Cytosolic 5'-nucleotidase III (NT5C3): gene sequence variation and functional genomics

Abstract

Background: 5'-Nucleotidases play a critical role in nucleotide pool balance and in the metabolism of nucleoside analogs such as gemcitabine and cytosine arabinoside (AraC). We previously performed an expression array association study with gemcitabine and AraC cytotoxicity using 197 human lymphoblastoid cell lines. One gene that was significantly associated with gemcitabine cytotoxicity was a nucleotidase family member, NT5C3. Very little is known with regard to the pharmacogenomics of this family of enzymes.

Methods: We set out to identify common genetic variation in NT5C3 by resequencing the gene and to determine the effect of that variation on NT5C3 protein function and potential effect on response to cytidine analogs. We identified 61 NT5C3 polymorphisms, 48 of which were novel, by resequencing 240 ethnically defined DNA samples. Functional studies were performed with one nonsynonymous (G847C, Asp283His) and four synonymous cSNPs (T9C, C276T, T306C, and G759A),as well as three combined variants (T276/His283, T276/C306, T276/C9).

Results: The His283 and T276/His283 constructs showed decreased levels of enzyme activity and protein. Substrate kinetic analysis showed no significant differences in Km values between wild type and His283 when cytidine monophosphate, AraCMP, and GemMP were used as substrates. An association study between single nucleotide polymorphisms (SNPs) and NT5C3 expression in the 240 cell lines from which DNA was extracted to resequence NT5C3 identified four SNPs that were significantly associated with NT5C3 expression. Electrophoretic mobility shift assays showed that two of those SNPs, I4(-114) and I6(9), altered DNA-protein binding patterns. These findings suggest that genetic variation in NT5C3 might affect protein function and potentially influence drug response.

Figure 1

Human NT5C3 genetic polymorphisms. Arrows indicate the locations of polymorphisms, with different colors indicating minor allele frequencies. Black rectangles represent portions of exons encoding the open-reading frame (ORF). Open rectangles represent portions of exons encoding 5’ and 3’-UTRs. AA is African-American, CA is Caucasian-American, HCA is Han Chinese-American, MA is Mexican-American, and indel is an insertion/deletion event.

Figure 2

NT5C3 allozyme functional genomics. (A) Western blot analysis with anti-HA antibody for four different NT5C3 isoforms. Expression constructs encoding 4 different transcripts were transfected into COS-1 cells. Cells were then lyzed and cell supernatant (S) and pellet (P) were used for the analysis as indicated. (B) Representative Western blot analysis for the nonsynonymous and synonymous cSNPs studied. (C) Levels of NT5C3 allozyme activity and immunoreactive protein as a percentage of WT. Each bar represents the average of 6 independent transfections (mean±SE). All values were corrected for transfection efficiency. (D) Correlation between average levels of NT5C3 immunoreactive protein and enzyme activity.

Figure 3

(A) NT5C3 expression levels in the 240 lymphoblastoid cells studied. Each dot represents one individual sample. Expression level was plotted against each ethnic group. ANOVA analysis was performed to compare the expression level among the 4 ethnic groups. (B) EMSA experiments showing “shifts” observed with I4(-114) and I6(9) WT and variant nucleotides in the presence of nuclear extract from the four cell lines indicated. (C) Quantile boxplot of NT5C3 expression based on genotype at the I6(9) and I4(-114) SNPs.

Figure 4

Human NT5C3 homology model. Three NT5C3 crystal structures are available (PDB accession numbers 2CN1, 2JGA, and 2VKQ). We used 2VKQ because it has the highest resolution of the 3 structures available. This structure contains residues 14-286 (isoform 3), and has BeF₃ and Mg ions in the active site. We studied the 297 amino acid isoform (isoform 2) since our lymphoblastoid cell lines express that isoform. Therefore, our nonsynonymous SNP is at position 283 in the 297 amino acid isoform but is located at position 272 in the 286 amino acid isoform. (A) The NT5C3 molecule is shown as a blue ribbon structure. Bound in the active site are magnesium (dark gray) and BeF₃^- (the pink beryllium is barely visible) with yellow fluorine ions, drawn as space-filling spheres. The side chain of Asp272 is also drawn as space-filling spheres, with green carbon atoms. (B) Closeup of Asp272 and His272 superposition to show that the surface solvent-exposed location of Asp272 provides adequate space for His272 such that it can also form a hydrogen bond with Ser274, although from a slightly different direction.