Secretion and N-Linked Glycosylation Are Required for Prostatic Acid Phosphatase Catalytic and Antinociceptive Activity

Department of Cell and Molecular Physiology, Neuroscience Center, University of North Carolina, Chapel Hill, North Carolina, United States of America.
PLoS ONE (Impact Factor: 3.23). 02/2012; 7(2):e32741. DOI: 10.1371/journal.pone.0032741
Source: PubMed


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.

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Available from: Jacqueline L Norris, Mar 18, 2014
    • "). It has been shown by Hurt et al. (2012) that mutation of N-linked residues closer to the active site has a greater effect on protein activity and stability. Therefore, the modest effect we observe could be explained by the fact that all the five glycosites are located far from the active site cleft as shown in the homology model of Tr7 (Supp. "
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    ABSTRACT: A mutant Trypanosoma rangeli sialidase, Tr7, expressed in Pichia pastoris, exhibits significant trans-sialidase activity, and has been used for analytical-scale production of sialylated human milk oligosaccharides. Mass spectrometry-based site-specific N-glycoprofiling of Tr7 showed that heterogeneous high-mannose type N-glycans were present at all the five potential N-linked glycosites. N-linked glycans in Tr7 were predominantly neutral oligosaccharides with compositions Man8-16GlcNAc2, but also mono- and di-phosphorylated oligosaccharides in the forms of Man9-15P1GlcNAc2 and Man9-14P2GlcNAc2, respectively. Some phosphorylated N-linked glycans further contained an additional HexNAc, which has not previously been reported in P. pastoris-expressed proteins. We compiled a method pipeline that combined hydrophilic interaction liquid chromatography enrichment of glycopeptides, high accuracy mass spectrometry and automated interpretation of the mass spectra with in-house developed “MassAI” software, which proved efficient in glycan site microheterogeneity analysis. Functional analysis showed that the deglycosylated Tr7 retained more than 90% of both the sialidase and trans-sialidase activities relative to the glycosylated Tr7.
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    • "This extracellular adenosine leads to a decrease in chronic pain by activating A1R in nociceptive neurons (Zylka et al., 2008). sPAP is glycosylated at three asparagine residues (N62, N188, N301) and has potent antinociceptive effects when administered to mice (Hurt et al., 2012a). Secretion and post-translational carbohydrate modifications were found to be required for PAP protein stability and catalytic activity. "
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    • "Here, we sought to determine if the adenosine receptor dependent antinociceptive effects of acupuncture could be exploited and greatly extended by injecting hPAP into an acupuncture point. When combined with a recombinant version of hPAP [18], this approach could be utilized to more effectively treat chronic pain in localized regions of the body without acupuncture needle stimulation. "
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