Establishment of blood glycosidase activities and their excursions in sepsis

Abstract

Glycosidases are hydrolytic enzymes studied principally in the context of intracellular catabolism within the lysosome. Therefore, glycosidase activities are classically measured in experimentally acidified assay conditions reflecting their low pH optima. However, glycosidases are also present in the bloodstream where they may retain sufficient activity to participate in the regulation of glycoprotein half-lives, proteostasis, and disease pathogenesis. We have, herein, established at physiological pH 7.4 in blood plasma and sera the normal ranges of four major glycosidase activities essential for blood glycoprotein remodeling in healthy mice and humans. These activities included β-galactosidase, β-N-acetylglucosaminidase, α-mannosidase, and α-fucosidase. We have identified their origins to include the mammalian genes Glb1, HexB, Man2a1, and Fuca1. In experimental sepsis, excursions of glycosidase activities occurred with differences in host responses to discrete bacterial pathogens. Among similar excursions in human sepsis, the elevation of β-galactosidase activity was a prognostic indicator of increased likelihood of patient death.

Fig. 1.

β-galactosidase activity and genetic source(s) in murine blood plasma and sera. (A) β-galactosidase assay used to determine specific activity at pH 7.4 compared to pH 4.3 with and without the addition of the inhibitor N-(n-nonyl)-DGJ (C_f = 1 mM). (B) Standard curve of fluorescence measured from serial dilutions of the fluorescent reporter 4-methylumbelliferone (4-MU) used to convert 4-MU fluorescence into units of enzyme activity at 60 min following assay start. A unit was defined as 1 μmole of substrate hydrolyzed per minute. (C) β-galactosidase activity in plasma and serum from wild-type mice (n = 14). (D) β-galactosidase activity in the plasma and serum of wild-type male and female littermates (n = 7 per sex). (E) β-galactosidase activity in plasma and serum of Glb1-null mice compared to wild-type littermates at pH 7.4 (n = 12 per genotype). (F) β-galactosidase activity in the plasma and serum of Glb1-null male and female mice at pH 7.4 compared to wild-type littermates (n = 6 per sex). Data are presented as means ± SD. Unpaired Mann–Whitney nonparametric statistical significance is denoted by *P < 0.05, **P < 0.01, or ***P < 0.001.

Fig. 2.

β-N-acetylglucosaminidase activity and genetic source(s) in murine blood plasma and sera. (A) β-N-acetylglucosaminidase assay used to determine specific activity at pH 7.4 compared to pH 4.7 with and without the addition of the inhibitor ADN (C_f = 0.25 mM). (B) Standard curve of fluorescence measured from serial dilutions of the enzyme product and fluorescent reporter 4-MU used to convert 4-MU fluorescence into units of enzyme activity at 60 min following assay start. A unit was defined as 1 μmole of substrate hydrolyzed per min. (C) β-N-acetylglucosaminidase activity in plasma and serum from wild-type mice (n = 12). (D) β-N-acetylglucosaminidase activity in the plasma and serum of wild-type male and female littermates (n = 6 per sex). (E) β-N-acetylglucosaminidase activity in plasma and serum of HexB-null mice compared to wild-type littermates at pH 7.4 (n = 10 per genotype). (F) β-N-acetylglucosaminidase activity in the plasma and serum of male and female HexB-null mice at pH 7.4 compared to wild-type littermates (n = 5 per sex). Assay background was 1 mU/L or less. Data are presented as means ± SD. Unpaired Mann–Whitney nonparametric statistical significance is denoted by *P < 0.05, **P < 0.01, or ***P < 0.001.

Fig. 3.

α-Mannosidase activity and genetic source(s) in murine blood plasma and sera. (A) α-Mannosidase assay used to determine specific activity at pH 7.4 compared to pH 4.3 with and without the addition of the inhibitor SW (C_f = 0.25 mM). (B) Standard curve of fluorescence measured from serial dilutions of the enzyme product and fluorescent reporter 4-MU used to convert 4-MU fluorescence into units of enzyme activity at 60 min following assay start. A unit was defined as 1 μmole of substrate hydrolyzed per minute. (C) α-Mannosidase activity in plasma and serum of wild-type mice (n = 26). (D) α-Mannosidase activity in the plasma and serum of wild-type male and female littermates (n = 13 per sex). (E) α-Mannosidase activity in plasma and serum of Man2a1-null mice compared to wild-type littermates at pH 7.4 (n = 12 per genotype). (F) α-Mannosidase activity in plasma and serum of male and female Man2a1-null mice at pH7.4 compared to wild-type littermates (n = 6 per sex). Data are presented as means ± SD. Unpaired Mann–Whitney nonparametric statistical significance is denoted by *P < 0.05, **P < 0.01, or ***P < 0.001.

