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The Enzymatic Activities of Nucleoside Triphosphate Diphosphohydrolases Towards Tenofovir Diphosphate and Their Spatial Distributions in Human Colorectal Tissue
Author(s) -
Seneviratne Herana,
Hendrix Craig,
Bumpus Namandje
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2021.35.s1.02825
Subject(s) - nucleoside triphosphate , enzyme , dephosphorylation , recombinant dna , nucleoside , biochemistry , chemistry , biology , nucleotide , phosphatase , gene
Tenofovir (TFV; prescribed as the tenofovir disoproxil fumarate prodrug) must be phosphorylated to form a competitive inhibitor of HIV, TFV‐diphosphate (TFV‐DP). Previously, we reported heterogeneity in TFV‐DP distribution in colorectal tissue (a putative site of HIV infection) sections collected from human participants receiving a TFV‐containing enema. This observed heterogeneity is likely multifactorial. TFV‐DP is structurally similar to ATP. It is known that nucleotidases such as nucleoside triphosphate diphosphohydrolases (NTPDases) dephosphorylate ATP. Therefore, we hypothesized that NTPDase‐mediated dephosphorylation plays a role in regulating TFV‐DP levels in colorectal tissue. To test this, we incubated recombinant NTPDase proteins (NTPDase 1, 3, 4, 5, and 8), individually, with TFV‐DP to determine the abilities of these enzymes to dephosphorylate TFV‐DP. Following incubation, TFV‐DP levels were measured using ultra‐high performance liquid chromatography tandem mass spectrometry. We observed TFV‐DP dephosphorylation in the presence of NTPDase 1 or 3 enzymes. Further, a malachite green phosphate assay that measures the release of inorganic phosphate was employed to further confirm the activities of NTPDase 1 and 3 towards TFV‐DP. Immunoblotting revealed that NTPDase 1 and 3 proteins are indeed expressed in human colorectal tissue lysates. To visualize the distributions of NTPDases across human colorectal tissue, we employed Matrix‐Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI). Initially, trypsin digestions on a range of recombinant NTPDases including 1 and 3 were carried out to detect and identify their tryptic peptides that could be targeted for detection in MALDI MSI. From these digestions, NTPDase 1 yielded peptides, SQHQETPVYLGATAGMR, SLSNYPFDFQGAR, and IITGQEEGAYGWITINYLLGK whereas NTPDase 3 exhibited the following peptides: YGIVLDAGSSR, GSGISSYGNNPQDVPR, and MFTVLTR. Five tryptic peptides corresponding to NTPDase 5 (which did not exhibit enzymatic activity towards TFV‐DP) were identified: LYTHSYLGFGLK, LHQPEEVQR, GSFYAFSYYYDR, QGAETVQGLLEVAK, and LATLGALETEGTDGHTFR. Next, the abovementioned MALDI MS methodology was applied to directly visualize tryptic peptides of NTPDase 1, 3 and 5 in human colorectal tissue sections. In these studies, human colorectal biopsies were collected and sectioned to 20‐micron thickness. Tissue sections were subjected to serial washing steps using ethanol. Trypsin and matrix (α‐cyano‐4‐hydroxycinnamic acid) were deposited onto the tissue using a robotic sprayer. We detected tryptic peptides, SQHQETPVYLGATAGMR, and LATLGALETEGTDGHTFR corresponding to NTPDase 1 and 5, respectively. The distribution profiles of NTPDase 1 and 5 were generated using the m/z values of above peptides. The identities of the detected peptides were validated using mass spectrometry‐based proteomics analyses. Thus, in this work, we established a MALDI MSI methodology for simultaneous visualization of tryptic peptides corresponding to NTPDases 1 and 5. Results from our in vitro studies suggest that NTPDase 1 and 3 may contribute to the regulation of TFV‐DP levels.