Abstract
Human carbonic anhydrase IX (CAIX), a unique member of the α carbonic anhydrase family, is a transmembrane glycoprotein with high enzymatic activity by which CAIX contributes to tumorigenesis through pH regulation. Due to its aberrant expression, CAIX is considered to be a marker of tumor hypoxia and a poor prognostic factor of several human cancers. Hypoxia-activated catalytic function of CAIX is dependent on posttranslational modification of its short intracellular domain. In this work, we have identified that C-terminal Ala459 residue, which is common across CAIX of various species as well as additional transmembrane isoforms, plays an important role in CAIX activation and in pH regulation. Moreover, structure prediction I-TASSER analysis revealed involvement of Ala459 in potential ligand binding. Using tandem mass spectrometry, Protein-L-isoaspartyl methyltransferase (PIMT) was identified as a novel interacting partner, further confirmed by an in vitro pulldown assay and an in situ proximity ligation assay. Indeed, suppression of PIMT led to increased alkalinization of culture media of C33a cells constitutively expressing CAIX in hypoxia. We suggest that binding of PIMT represents a novel intracellular signal required for enzymatic activity of CAIX with a potential unidentified downstream function.
Highlights
Carbonic anhydrase IX (CAIX) belongs to the α carbonic anhydrase family of zinc metalloenzymes that catalyze the reversible interconversion of carbon dioxide to form bicarbonate ions and protons [1]
We showed that a structural change at 459 position perturbs the ability of carbonic anhydrase IX (CAIX) to regulate pH, decreases its enzymatic activity and affects the cell migration
Using a proteomic-based approach, we found and subsequently proved the intracellular interaction of CAIX with cytoplasmic protein Protein-L-isoaspartyl methyltransferase (PIMT)
Summary
Carbonic anhydrase IX (CAIX) belongs to the α carbonic anhydrase family of zinc metalloenzymes that catalyze the reversible interconversion of carbon dioxide to form bicarbonate ions and protons [1]. GST-tagged CAIX-IC-wt recombinant protein was produced, isolated, immobilized on glutathione sepharose beads (Figure 5a, left panel) and incubated with hypoxic cell lysate Using this approach, we showed the binding capacity of PIMT protein expressed ligation assay (PLA, Figure 5b). Using this approach, we showed the binding capacity of PIMT protein expressed by cells of various cancer types and recombinant intracellular domain of CAIX (Figure 5a, right panel). GENEVESTIGATOR analysis of microarray data proved the expression of Pcmt in a broad spectrum of CA9-positive cancers (Figure 6b) We stained both proteins in CRC tissue samples and showed co-localization of PIMT with CAIX (Figure 6c). We did not elucidate the precise mechanism, we can conclude that hypoxia is involved in the regulation of PIMT and may contribute to its cancer-related functions
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