Fig. 4.

α-Fucosidase activity and genetic source(s) in murine blood plasma and sera. (A) α-Fucosidase assay used to determine specific activity at pH 7.4 compared to pH 5.5 with and without the addition of the inhibitor DFJ (C_f = 0.2 mM). (B) Standard curve of fluorescence measured from serial dilutions of the enzyme product and fluorescent reporter 4-MU used to convert 4-MU fluorescence into units of enzyme activity at 60 min following assay start. A unit was defined as 1 μmole of substrate hydrolyzed per minute. (C) α-Fucosidase activity in plasma and serum from wild-type mice (n = 20). (D) α-Fucosidase activity in the plasma and serum of wild-type male and female littermates (n = 10 per sex). (E) α-Fucosidase activity in plasma and serum of Fuca1-null mice at pH 7.4 compared to wild-type littermates. (n = 6 per genotype). (F) α-Fucosidase activity in plasma and serum of male and female Fuca1-null mice at pH 7.4 compared to wild-type littermates (n = 3 per sex). ND; not detected. Data are presented as means ± SD. Unpaired Mann–Whitney nonparametric statistical significance is denoted by *P < 0.05, **P < 0.01, or ***P < 0.001.

Fig. 5.

Glycosidase activities in mouse plasma at pH 7.4 during experimental sepsis. Mouse cohorts were infected with clinically isolated bacterial pathogens and analyzed for changes in plasma glycosidase activities at two times postinfection measured either in hours (h) or days (d) during the pathogenesis of sepsis compared to uninfected littermates at time 0 (t = 0). Activities relative to uninfected littermate cohorts were plotted and include (A) β-galactosidase activity (n = 8 per timepoint including four males and four females), which increased at later infection timepoints on average 600% in sepsis caused by ST, 120% by EC, 292% by SP, and 153% by MR infections, (B) β-N-acetylglucosaminidase activity (n = 8 per timepoint including four males and four females), which was increased on average 320% by ST, 145% by SP, and 146% by SA infections at the late sepsis timepoint, (C) α-mannosidase activity (n = 8 per timepoint including four males and four females), which on average was significantly elevated to 213% of normal in later stage sepsis caused by ST infection, 141% of normal in early sepsis caused by MR infection, and decreased to 87% of normal at the late sepsis timepoint caused by SA infection, and (D) α-fucosidase activity (n = 8 per timepoint including four males and four females), which on average increased at the late sepsis timepoint to 141% in ST, 151% in SA, and 124% in MR infections, while decreasing to 82% of normal at the later sepsis timepoint and 84% of normal in the early sepsis timepoint among EC and SA infections, respectively. Data are presented as means ± SD. Unpaired Mann–Whitney parametric statistical significance is denoted by *P < 0.05, **P < 0.01, or ***P < 0.001.

Fig. 6.

Glycosidase activities measured at pH 7.4 in healthy and septic humans. (A)–(D) Glycosidase enzyme activities indicated were assayed identically to the murine studies among individual healthy humans (n = 42) and human patients diagnosed with sepsis (n = 88). A fraction of samples (n = 6 for each glycosidase activity) were further compared in the presence of the relevant glycosidase inhibitor as indicated. Activities were further compared among males and females and all activities are presented using µU or mU per gram of plasma protein to control for fluid therapy provided to all septic patients prior to blood draws. A unit was defined as 1 μmole of substrate hydrolyzed per min. Average activities are denoted with numbers above the dot plots. (E) and (F) Glycosidase activities plotted among patients whom either survived or expired during sepsis are plotted and compared. Data are presented as means ± SD. Unpaired Mann–Whitney nonparametric statistical significance is denoted by *P < 0.05, **P < 0.01, or ***P < 0.001.