Phytase properties and locations in tissues of transgenic pigs secreting phytase in the saliva1

Abstract

A transgenic Cassie (CA) line of Yorkshire (YK) pigs was developed using a transgene composed of the mouse parotid secretory protein promoter linked to the Escherichia coli phytase gene integrated in chromosome 4. Previous studies documented that salivary secretion of phytase was sufficient to enable efficient digestion of plant feed phytate P. In the present study the catalytic properties and tissue distribution of the phytase in CA pigs were determined by a combination of enzymatic assays, immunohistochemistry, and immunoblots of tissue samples. The E. coli phytase had a mass of 44.82 kDa whereas the phytase secreted in CA saliva had a mass of 52.42 kDa as a result of glycosylation of the enzyme in the parotid gland. Despite the difference in size, the 2 enzymes exhibited similar substrate specificities, and substrate affinity ( K: m) and maximum hydrolytic activity ( V: max) catalytic properties. Phytase assays showed that the enzyme was present at high specific activity in the salivary glands with low activity in the soft palate and essentially none in the kidney, lean (muscle), liver, or skin of CA pigs and none in YK pigs. This conclusion was supported by immunoblot analysis using a polyclonal anti-phytase antibody. Immunohistochemical analysis of 83 different tissue locations of CA and YK pigs confirmed the ubiquitous presence of phytase in serous cells of the salivary glands and the localized presence of phytase in both serous and mixed cell types in the submucosal glands of the oropharynx; in the pharynx, tonsils, and esophagus; in some Bowman's glands in the nasal mucosa and eustachian tube; and in the prostate gland of CA boars. Furthermore, it showed the absence of phytase from the kidney, lean, liver, and skin of CA pigs. Phytase was not detected in any of the conventional YK tissues tested. The phytase was found to be glycosylated with the allergenic galactose-α-1,3-galactose (α-gal) epitope by immunoblotting using α-gal specific monoclonal antibodies. Galactose-α-1,3-galactose glycosylation of proteins is a common feature of pork and other red meats. The α-gal epitope was shown to be associated with a few proteins in muscle and skin but with the greatest number of proteins in kidney and parotid tissues of CA and YK pigs. The absence of phytase from the major food tissues and the displacement of other α-gal glycosylated proteins in the parotid glands by α-gal glycosylated phytase in conjunction with previously published data support the contention that expression of the novel phytase has minimal influence on pork quality and safety.

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

“…We then adopted a previous strategy of using the peptide N -glycosidase-F enzyme in the complete cleavage of N -glycosylation for clarification of roles of the N -glycosylation in modulation of phytase and acid AP functionality in the transgenic phytase pig [ 36 ]. We conducted a dose-response experiment with the weaned porcine jejunal homogenates and observed sigmoidal responses in the residual AP activity with gradient treatment doses (U/mg jejunal homogenate protein) of the peptide N -glycosidase F (PNGase-F) enzyme cocktail under the in vitro incubations of pH of 7.4 at 37 °C, clearly defining the optimal doses of the PNGase-F enzyme cocktail required for the complete cleavage of N -glycosylation in vitro treatments in this study ( Figure 5 ).…”

“…The gel band-c shown in the Figure 2 represented the pre-mature IAP MW at about 56 kDa, which is consistent with the coding AA-based predicting of MW for the porcine IAP isoforms at about 53 kDa. Forsberg et al [ 36 ]. reported that an extra 7.6 kDa glycan was added to phytase in the transgenic phytase pigs primarily due to the N -glycosylation.…”

“…We then investigated the role of the N -glycosylation in modulation of the weaned porcine intestinal AP isoform functionality through using the PNGase-F enzyme cocktail for the complete cleavage of N -glycosylation [ 36 ]. The recombinant PNGase-F enzyme is an amidase that only hydrolyzes the glycosyl-amine linkage between the N -acetyl-ß-glucosamine and the asparagine-formed N -glycosylation of the AP and leaves the oligosaccharide glycan moiety intact [ 58 , 67 ].…”

“…It is a common belief that N - and O -glycosylation is limited in bacteria [ 40 ]. This was evident from our previous study that a bacterial phytase was not N -glycosylated when overexpressed in E. coli , whereas this bacterial phytase was heavily N -glycosylated when expressed in the transgenic phytase pig [ 36 ]. Here we also reported the overexpression of the porcine intestinal AP (IAP) isoform-X1 (IAPX1) in an endotoxin-free E. coli strain, referred to as the ClearColiBL21 (DE3) as developed by Mamat et al [ 41 ].…”

“…Through a using N -glycosylation inhibitor, López-Posadas et al [ 35 ] demonstrated that enterocytic oxidative stress induced increases in TNAP activity and this was mediated via up-regulating N -glycosylation. Forsberg et al [ 36 ] reported the use of the peptide N -glycosidase F enzyme in the complete cleavage of N -glycosylation for clarification of roles of the N -glycosylation in phytase and acid AP functionality of their bacterial phytase expressed in the transgenic phytase pig. Weaning-associated reduction in the intestinal AP digestive capacity could have been partially attributed to the villus enterocytic atrophy and further linked to insufficient enteral nutrient supply; whereas the role of glycosylation in modulation of the AP functionality still needs to be clarified along the intestinal longitudinal axis in the weaned pig [ 6 , 37 ].…”