulting in an increased pKa as the proton donor. (B) Sequence comparison of HYAL1, PH20, and venom hyaluronidase orthologs. The HYAL1 sequences are from Homo sapiens (UniProt ID: Q12794), Bos taurus (UniProt ID: Q5E985), Terrapene carolina (UniProt ID: A0A674IY09), and Xenopus laevis (UniProt ID: A0A1L8GP88). The PH20 sequences are from H. sapiens (UniProt ID: P38567), B. taurus (UniProt ID: Q2YDK3), T. carolina (UniProt ID: A0A674IDS3), and X. laevis (UniProt ID: Q8UVT7). The venom hyaluronidase sequences are from Loxosceles intermedia (UniProt ID: R4J7Z9), Apis mellifera (UniProt ID: Q08169), Agkistrodon contortrix (NCBI ID: JAS04369.1), and Tityus serrulatus (NCBI ID: AHF72517.1). (C) Active-site structures of HYAL1, PH20, and bee venom hyaluronidase (A. Mellifera), depicting the proximity of the β3-loop and β3′-β3″ hairpin to the catalytic Glu residue. HYAL1 (PDB ID: 2PE4), PH20 (AlphaFold structure), and bee venom hyaluronidase (PDB ID: 1FCU).
Fig 2.
pH-dependent activity analysis of HYAL1 Ala132 mutants.
(A) Substrate gel assay results at pH 4, 6, and 7 and Western blot analysis for HYAL1 WT and mutants. The blot was probed with an anti-His-tag monoclonal antibody. A representative set of experiments is shown. (B) Densitometric analysis of substrate gel assay. The activity of HYAL1 WT at pH 4 is set as 100%. Data presented are the means ± S.D. of three experiments.
Fig 3.
pH-dependent activity analysis of HYAL1 β3-loop and β-hairpin mutants.
(A) Substrate gel assay results at pH 4, 6, and 7 and Western blot analysis for HYAL1 WT and mutants. The blot was probed with an anti-His-tag monoclonal antibody. A representative set of experiments is shown. (B) Densitometric analysis of substrate gel assay. The activity of HYAL1 WT at pH 4 is set as 100%. Data presented are the means ± S.D. of three experiments.
Fig 4.
Characterization of HYAL1 β3-loop and β-hairpin mutants with double substitutions.
(A) Substrate gel assay results at pH 4, 6, and 7 and Western blot analysis for HYAL1 WT and mutants. The blot was probed with an anti-His-tag monoclonal antibody. A representative set of experiments is shown. (B) Densitometric analysis of substrate gel assay. The activity of HYAL1 WT at pH 4 is set as 100%. Data presented are the means ± S.D. of three experiments.
Fig 5.
Effect of double and triple substitutions in the β3-loop, β-hairpin, and Ala132 on HYAL1 activity.
(A) Substrate gel assay results at pH 4, 6, and 7 and Western blot analysis for HYAL1 WT and mutants. The blot was probed with an anti-His-tag monoclonal antibody. A representative set of experiments is shown. (B) Densitometric analysis of substrate gel assay. The activity of HYAL1 WT at pH 4 is set as 100%. Data presented are the means ± S.D. of three experiments.
Fig 6.
Effect of non-acidic substitutions in the β3-loop, β-hairpin, and Glu149 on PH20 activity.
(A) Substrate gel assay results at pH 4, 6, and 7 and Western blot analysis for PH20 WT and mutants. The blot was probed with an anti-His-tag monoclonal antibody. A representative set of experiments is shown. (B) Densitometric analysis of substrate gel assay. The activity of PH20 WT at pH 6 is set as 100%. Data presented are the means ± S.D. of three experiments.
Fig 7.
Specific activity of HYAL1 and PH20 WT and single mutants at pH 4 and 7.
Enzyme activity was measured by incubating 0.03% (w/v) HA solution with enzyme mixture at 37°C for 45 min, followed by addition of acidic albumin solution at pH 3.75 and a 10-min incubation at 25°C. Turbidity absorbance was measured at 600 nm. Data presented are the means ± S.D. of three experiments.