The impact of pH on proteolytic activity in wound fluid: Implications for acid therapy

Abstract

Wound healing necessitates a balance between synthesis and breakdown of extracellular matrix components, which is tightly regulated by proteases and their inhibitors. While studies have demonstrated that citric and acetic acid treatments enhance healing in recalcitrant wounds, the underlying proteolytic mechanisms remain elusive. In this study, we systematically evaluated changes in the proteolytic activity of murine wound fluid upon acidification. A library of 228 synthetic peptides served as reporters of protease activity at pH 7.4, pH 5.0, and pH 3.5. The peptide digestion patterns differed at each pH, revealing that proteases active at pH 7.4 are inactivated at pH 3.5. Notably, cathepsin D emerged as the dominant active enzyme at pH 3.5, and its activity was inhibited by pepstatin. Using a fluorogenic substrate, we quantified cathepsin D activity across varying pH levels and demonstrated optimal activity between pH 3.0 and 3.8. This activity was detectable as early as 1 day postinjury and persisted over the following 10 days. Importantly, human wound fluid exhibited the same activity profile, validating the mouse model as a relevant system for studying acid-mediated wound healing processes.

Keywords: MSP-MS; acid treatment; aspartic acid protease; fluorogenic substrate; pepstatin; proteolytic activity; wound fluid.

Figure 1

Overview of the substrate profiling studies with murine wound fluid at pH 3.5, 5.0, and 7.4 and the development of fluorogenic reporter substrates.

Figure 2

pH-dependent cleavage of YTRLNGEAVnFnSK by murine wound fluid proteases over time.A, peptide was cleaved between S and K at pH 7.4, and the cleavage site is indicated by ∗. The cleavage product (highlighted in bold font) was quantified by LC–MS/MS after 0-, 15-, 60-, and 120-min incubation. B, a second cleavage product was detected and quantified at pH 7.4. C and D, cleavage products quantified at pH 5.0. E and F, cleavage products quantified at pH 3.5. All assays were performed in four technical replicates using a pooled wound fluid sample from three mice. Lowercase n corresponds to norleucine.

Figure 3

Quantitative multiplex substrate profiling of murine wound fluid.A, distribution of murine wound fluid protease cleavage sites within 14-mer peptides. B, the Venn diagram shows the number of cleavage sites shared between the three different pH conditions.

Figure 4

iceLogo plots illustrating the cleavage profiles. Murine wound fluid samples taken 5 days after injury were assayed at A, pH 3.5, B, 5.0, and C, 7.4. Data represent a pooled wound sample from three mice assayed in quadruplicate reactions. The red arrow indicates the site of cleavage. iceLogo plots the relative frequencies of amino acid residues at each of the P4 to P4′ positions of the cleaved peptides, where the height of each amino acids represents “percent difference,” defined as the difference in frequency for an amino acid appearing in the “experimental dataset” relative to the “reference dataset.” Ranked preferred amino acids are shown above the midline, whereas unpreferred amino acids are represented below the midline.

Figure 5

Comparation of proteolytic activity in murine wound fluid by MSP-MS, 5 and 10 days postinjury.A, number of cleavage sites detected at pH 3.5 in wound fluid 5 and 10 days postinjury. B and C, quantification of the time-dependent accumulation of select N-terminal and C-terminal fragments. D, normalized intensity of proteolytic cleavage products in the presence and absence of pepstatin. Each rectangle represents the average intensity of a cleaved peptide product (n = 4 technical replicates) normalized to the intensity of the same cleaved peptide that was quantified in the day 5 sample. The lighter color indicates that the cleaved peptide decreased in intensity, whereas the darker color indicates that the peptide increased. MSP-MS, multiplex substrate profiling by mass spectrometry.

Figure 6

Murine wound fluid protease activity.A, protease activity in acidified wound fluid (pH 3.5) from day 1 to 10 in the absence and presence of pepstatin. B, superposition of pH curves for murine wound fluid from day 5 and 10 postinjury using GKPILFFRL substrate. Each data point corresponds to technical triplicates from one pooled wound fluid sample derived from three mice.

Figure 7

Human wound fluid protease activity.A, activity profile of wound fluid proteases using the GKPILFFRL substrate when assayed across different pH conditions. B, inhibition profile of acid protease. Assays were performed in the absence and presence of 10 μM pepstatin, 1 mM EDTA, 100 μM marimastat, 1 mM AEBSF, 10 μM E64, and 10 μM CFZ inhibitors. C, activity profile of wound fluid proteases using the PLGLdAR substrate when assayed across different pH conditions. D, inhibition profile of MMP-2 protease in wound fluid. Each bar corresponds to one assay performed in triplicate wells. ∗∗p < 0.01; ∗∗∗∗p < 0.0001 using one-way ANOVA followed by Dunnett’s multiple comparisons test versus control. AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; CFZ, carfilzomib; E64, trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane; MMP-2, matrix metalloproteinase 2.

Figure 8

Human wound fluid protease activity.A, protease activity assays of human wound fluid proteases using RPKPVEvWR as substrate when assayed across different pH conditions. B, inhibition profile of acid protease. Assays were performed in the absence and presence of 10 μM pepstatin, 1 mM EDTA, 100 μM marimastat, 1 mM AEBSF, 10 μM E64, and 10 μM CFZ inhibitors at pH 3.5. C, inhibition profile of serine proteases in wound fluid at pH 7.4. Each bar corresponds to one assay performed in triplicate wells. ∗∗∗∗p < 0.0001 using one-way ANOVA followed by Dunnett’s multiple comparisons test versus control. AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; CFZ, carfilzomib; E64, trans-epoxysuccinyl-l-leucylamido(4-guanidino)butane.