IQGAP-2: a novel interacting partner with the human colonic thiamin pyrophosphate transporter

Abstract

The human colonic thiamin pyrophosphate transporter (hcTPPT) mediates the uptake of the microbiota-generated and phosphorylated form of vitamin B₁ (i.e., thiamin pyrophosphate) in the large intestine. Expression of hcTPPT along the absorptive tract is restricted to the large intestine, and the transporter is exclusively localized at the apical membrane domain of the polarized epithelial cells/colonocytes. Previous studies have characterized different physiological/pathophysiological aspects of the hcTPPT system, but nothing is currently known on whether the transporter has interacting partner(s) that affect its physiology/biology. We addressed this issue using a Y2H to screen a human colonic cDNA library and have identified three putative interactors, namely IQGAP-2, SNX-6, and DMXL-1. Focusing on IQGAP-2 (whose expression in human colonocytes is the highest), we found (using fluorescent microscopy imaging and coimmunoprecipitation approaches) the putative interactor to colocalize with hcTPPT and to directly interact with the transporter. Also, overexpressing IQGAP-2 in NCM460 cells and in human primary differentiated colonoid monolayers was found to lead to significant (P < 0.01) induction in TPP uptake, while knocking down (using gene-specific siRNAs) caused significant (P < 0.01 and P < 0.05) decrease in uptake. Furthermore, overexpressing IQGAP-2 in NCM460 cells was found to lead to a significant enhancement in hcTPPT protein stability. Finally, we found the expression of IQGAP-2 to be markedly suppressed in conditions/factors that negatively impact colonic TPP uptake. These results identify the IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and show that this interaction has physiological and biological consequences.NEW & NOTEWORTHY This study reports on the identification of IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and how that impacts the transporter's physiology and cell biology.

Keywords: IQGAP-2; accessory proteins; colonic uptake; human colonocytes; thiamin pyrophosphate.

Graphical abstract

Figure 1.

Identification of putative interactors with the hcTPPT in human colonic epithelial cells. A: sequence alignment of the COOH-terminal tail (aa 667–710) of the hcTPPT compared with other mammals (performed using the PRALINE program). B: confirmation of interaction between the hcTPPT protein and the three identified putative protein interactors (i.e., DMXL-1, SNX-6, and IQGAP-2) by “Y-1-by-1” assay (see materials and methods). Yeast cells were grown in selective media DO-2 (which lacks tryptophan and leucine) and DO-3 (which lacks tryptophan, leucine, and histidine). The DO-2 selective medium was used as a growth control and to validate the presence of the bait and prey vectors in yeast; the DO-3 selective medium was used to confirm interaction between bait and prey clones. i: positive control [interaction between the bait (pB27-SMAD) and the prey (pP6-SMURF)]. ii: negative control (the bait contains pB27-hcTPPT, whereas the prey was an empty pP6 vector). iii, v, and vii: negative control (the bait was empty pB27 vector, and the pray was pP6-DMXL-1, pP6-SNX-6, or pP6-IQGAP-2). iv, vi, and viii: positive interactions between the bait pB27-hcTPPT protein and the prey containing individual putative interactor(s) (i.e., pP6-DMXL-1, pP6SNX-6, and pP6-IQGAP-2, respectively).

Figure 2.

Relative mRNA expression of the three putative interactors with the hcTPPT (i.e., SNX-6, IQGAP-2, and DMXL-1) in human intestinal epithelial NCM460 cells (A) and human primary differentiated colonoid monolayers (B). mRNA levels were determined by RT-qPCR. Data were normalized relative to β-actin and presented as means ± SE of at least 3 or 4 separate experiments. Statistical analysis was performed using Student’s t test. *P < 0.05; **P < 0.01.

Figure 3.

Cellular colocalization of hcTPPT and IQGAP-2 in human primary differentiated colonoid monolayers. Representative confocal microscopy images (performed as described in materials and methods) showing fluorescent signals for IQGAP-2 (A), hcTPPT (B), overlay of A and B (C), and nucleus (D). Images were taken from multiple fields across at least three independent experiments.

