Functional analysis of recombinant buffalo lactoferrin and monoferric lobes and their cytotoxic effect on buffalo mammary epithelial cells

Abstract

Lactoferrin (Lf) has been involved in diverse type of cellular activities and its biochemical properties are species specific. Lf is a bilobal molecule in which each lobe binds with one Fe 2? /Fe 3? ion. A lot of physiological effects of Lf are regulated by its iron binding and release properties; however these properties are species-specific. To understand the ironbinding, thermal stability and cytotoxic effect of buffalo Lf (buLf) and contribution of individual Nand C-terminal lobes therein, buLf and the truncated monoferric lobes were expressed in Kluyveromyces lactis or Pichia pastoris yeast expression systems. The iron-uptake/release behavior and thermal stability of recombinant buLf was observed similar to the Lf purified from buffalo milk. Supplementation of recombinant buLf to the buffalo mammary epithelial cells (BuMEC) culture decreased their proliferation and the cell viability in a dose dependent manner. The cell growth decreased by 37% at 1.0 mg/ml Lf. C-lobe decreased the viability of BuMEC by 15% at 1 mg/ml. The C-lobe showed greater cytotoxic effect against BuMEC in comparison to N-lobe. buLf caused a reduced expression of the casein in BuMEC. At 1.0 mg/ml of buLf, CSN2 transcript level was reduced by 74% and 78% in the normal and hormone free media, respectively. The expression of IL-1b gene in BuMEC increased by 4-5 fold in the presence of 1.0 mg/ml of Lf. The effect was similar to that observed in the involutory mammary gland, suggesting the role of elevated level of Lf in remodeling of buffalo mammary tissue during involution.

N, S-Doped Carbon Dots Prepared by Peanut Protein Isolates and Cysteamine as Highly Sensitive Fluorescent Sensors for Fe2+, Fe3+ and Lactoferrin

“…LF is a multifunctional protein with varying activities: promoting intestinal iron absorption, anti-inflammation, antibacterial, etc. [ 17 , 18 ]. This protein is an iron-binding glycoprotein with two lobes, which can reversibly bind to Fe 2+ /Fe 3+ [ 13 ].…”

“…LF has a polypeptide chain with about 690 amino acid residues and forms a basically symmetrical bilobal structure, while each lobe can reversibly bind to Fe 2+ /Fe 3+ [ 1 , 17 , 18 ]. It was reported that iron binding can obviously affect the biological functions of LF.…”