Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has\r\npotent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required\r\nfor hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a\r\nseries of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell\r\nlysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6ââ?¬â??7 mg protein/L). This\r\nprotein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic\r\npain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels\r\nand were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in\r\nvivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the\r\nimportance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This ââ?¬Ë?ââ?¬Ë?cellularââ?¬â?¢Ã¢â?¬â?¢\r\nconstruct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and\r\npost-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our\r\nfindings indicate that recombinant hPAP can be produced in Pichiaââ?¬â?a yeast strain that is used to generate biologics for\r\ntherapeutic purposes.
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