Deglycosylation Differentially Regulates Weaned Porcine Gut Alkaline Phosphatase Isoform Functionality along the Longitudinal Axis

Gut alkaline phosphatases (AP) dephosphorylate the lipid moiety of endotoxin and other pathogen-associated-molecular patterns members, thus maintaining gut eubiosis and preventing metabolic endotoxemia. Early weaned pigs experience gut dysbiosis, enteric diseases and growth retardation in association with decreased intestinal AP functionality. However, the role of glycosylation in modulation of the weaned porcine gut AP functionality is unclear. Herein three different research approaches were taken to investigate how deglycosylation affected weaned porcine gut AP activity kinetics. In the first approach, weaned porcine jejunal AP isoform (IAP) was fractionated by the fast protein-liquid chromatography and purified IAP fractions were kinetically characterized to be the higher-affinity and lower-capacity glycosylated mature IAP (p < 0.05) in comparison with the lower-affinity and higher-capacity non-glycosylated pre-mature IAP. The second approach enzyme activity kinetic analyses showed that N-deglycosylation of AP by the peptide N-glycosidase-F enzyme reduced (p < 0.05) the IAP maximal activity in the jejunum and ileum and decreased AP affinity (p < 0.05) in the large intestine. In the third approach, the porcine IAP isoform-X1 (IAPX1) gene was overexpressed in the prokaryotic ClearColiBL21 (DE3) cell and the recombinant porcine IAPX1 was associated with reduced (p < 0.05) enzyme affinity and maximal enzyme activity. Therefore, levels of glycosylation can modulate plasticity of weaned porcine gut AP functionality towards maintaining gut microbiome and the whole-body physiological status.

Automated summary

Research has shown that the glycosylation level of gut alkaline phosphatases (AP) in weaned pigs can regulate their functionality, maintaining gut microbiome balance and preventing metabolic endotoxemia. Three different research approaches demonstrated that deglycosylation reduces the activity and affinity of gut AP in weaned pigs. These findings suggest that the glycosylation level can modulate the functionality of gut AP in weaned pigs.