title: Phytase properties and locations in tissues of transgenic pigs secreting phytase in the saliva.
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.
authors.affiliation: Departments of Molecular and Cellular Biology cforsber@uoguelph.ca.
authors.forename: C W
authors.initials: CW
authors.lastname: Forsberg
authors.affiliation: Departments of Molecular and Cellular Biology.
authors.forename: R G
authors.initials: RG
authors.lastname: Meidinger
authors.affiliation: Departments of Molecular and Cellular Biology.
authors.forename: D
authors.initials: D
authors.lastname: Murray
authors.affiliation: Pathobiology.
authors.forename: N D
authors.initials: ND
authors.lastname: Keirstead
authors.affiliation: Pathobiology.
authors.forename: M A
authors.initials: MA
authors.lastname: Hayes
authors.affiliation: Animal and Poultry Science.
authors.forename: M Z
authors.initials: MZ
authors.lastname: Fan
authors.affiliation: Chemistry, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: Jeyabarathy
authors.initials: J
authors.lastname: Ganeshapillai
authors.affiliation: Chemistry, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: M A
authors.initials: MA
authors.lastname: Monteiro
authors.affiliation: Departments of Molecular and Cellular Biology.
authors.forename: S P
authors.initials: SP
authors.lastname: Golovan
authors.affiliation: Departments of Molecular and Cellular Biology.
authors.forename: J P
authors.initials: JP
authors.lastname: Phillips
mesh_terms: D010832:6-Phytase / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism; D008297:Male; D018482:Muscle, Skeletal / Q000378:metabolism; D010159:Palate / Q000378:metabolism*; D010306:Parotid Gland / Q000378:metabolism*; D012463:Saliva / Q000378:metabolism*; D012469:Salivary Glands / Q000378:metabolism*; D013552:Swine / Q000235:genetics / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D010832:6-Phytase
keywords: allergen; galactose-α-1,3-galactose; phytase; porcine; tissue; transgene