Dietary N-Glycans from Bovine Lactoferrin and TLR Modulation

Abstract

Scope: Bovine lactoferrin (bLF) is an ingredient of food supplements and infant formulas given its antimicrobial and antiviral properties. We modified bLF enzymatically to alter its N-glycosylation and to isolate the glycan chains. The aims of this study include (1) to evaluate whether such derivates induce responses via pattern recognition receptors namely Toll-like receptors (TLRs) and (2) to relate those responses to their different glycosylation profiles.

Methods and results: The unmodified and modified bLF fractions are incubated with reporter cell lines expressing pattern recognition receptors. Afterwards, we screen for TLRs and analyze for nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) activation. Activation of reporter cell lines show that signaling is highly dependent on TLRs. The activation pattern of bLF is reduced with the desialylated form and increased with the demannosylated form. In reporter cells for TLR, bLF activate TLR-4 and inhibit TLR-3. The isolated glycans from bLF inhibit TLR-8. TLR-2, TLR-5, TLR-7, and TLR-9 are not significantly altered.

Conclusion: The profile of glycosylation is key for the biological activity of bLF. By understanding how this affects the human defense responses, the bLF glycan profile can be modified to enhance its immunomodulatory effects when used as a dietary ingredient.

Keywords: N-glycosylation; NF-κB; bovine lactoferrin; toll-like receptors.

Figure 1

bLF stimulates THP1 MD2 CD14 via TLRs. The cell lines THP1 MD2 CD14 and THP1 MD2 CD14 with a truncated MyD88 adaptor were stimulated with 2 mg mL⁻¹ of bLF after which NF‐κB activation was measured by spectrophotometry at 650 nm. The release of NF‐κB after bLF incubation was compared with the release of NF‐κB of nontreated cells. NF‐κB release is expressed in arbitrary units. Data are represented as median with interquartile range (n = 5). Statistical differences were measured using Mann–Whitney test (**p < 0.01, ***p < 0.001).

Figure 2

The absence of sialic acid has an effect on the release of NF‐κB. THP1 MD2 CD14 cells were stimulated with 2 mg mL⁻¹ of bLF without sialic acid (A) and bLF with 25% reduced mannose on the N‐glycan chains(B). NF‐κB activation was measured by spectrophotometry at 650 nm. NF‐κB release is expressed in arbitrary units. Data are represented as median with interquartile range (n = 5). Statistical differences were measured using Mann–Whitney test (**p < 0.01).

Figure 3

Inhibitory effects of bLF proteins on HEK‐ TLR‐3 cells. The cells were coincubated with the bLF structures and with the specific agonist for TLR‐3 Poly(I:C) LMW (5 μg mL⁻¹). NF‐κB release was measured by spectrophotometry at 650 nm. Median and interquartile range of activation is plotted as NF‐κB release (n = 3). Statistical differences were calculated with Mann–Whitney test (**p < 0.01).

Figure 4

bLF glycosylation pattern influence in the release of NF‐κB in HEK‐Blue TLR‐4. Cells were incubated with bLF proteins at 2 mg mL⁻¹ and isolated N‐glycans and desialylated and demannosylated N‐glycans at 1.2 mg mL⁻¹. Culture medium served as negative control. NF‐κB release is expressed in arbitrary units. NF‐κB activation was measured by spectrophotometry at 650 nm. Data are represented as median with interquartile range (n = 5). Statistical differences were measured using Mann–Whitney test (***p < 0.001).

Figure 5

bLF proteins structural influence in the release of NF‐κB in HEK‐Blue TLR‐4. The effect of Polymyxin B blockade at 100 μg mL⁻¹ was confirmed by coincubation of this cationic compound with LPS at 10 μg mL⁻¹ (A). LPS neutralization was also achieved when Polymixyn B was coincubated with bLF (B), the desialylated bLF (C), and demannosylated bLF (D) at a concentration of 2 mg mL⁻¹. Culture medium served as negative control. NF‐κB activation was measured by spectrophotometry at 650 nm. The statistical differences were measured with unpaired t‐test with (n = 4) (**p < 0.01, ***p < 0.001).

Figure 6

bLF proteins glycosylation profile influences the release of NF‐κB in HEK‐Blue TLR‐4. Cells were incubated with bLF proteins at 2 mg mL⁻¹ and Polymyxin B at 100 μg mL⁻¹. The statistical differences were measured with unpaired t‐test (n = 4) (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7

Inhibitory effects of isolated bLF N‐glycans on HEK‐TLR‐8 cells. The cells were coincubated with the bLF structures and with the specific agonist for TLR‐8 ssRNA40 (50 μg mL⁻¹). NF‐κB was measured by spectrophotometry at 650 nm. Median and interquartile range of activation is plotted as NF‐κB release (n = 5). Statistical differences were calculated with Mann–Whitney test (*p < 0.05).