title: Secretion and N-linked glycosylation are required for prostatic acid phosphatase catalytic and antinociceptive activity.
abstract: Secretory human prostatic acid phosphatase (hPAP) is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice. Currently, it is unknown if these N-linked residues are required for hPAP protein stability and activity in vitro or in animal models of chronic pain. Here, we expressed wild-type hPAP and a series of Asn to Gln point mutations in the yeast Pichia pastoris X33 then analyzed protein levels and enzyme activity in cell lysates and in conditioned media. Pichia secreted wild-type recombinant (r)-hPAP into the media (6-7 mg protein/L). This protein was as active as native hPAP in biochemical assays and in mouse models of inflammatory pain and neuropathic pain. In contrast, the N62Q and N188Q single mutants and the N62Q, N188Q double mutant were expressed at lower levels and were less active than wild-type r-hPAP. The purified N62Q, N188Q double mutant protein was also 1.9 fold less active in vivo. The N301Q mutant was not expressed, suggesting a critical role for this residue in protein stability. To explicitly test the importance of secretion, a construct lacking the signal peptide of hPAP was expressed in Pichia and assayed. This "cellular" construct was not expressed at levels detectable by western blotting. Taken together, these data indicate that secretion and post-translational carbohydrate modifications are required for PAP protein stability and catalytic activity. Moreover, our findings indicate that recombinant hPAP can be produced in Pichia--a yeast strain that is used to generate biologics for therapeutic purposes.
authors.affiliation: Department of Cell and Molecular Physiology, Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina, United States of America.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Hurt
authors.forename: Brendan J
authors.initials: BJ
authors.lastname: Fitzpatrick
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Norris-Drouin
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Zylka
mesh_terms: D000135:Acid Phosphatase; D000700:Analgesics / Q000378:metabolism / Q000627:therapeutic use*; D000818:Animals; D006031:Glycosylation; D006801:Humans; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010146:Pain / Q000188:drug therapy; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000235:genetics / Q000502:physiology; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry / Q000378:metabolism* / Q000627:therapeutic use
chemical_list: D000700:Analgesics; D000135:Acid Phosphatase; C428439:prostatic acid phosphatase; D017027:Protein Tyrosine Phosphatases