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

Abstract

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.

Modulation of Porcine Gut Microbiota and Microbiome: Hologenomic, Dietary, and Endogenous Factors

“…As a group of the major endogenous digestive enzymes, gut alkaline phosphatases (APs), including the intestinal AP isoform (IAP) and the tissue non-specific AP isoform (TNAP), are expressed on the gut apical membrane along the porcine small-large intestinal tract [ 35 ]. Gut APs contribute to the dephosphorylation of the lipid moiety of endotoxin and other pathogen-associated-molecular pattern (PAMP) member molecules such as the endotoxin lipopolysaccharides (LPSs) and triphosphate nucleotides, in promoting a balanced gut commensal microbiota and microbiome, thus maintaining gut eubiosis and preventing inflammation and metabolic endotoxemia [ 35 ]. Previous studies showed that weaning-associated growth retardation and gut mucosal villus atrophy were associated with decreased gut AP digestive capacity and affinity in the pig [ 36 ].…”

“…Previous studies showed that weaning-associated growth retardation and gut mucosal villus atrophy were associated with decreased gut AP digestive capacity and affinity in the pig [ 36 ]. In this Special Issue, Yin et al [ 35 ] evidently reviewed that gut AP affinity is more limiting and is the bottleneck in the dephosphorylation of gut luminal PAMP compounds. They further showed that N -deglycosylation of IAP and TNAP along the porcine small–large intestinal longitudinal axis could effectively modulate the plasticity of weaned porcine gut AP functionality in terms of the maximal activity and affinity of these AP enzymes.…”