Figure 4.

Coimmunoprecipitation (co-IP) of IQGAP-2 with the hcTPPT in human colonic epithelial NCM460 cells. Cells stably expressing the hcTPPT-GFP were transiently transfected with IQGAP-2-mCherry plasmid, lysed (using lysis buffer), and then subjected to co-IP using anti-IQGAP-2 antibodies (Ab). The co-IP complex was then separated into NuPAGE 10% Bis-Tris minigel and analyzed by Western blot analysis using anti-hcTPPT antibodies. For controls, co-IP complex was obtained from nontransfected NCM460 cells (with or without anti-mouse IgG1 antibody) that stably express hcTPPT-GFP. All co-IP studies were performed on three different occasions using three different cell lysate preparations.

Figure 5.

Effect of overexpressing the IQGAP-2 protein in human colonocytes on carrier-mediated [³H]-TPP uptake. Human colonic epithelial NCM460 cells (A) and human primary differentiated colonoid monolayers (B) were transiently cotransfected with hcTPPT-GFP and IQGAP-2-mCherry plasmid. Control cells were transfected with hcTPPT-GFP alone. Carrier-mediated [³H]-TPP uptake (0.23 μM; 10 min; 37°C) was determined 48 h posttransfection. Data are means ± SE of at least 3 or 4 separate determinations. Statistical analysis was performed using Student’s t test. *P < 0.01.

Figure 6.

Effect of knocking down the IQGAP-2 protein in human colonocytes on carrier-mediated [³H]-TPP uptake. A: validation of the knockdown: human colonic epithelial NCM460 cells were transfected with the human IQGAP-2 gene-specific siRNAs or with negative control siRNAs. Effect of knocking down IQGAP-2 on level of expression of IQGAP-2 protein (i), IQGAP-2 mRNA (ii), and hcTPPT mRNA (iii). Protein level was determined by Western blot analysis; mRNA levels were determined by RT-qPCR (protein and mRNA levels were normalized relative to β-actin). B: effect of knocking down IQGAP-2 on carrier-mediated [³H]-TPP uptake in NCM460 cells (i) and human primary differentiated colonoid monolayers (ii). Data presented are means ± SE of at least 4 or 5 separate determinations. Statistical analysis was performed using Student’s t test. *P < 0.05; **P < 0.01.

Figure 7.

Effect of IQGAP-2 on stability of the hcTPPT protein and on [³H]-TPP uptake by human colonic epithelial NCM460 cells. Cells were cotransfected with hcTPPT-GFP and IQGAP-2-mCherry, then (48 h later) treated (for 6 and 12 h) with cycloheximide (100 μg/mL) as previously described (32). A: cell lysates were then run in NuPAGE 4–12% Bis-Tris mini protein gel and analyzed by Western blot analysis using anti-hcTPPT and anti-β-actin antibodies (4967S) as described in materials and methods. The level of the hcTPPT protein was normalized relative to β-actin. B: carrier-mediated [³H]-TPP uptake by cells that were treated (for 6 and 12 h) with cycloheximide (100 μg/mL) (see materials and methods). Data are means ± SE of at least 4 or 5 separate determinations. Statistical analysis was performed using Student’s t test. *P < 0.01.

Figure 8.

Effect of conditions/factors that negatively impact colonic TPP uptake on level of expression of IQGAP-2 mRNA in human colonocytes. A and B: level of expression of IQGAP-2 mRNA in human primary differentiated colonocyte monolayers subjected to hypoxia (A) (27), proinflammatory cytokine TNF-α (B) (26), and bacterial LPS (C) (29). mRNA levels were determined by RT-qPCR, and data were normalized relative to β-actin. Data are means ± SE of at least 3 or 4 separate experiments performed on separate occasions. Statistical analysis was performed using Student’s t test. *P< 0.05 and **P < 0.01.