Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX

Abstract

Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regarding the expression and crystallization of the enzyme. Currently, only one baculovirus-produced CA IX structure in complex with a nonspecific CA inhibitor, acetazolamide, is available in Protein Data Bank. We have developed an efficient system for the production of the catalytic domain of CA IX in methylotrophic yeast Pichia pastoris. The produced protein can be easily crystallized in the presence of inhibitors, as we have demonstrated for several 2-thiophene-sulfonamide compounds. We have also observed significant differences in the binding mode of chemically identical compounds to CA IX and CA II, which can be further exploited in the design of CA IX-specific inhibitors.

Inhibition of carbonic anhydrase IX targets primary tumors, metastases, and cancer stem cells: Three for the price of one

“…The wild type catalytic domain, in agreement with previously described light scattering experiments, crystallized as a dimer whose formation was mediated by an intermolecular disulfide bond (Figure ) . Interestingly, also when mutating the cysteine involved in the formation of the intermolecular disulfide (single mutant C174S and double mutant C174S, N346Q), the protein formed in the crystal a dimer, stabilized by several hydrogen bonds and numerous van der Waals interactions . Two other human α‐CAs have been reported to be dimeric, hCA VI and hCA XII .…”

“…The structure of the wild type domain was solved at very low resolution (3.2 Å), whereas that of the mutant was solved at higher resolution (2.2 Å), allowing to obtain detailed structural information on the enzyme active site. Subsequently, in 2015 Leitans and co‐workers reported the structure of a double mutant of this domain (C174S, N346Q), expressed into yeast ( Pichia pastoris ) and complexed with three different sulfonamide inhibitors . Finally, in 2016 the crystal structure of another engineered form of the CA IX catalytic domain, expressed in E. coli , was reported .…”

“…25 Due to the difficulties in the production of high amount of CA IX extracellular region in vitro and in the obtainment of well-ordered crystals, only the CA domain of this protein has been so far structurally characterized by Xray diffraction studies, with five structures actually deposited in the Protein Data Bank (PDB codes 3IAI, 5DVX, 5FL4, 5FL5, 5FL6). [26][27][28] The first structure was determined in 2009 in complex with acetazolamide (a classic CA inhibitor [CAI]) by our group, which expressed both a wild type and a mutated form (C174S) of the CA domain, using the baculovirus-insect cell expression system. 26 The structure of the wild type domain was solved at very low resolution (3.2Å), whereas that of the mutant was solved at higher resolution (2.2Å), allowing to obtain detailed structural information on the enzyme active site.…”

“…Subsequently, in 2015 Leitans and co-workers reported the structure of a double mutant of this domain (C174S, N346Q), expressed into yeast (Pichia pastoris) and complexed with three different sulfonamide inhibitors. 27 Finally, in 2016 the crystal structure of another engineered form of the CA IX catalytic domain, expressed in E. coli, was reported. 28 The latter form of the enzyme was designed to mimic the hCA II surface, the most easily expressed and crystallized CA isoform, through the introduction of six surface mutations (C174S, L183S, M350S, A213K, A258K, and F259Y), which were able to reduce at the same time protein aggregation and surface hydrophobicity, thus facilitating the crystallization process.…”

Expression of Carbonic Anhydrase I in Motor Neurons and Alterations in ALS

“…Homozygous CA9 -knockout mice have gastric hyperplasia and vacuolar degenerative changes in the brain with behavioral defects in locomotor activity and memory test [31,32]. On the other hand, CA9 is highly expressed in several cancers [33]. Patients with a mutation in CA12 exhibit hyponatremia and hyperchlorhydria [34].…”