Fluid-Phase Endocytosis and Lysosomal Degradation of Bovine Lactoferrin in Lung Cells

Abstract

The iron-binding protein lactoferrin and the cell-penetrating peptides derived from its sequence utilise endocytosis to enter different cell types. The full-length protein has been extensively investigated as a potential therapeutic against a range of pathogenic bacteria, fungi, and viruses, including SARS-CoV-2. As a respiratory antiviral agent, several activity mechanisms have been demonstrated for lactoferrin, at the extracellular and plasma membrane levels, but as a protein that enters cells it may also have intracellular antiviral activity. Characterisation of lactoferrin's binding, endocytic traffic to lysosomes, or recycling endosomes for exocytosis is lacking, especially in lung cell models. Here, we use confocal microscopy, flow cytometry, and degradation assays to evaluate binding, internalisation, endocytic trafficking, and the intracellular fate of bovine lactoferrin in human lung A549 cells. In comparative studies with endocytic probes transferrin and dextran, we show that lactoferrin binds to negative charges on the cell surface and actively enters cells via fluid-phase endocytosis, in a receptor-independent manner. Once inside the cell, we show that it is trafficked to lysosomes where it undergoes degradation within two hours. These findings provide opportunities for investigating both lactoferrin and derived cell-penetrating peptides activities of targeting intracellular pathogens.

Keywords: endocytosis; intracellular trafficking; lactoferrin; lysosomal degradation.

Figure 1

Viability analysis of bLF incubated with A549 over 24 h. A549 cells were incubated with bLF, diluent control (dH₂O), staurosporine, or 0.2% Triton X-100 for 24 h in serum-containing medium before analysis using CellTitre Blue metabolic assay. Error bars represent SEM.

Figure 2

bLF cell binding and competition. Cells were incubated with 250 nM bLF647 in the presence of potential competitors for 1 h at 4 °C before being analysed by confocal microscopy (A) or flow cytometry (B). Scale bar = 50 µm, error bars represent SEM. Flow cytometry represents the mean of three independent experiments performed in duplicate; fluorescence intensity is measured using the median and normalised to the control. Statistical analysis was performed using a one-way ANOVA (F (6, 14) = 17.13, p < 0.0001) with a Dunnett post hoc analysis of control versus samples * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Kinetic analysis of the uptake of bLF647 in A549 cells. Uptake in A549 cells of bLF647, Dex647, and Tf488 analysed against time (A) or concentration (B). Cells were incubated with 250 nM bLF647, 50 µg/mL Dex647, or 20 nM Tf488 for between 10 min and 6 h; or cells were incubated with differing concentrations of bLF647, Dex647, and Tf488 for 15 min, and analysed by flow cytometry. Data represents the mean of three independent experiments with fluorescence intensity calculated as the mean from a duplicate of median cell intensity values. Error bars represent SEM.

Figure 4

Trafficking of bLF647, Tf488, and Dex647 to lysosomes by confocal microscopy. Cells were incubated with 500 nM bLF647 (A), 20 nM Tf488 (B), or 100 µg/mL Dex647 (C) for 1 h in SFM, washed in serum-containing medium, and incubated for the chase period before being washed and imaged by confocal microscopy. Lysosomes (green, middle row) were prelabelled using a pulse–chase protocol. Arrows represent endocytic structures with colocalised bLF and endolysosomal structures (white), arrow heads indicate bLF647 endocytic structures not colocalised with the lysosome. Scale bar = 10 µm, representative images from three independent experiments quantified in Figure 5. See Supplementary Figures S2–S4 for uncropped images.

Figure 5

Quantification of trafficking. Cells were incubated with 500 nM bLF647, 100 µg/mL Dex647, or 20 nM Tf488 for 1 h in serum-free medium before being washed and incubated for between 0 and 24 h in serum-containing medium and imaged by confocal microscopy (A), flow cytometry (B), or Western blotting ((C,D), 1 µM unlabelled bLF). To obtain relative total uptake (A), 10 images were quantified from each independent experiment and normalised to the 0 h chase period. Flow cytometry represents the mean of the median fluorescence from each independent experiment performed in duplicate (N = 3).