Aldo-keto reductase family 1 member B8 is secreted via non-classical pathway

Abstract

Mouse aldo-keto reductase family 1 member B8 (AKR1B8) has the highest similarity to human aldo-keto reductase family 1 member B10 (AKR1B10), a secretory protein through lysosomes-mediated non-classical secretory pathway. To identify whether AKR1B8 is secreted through the same pathway, we carried out this study. Self-developed sandwich ELISA and western blot were used to detect AKR1B8 in cells and culture medium of CT-26 murine colon carcinoma cells. AKR1B8 releases in an independent manner to Brefeldin A, an inhibitor of ER-to-Golgi classical secretion pathway. Several factors, which are involved in the non-classical secretion pathway, such as temperature, ATP and calcium ion, regulated AKR1B8 secretion from mouse colorectal cancer cells CT-26. Lysosomotropic NH4Cl increased AKR1B8 secretion, and AKR1B8 was located in isolated lysosomes. Therefore, AKR1B8 is a new secretory protein through the lysosomes-mediated non-classical pathway.

Keywords: AKR1B8; Aldose reductase; aldose reductase family 1 member B8; non-classical pathway; secretion.

Figure 1

Preparations of AKR1B8 protein and antibody. A. Purification of AKR1B8 protein with different concentrations was analyzed by Coomassie blue staining. B. Purification of rabbit anti-AKR1B8 antibody was analyzed by Coomassie blue staining. C. Specificity of AKR1B8 antibody to AKR1B8 (FR1) and not to mAR and MVDP was analyzed by western blot.

Figure 2

AKR1B8 release from CT-26 cells. A. AKR1B8 secreted from CT-26 cells in medium was detected by western blot. B. AKR1B8 activity. Cells (1 × 10⁷) as indicated were seeded into six-cm dishes overnight and then fed with 2 ml of serum-free medium for 30 min. Medium was collected and subjected to an enzyme activity assay as described in the Materials and methods section. *p < 0.01 vs Fresh Medium.

Figure 3

AKR1B8 secretes via a non-classical pathway. (A) Signal-peptide analyses in AKR1B8. Amino acid sequence of full-length mouse AKR1B8 was inputted into the SignalP 3.0 server (http://www.cbs.dtu.dk/services/SignalP/), and neural networks (NN) and hidden Markov models (HMM) were run. Both SignalP-NN (A) and SignalP-HMM (B) results showed no signal peptide in the AKR1B8 sequence. (B) Brefeldin (A) cells (2.5 × 10⁵) were cultured in six-well plates overnight and then expose to the ER-to-Golgi protein transport inhibitor brefeldin A at 10 μg/ml in fresh serum-free medium for 8 h. Medium was collected for Western blotting. Vimentin was detected by Western blotting as a positive control.

Figure 4

AKR1B8 secretion is affected by nonclassical secretory pathway-related factors. A. Temperature. cells (2.5 × 10⁵) were seeded into six-well plates overnight and then exposed to the temperature (°C) indicated for 30 min. The cells were fed with fresh serum-free medium at the same temperature for 30 min, and medium was collected for Western blotting as described in the Materials and methods. B. ATP and Calcium. Cells (2.5 × 10⁵) were incubated in six-well plates overnight and then exposed to 1 mM Ca²⁺ ions or 2 µM Ionomycin in serum-free medium for 2 h. At 30 min before harvest, ATP (1 mM) was added or not, and the medium was subjected to Western blot analysis.

Figure 5

Lysosomes are involved in AKR1B8 secretion. A. AKR1B8 secretions were enhanced by NH₄Cl treatment. B. AKR1B8 was present inside the isolated lysosomes by western blotting analysis.