Arsenite Regulates Prolongation of Glycan Residues of Membrane Glycoprotein: A Pivotal Study via Wax Physisorption Kinetics and FTIR Imaging

Abstract

Arsenic exposure results in several human cancers, including those of the skin, lung, and bladder. As skin cancers are the most common form, epidermal keratinocytes (KC) are the main target of arsenic exposure. The mechanisms by which arsenic induces carcinogenesis remains unclear, but aberrant cell proliferation and dysregulated energy homeostasis play a significant role. Protein glycosylation is involved in many key physiological processes, including cell proliferation and differentiation. To evaluate whether arsenite exposure affected protein glycosylation, the alteration of chain length of glycan residues in arsenite treated skin cells was estimated. Herein we demonstrated that the protein glycosylation was adenosine triphosphate (ATP)-dependent and regulated by arsenite exposure by using Fourier transform infrared (FTIR) reflectance spectroscopy, synchrotron-radiation-based FTIR (SR-FTIR) microspectroscopy, and wax physisorption kinetics coupled with focal-plane-array-based FTIR (WPK-FPA-FTIR) imaging. We were able to estimate the relative length of surface protein-linked glycan residues on arsenite-treated skin cells, including primary KC and two skin cancer cell lines, HSC-1 and HaCaT cells. Differential physisorption of wax adsorbents adhered to long-chain (elongated type) and short-chain (regular type) glycan residues of glycoprotein of skin cell samples treated with various concentration of arsenite was measured. The physisorption ratio of beeswax remain/n-pentacosane remain for KC cells was increased during arsenite exposure. Interestingly, this increase was reversed after oligomycin (an ATP synthase inhibitor) pretreatment, suggesting the chain length of protein-linked glycan residues is likely ATP-dependent. This is the first study to demonstrate the elongation and termination of surface protein-linked glycan residues using WPK-FPA-FTIR imaging in eukaryotes. Herein the result may provide a scientific basis to target surface protein-linked glycan residues in the process of arsenic carcinogenesis.

Keywords: focal-plane-array-based FTIR; glycosylation; synchrotron-radiation-based FTIR; wax physisorption kinetics.

Figure 1

The representative normalized FTIR reflectance spectra of cell samples on low-e slides after arsenite exposure by co-cultured with sodium arsenite (0 and 5 μM) in the range of 3600–900 cm⁻¹; the inset plot showed the amide A band of cell samples without spectral normalization in the range of 3500–3000 cm⁻¹. All FTIR reflectance spectra were acquired by using a FTIR spectrometer coupled with an infrared integrating sphere.

Figure 2

Peak height images of amide A band for KC, HaCaT and HSC-1 cell samples in the field of view of 100 × 100 µm² were presented after treating with sodium arsenite using SR-FTIR microspectroscopy. The FTIR images of peak height of amide A were collected at 3294 cm⁻¹ for cell samples (representative data were shown; 3 repeated experiments, 3 replicates, at least 10 cells were measured).

Figure 3

A representative profile of average amount of wax adsorbent remaining adhered on the cell surface of a single cell (blue and red column, left axis) acquired by WPK-FPA-FTIR imaging and cell viabilities (purple line, right axis with an inner ticks) after treating with a variety of concentrations of sodium arsenite and co-cultured with oligomycin (om) and sodium arsenite. (a,d) KC; (b,e) HSC-1; and (c,f) HaCaT cells. The glycan residues population ratio (long-chain/short-chain; green column, right axis with an outer ticks) was used to estimate the ratio of beeswax remaining/n-pentacosane remaining (BR/PR) in different cultured conditions.

Figure 3

A representative profile of average amount of wax adsorbent remaining adhered on the cell surface of a single cell (blue and red column, left axis) acquired by WPK-FPA-FTIR imaging and cell viabilities (purple line, right axis with an inner ticks) after treating with a variety of concentrations of sodium arsenite and co-cultured with oligomycin (om) and sodium arsenite. (a,d) KC; (b,e) HSC-1; and (c,f) HaCaT cells. The glycan residues population ratio (long-chain/short-chain; green column, right axis with an outer ticks) was used to estimate the ratio of beeswax remaining/n-pentacosane remaining (BR/PR) in different cultured conditions.

Figure 4

Characteristic IR absorbance of n-pentacosane remaining (PR) and beeswax remaining (BR) adhered on cell samples with (w) and without (w/o) sodium arsenite (As³⁺) exposure after a dewaxing procedure via xylene washing, using the WPK-FPA-FTIR imaging technique. The IR absorbance spectrum data was determined relative to that of the corresponding non-waxed cell sample.

Figure 5

The schematic WPK-FPA-FTIR imaging procedure was demonstrated for estimating the amount of wax adsorbent remaining on the cell sample surface using IR absorbance of wax adsorbent in the range of 3000–2800 cm⁻¹.