Ketohexokinase: expression and localization of the principal fructose-metabolizing enzyme

Abstract

Ketohexokinase (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and Western blotting of rodent tissues, including those from mouse knockouts, coupled with RT-PCR assays, we determined the distribution of the splice variants. The highly expressed KHK-C isoform localized to hepatocytes in the liver and to the straight segment of the proximal renal tubule. In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of other tissues, and by Western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from experimental artifact.

Figure 1

PCR analysis of splice variant usage. (A) Schematic of the assay. (B) Typical profile obtained. Using heart cDNA, we identified the uncut PCR product (285 bp), the 3− splice variant (150 bp), and an additional aberrantly spliced product (227 bp).

Figure 2

Western blot of recombinant mouse proteins and tissue lysates. (A) N-terminal antibody. Tissue and cell extracts as follows: L (liver), K (kidney), S (spleen), B (brain), H (HepG2). Induced Escherichia coli extracts expressing the KHK-3C or KHK-3− protein. The 30-kDa band is indicated with an arrow. (B) C-terminal antibody. E. coli extracts were either uninduced (−) or induced (+) with isopropyl β-d-1-thiogalactopyranoside to express the KHK-3C, -3A or -3− proteins. The 30-kDa band is indicated with an arrow. (C) KHK-C–specific antibody. (D) N-terminal antibody on Khk^3a/3a mouse brain tissue extracts (1), wild-type brain tissue extracts (2), Khk^3a/3a mouse liver tissue extracts (3), and wild-type liver tissue extracts (4). The 30-kDa band is indicated with an arrow.

Figure 3

KHK localization in mouse kidney. (A,B) N-terminal antibody, precompeted with control E. coli extract. (C,D) Same antibody, precompeted with E. coli extract containing mouse KHK-A. (E,F) Aldehyde reductase (proximal tubule marker). (G,H) N-terminal antibody, tissue from Khk^Δ/Δ mice.

Figure 4

KHK localization in mouse liver (N-terminal antibody). (A) liver lobe, low power, (B) high-power view.

Figure 5

KHK localization in mouse kidney (A,B) and liver (C,D), using Carnoy's fixative (N-terminal antibody).

Figure 6

Silver-enhanced immunogold detection of KHK in liver (N-terminal antibody). (A) Khk^Δ/Δ knockout animal. (B) Wild-type animal. (C) Wild-type animal, primary antibody omitted.

Figure 7

KHK immunostaining of mouse pancreas (N-terminal antibody). (A) Normal mouse pancreas. (B) Pancreas of homozygous Khk^Δ/Δ knockout mouse.

Figure 8

Immunohistochemical staining of rat tissues, using the N-terminal antibody. (A,B) Kidney. (C,D) Liver. (E) Pancreas.