The origin and evolution of human glutaminases and their atypical C-terminal ankyrin repeats

Abstract

On the basis of tissue-specific enzyme activity and inhibition by catalytic products, Hans Krebs first demonstrated the existence of multiple glutaminases in mammals. Currently, two human genes are known to encode at least four glutaminase isoforms. However, the phylogeny of these medically relevant enzymes remains unclear, prompting us to investigate their origin and evolution. Using prokaryotic and eukaryotic glutaminase sequences, we built a phylogenetic tree whose topology suggested that the multidomain architecture was inherited from bacterial ancestors, probably simultaneously with the hosting of the proto-mitochondrion endosymbiont. We propose an evolutionary model wherein the appearance of the most active enzyme isoform, glutaminase C (GAC), which is expressed in many cancers, was a late retrotransposition event that occurred in fishes from the Chondrichthyes class. The ankyrin (ANK) repeats in the glutaminases were acquired early in their evolution. To obtain information on ANK folding, we solved two high-resolution structures of the ANK repeat-containing C termini of both kidney-type glutaminase (KGA) and GLS2 isoforms (glutaminase B and liver-type glutaminase). We found that the glutaminase ANK repeats form unique intramolecular contacts through two highly conserved motifs; curiously, this arrangement occludes a region usually involved in ANK-mediated protein-protein interactions. We also solved the crystal structure of full-length KGA and present a small-angle X-ray scattering model for full-length GLS2. These structures explain these proteins' compromised ability to assemble into catalytically active supra-tetrameric filaments, as previously shown for GAC. Collectively, these results provide information about glutaminases that may aid in the design of isoform-specific glutaminase inhibitors.

Keywords: X-ray crystallography; cancer; glutaminase; human; isoform; metabolism.

Figure 1.

Sequence and architecture analysis of bacterial and eukaryotic glutaminases. The amino acid sequence corresponding to the glutaminase domain of the GLS gene (Ile²²¹-Arg⁵⁴⁴) was used as input of blastp search against the non-redundant protein sequences (nr) database (E value ≤ 0.0001). The sequences obtained were analyzed by number of amino acids (aa); the normal distribution of the sequence length for bacterial and eukaryote glutaminases are, respectively, represented in A and B, with the respective architectures indicated in rectangles; in parentheses, the number of sequences containing the corresponding architecture/the total number of sequences contained in the referred region of the distribution plot. C, cladogram based on architectural organization obtained from a maximum likelihood phylogenetic reconstruction approach. D, the generated phylogenetic tree and respective alignment were used to reconstruct ancestral protein sequences for specific nodes (ancestral chordates (1); nematodes (2); arthropodes (3); and fungi (4)) using maximum parsimony. The obtained ancestral sequences were aligned with the human GLS (KGA and GAC) and GLS2 (GAB and LGA) and the sequence similarity displayed as a heatmap of pairwise distances constructed in SDT using MUSCLE alignment. The obtained result suggests that GLS is the most primitive gene, which has been further duplicated to generate GLS2. cNMP, cyclic nucleotide-binding domain; SRPBCC, START/RHOαC/PITP/Bet v1/CoxG/CalC.

Figure 2.

Evolution of glutaminase intron-exon structure. A, protein sequence corresponding to human GLS exon 14 was used as a query of translated blast (tblastn) searches against available genomes from C. intestinalis (Tunicate), B. floridae (Cephalochordate), P. marinus (Cyclostomata), C. milii (Chondrichthyes), D. rerio (Actinopterygii), and X. tropicalis (Amphibia). After an initial Reciprocal Best Hit region was found, the correct position of the exon was determined by pairwise LALIGN between sequences. Region downstream genomic sequence homologous to exon 14 were also evaluated by LALIGN against human GAC-exclusive exon (exon 15). As both P. marinus glutaminases are incomplete, available exons were found in contig using LALIGN approach. The human genes are represented at the top branch (Amniota). The thin red bars represent the homologous region to the GLS exon 14 and the light blue boxes indicate homologous regions to the exon 15 of human GLS. The introns, represented as dashed lines, are not drawn to scale and the omitted sections are indicated by two bars (‖). The asterisk (*) indicates that the deposited sequences are incomplete. B, a search for retrotransposition evidences in the genomic region comprehending exon 15 and introns 14 and 15 of human GLS using TranspoGene resulted in the identification of various transposable elements in the intron 15; however, no consensus matching elements were found in the intron 14.

Figure 3.

Overall structural features of ANK domain of glutaminases. A, schematic representation of three ANK repeats (ANK1 to ANK3) present as a C-terminal domain of KGA and GLS2. Both ANK repeats are very similar in sequence as well as in the overall structure. B, alignment of the glutaminase ANK repeats and the consensus sequence (shown on top), proposed in Ref. . Highly conserved residues are capitalized and in red, semiconserved residues are colored cyan and not capitalized. Residues involved in the dimer interface are indicated by a bar. ANK2 in both KGA and GLS2 contains an extra surface-exposed lysine, which is indicated by the + signal. On the right, the superposition of ANK1, ANK2, and ANK3 is represented. C, dimer interface and associated interaction of ANK repeats. The side chains of the motifs DYD (left) and DRW (right) are represented in sticks. D, representation of sequence conservation of glutaminases ANK from eukaryotes and E, bacteria. The size of the letters is proportional to the degree of conservation of residues. The motifs DYD and DRW are highlighted. The residues in red shows the 100% conservation throughout the alignment. Residues are numbered according to human KGA.

Figure 4.

Multidomain structure of KGA and biophysical characterization of KGA, GAC, and ΔC. A, electron density and final model for the complete tetramer of KGA. B, planar representation, dividing the tetramer into front and back halves to show the position of two ANK dimers at the same side of the plane. C, upper view of the tetramer, indicating the close contact between the ANK1 outer helix and the helix H1 of the N-terminal EF-hand like domain. D, the filament interface between two N-terminal domains (region delimited by gray), and by which the single strand polymer of GAC (PDB code 4jkt) grows (23), is occluded in KGA because of the presence of the ankyrin dimers. E, the Stokes radii calculated from the size exclusion chromatographic profiles of KGA, GAC, and ΔC (KGA lacking the ANK repeats), indicating that ANK repeats prevent the self-assembly of glutaminases into the higher supra-tetrameric filament forms. F, the comparative enzymatic efficiency of KGA, GAC, and ΔC toward glutamine in the presence of 20 mm phosphate. The lack of ANK repeats enhances the catalytic efficiency of KGA. Red lines indicate mean ± S.D.

Figure 5.

Low-resolution solution model for full-length GLS2 (GAB/LGA). Intensity (left panel) and pair-distance (right panel) distribution functions, obtained for GAB/LGA subject to SAXS experiments. B, orthogonal views of the superposition between the SAXS envelope and the collection of X-ray crystal structures from GLS2 glutaminase domain (PDB code 4BQM), N-terminal portion of human GLS (PDB code 3CZD) and GAB/LGA.ANK. C, box plot of the two perpendicular dimensions for GAB/LGA, large (L) and short (S) as taken from the cryo-EM micrographs (inset). KGA dimer and tetramer crystal structures projections were used as a reference for the dimensions of these two oligomeric states.