Abstract
Upregulation of carbonic anhydrase IX (CA IX) is associated with several aggressive forms of cancer and promotes metastasis. CA IX is normally constitutively expressed at low levels in selective tissues associated with the gastrointestinal tract, but is significantly upregulated upon hypoxia in cancer. CA IX is a multi-domain protein, consisting of a cytoplasmic region, a single-spanning transmembrane helix, an extracellular CA catalytic domain, and a proteoglycan-like (PG) domain. Considering the important role of CA IX in cancer progression and the presence of the unique PG domain, little information about the PG domain is known. Here, we report biophysical characterization studies to further our knowledge of CA IX. We report the 1.5 Å resolution crystal structure of the wild-type catalytic domain of CA IX as well as small angle X-ray scattering and mass spectrometry of the entire extracellular region. We used matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry to characterize the spontaneous degradation of the CA IX PG domain and confirm that it is only the CA IX catalytic domain that forms crystals. Small angle X-ray scattering analysis of the intact protein indicates that the PG domain is not randomly distributed and adopts a compact distribution of shapes in solution. The observed dynamics of the extracellular domain of CA IX could have physiological relevance, including observed cleavage and shedding of the PG domain.
Highlights
Carbonic anhydrases (CAs) are a family of metalloenzymes that catalyze the reversible hydration of CO2 in the presence of water, to HCO3− and H+ [1]
During our efforts to study the crystal structure of the entire extracellular domain (ECD) of carbonic anhydrase IX (CA IX), we observed spontaneous degradation of the protein by SDS-PAGE and visible precipitation of the purified protein. This degradation enabled crystallization of the soluble and stable catalytic domain of CA IX, while the remaining insoluble product was further characterized with mass spectrometry
The crystal structure of the CA domain we report is for wild-type CA IX to 1.53 Å resolution and we were able to resolve residues Asp137–Val394
Summary
Carbonic anhydrases (CAs) are a family of metalloenzymes that catalyze the reversible hydration of CO2 in the presence of water, to HCO3− and H+ [1]. CA IX has a number of post-translational modifications that have been identified: O-linked glycosylation by heparan or chondroitin sulfate glycosaminoglycan chains at Thr115 in the N-terminal of the PG domain [26,27], N-linked glycosylation of high mannose sugar chain at Asn346 localized on the catalytic domain [26], and three phosphorylation sites on CY region, namely Thr443, Ser448, and Tyr449 [28,29] The effect of these modifications on CA IX in pH regulation, cell adhesion, and tumor progression has not been fully understood, the perturbed glycosaminoglycan modification results in increased internalization and cytotoxicity of an antibody drug conjugate targeted to CA IX [27]. The constructs were coded for amino acids 1–414 (Uniprot ID Q16790) corresponding to the signaling peptide, PG domain, and the catalytic CA domain—similar to previous descriptions [21,26,38]
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