Lactoferrin

What is Lactoferrin?

Lactoferrin is an iron-binding diferric glycoprotein that comes from the transferrin family. Proteins from the transferrin family fulfil a key role in controlling the levels of free iron in the body. Lactoferrin transfers iron to different body cells after being absorbed in the small intestines, and helps to control the level of free iron in the blood and external secretions. Lactoferrin is continuously sequenced in the body, and can be found in most exocrine secretions and only at well-defined stages of cell differentiation; such as the granules of neutrophils. In humans, such exocrine secretions include saliva, tears, semen, vaginal fluids, gastrointestinal fluids, nasal mucosa and bronchial mucosa. The highest concentration of lactoferrin is found in human colostrum, and then human milk, followed by cow's milk. It is the second most abundant milk protein after Caseins.

Lactoferrin is also known for its antibacterial, antifungal, antiviral, antimicrobial, anti-oxidant, anti-inflammatory, antiparasitic, anti-allergic, and most importantly anticancerous properties. Due to its therapeutic potential, it has been isolated and studied from many sources; including human, bovine, chicken, goat, rabbit, camel, and duck. During an infection or an inflammatory condition, the levels of lactoferrin are raised in the body, making it a good biomarker for inflammatory conditions.

Overall Bovine Structure

Lobes

Lactoferrin consists of a single polypeptide chain that weighs about 78 kDa. It consists of 689 amino acids, split equally into two halves: the N-lobe and C-lobe. Its two lobes are connected by a 10-residue helical peptide. This 10-residue connection allows for lactoferrin to be a flexible molecule. Each lobe consists of a Fe⁺³ binding cleft.

N-lobe - Pink
C-lobe - Light Green
10 Residue Connecting Helix - Blue

Domains

Each lobe is further subdivided into two domains; which are designated as N1 and N2 domains of the N-lobe, and C1 and C2 domains of the C-lobe. The iron-binding sites sit deep in the clefts formed between the two domains.

N1 - Orchid
N2 - Red
C1 - Gold
C2 - Light Green
10 Residue Connecting Helix - Aqua

Secondary Structures

Lactoferrin is composed of 26 helices and 28 beta sheets.

Alpha-helices = Pale Green
Beta-sheets = yellow

Iron and the Body

Iron is a mineral that is naturally present in many foods, and is an essential bioelement for most forms of life. Its importance lies in its ability to mediate electron transfer. It is an essential component of hemoglobin, a protein that transfers oxygen from the lungs to the tissues, and of myoglobin, a protein that provides oxygen to the body's muscles. Iron is also necessary for growth, development, normal cellular functioning, and synthesis of some hormones and connective tissue.

While in healthy cells, or if being transported, iron ions are never found in a naked state, but are always tightly chelated, or bound, usually with a protein. Free iron is a loose cannon - chemically - and potentially toxic. Its ability to donate and accept electrons means that if iron is free within or outside of the cell, it can initiate unwanted oxidation or reduction reactions with other molecules. This can cause damage to a wide variety of cellular structures, and ultimately kill cells.

Iron-binding Sites

Iron = Orange

N-lobe (Light green)
Aspartic acid (60) = Red
Tyrosine (92) = Blue
Tyrosine (192) = Purple
Histidine (253) = Yellow
CO₃^-2 = Grey & Red

C-lobe (Pink)
Aspartic acid (395) = Red
Tyrosine (433) = Blue
Tyrosine (526) = Purple
Histidine (595) = Yellow
CO₃^-2 = Grey & Red

As mentioned above, lactoferrin has two Fe⁺³ binding sites: one in the N-lobe and one in the C-lobe. Each lobe binds iron in generally the same fashion, with different amino residues. The iron-binding site is situated deep in the cleft formed between the two domains in each lobe. Whenever iron is not bound in the active site, these domains are found to be in an 'open' conformation. Then, whenever iron binds to the active site, the domains move closer to each other and are viewed to be in a 'closed' conformation. Whenever iron is released, these domains move farther away to appear 'open' once more.

Whenever Fe⁺³ is not bound within the active site, and the domains are in an open position, certain residues are fully exposed within the cleft. These residues, together with other positively charged residues, provide a favorable environment for attracting a negatively charged CO₃^-2 ion into the cleft. Once the carbonate ion is bound within the cleft, iron is immediately attracted to it. This is because of the combined negative effects of the carbonate ion and a nearby alpha-helix. Once iron enters the cleft, the iron atom is coordinated to the active site via the carbonate ion and the OH groups of the hydrogen-bonded Tyrosines (Tyr433 & Tyr526); then, it binds with each. After these interactions, two other residues, His595 & Asp395, move closer to bind with the iron atom. In the end, 6 bonds are formed between the iron atom and the protein, as well as an overall conformation change of the protein due to new and different bonds that formed.

Iron can become unbound from lactoferrin through different means. One way is if the pH of the surrounding environment drops below 5.0, and another is via means of proteinases. The change in pH, and cleaving from proteinases, alters amino acid residues throughout the protein and causes conformational changes. These changes then affect the binding properties of lactoferrin, which ultimately causes the release the iron.

References

Centers for Disease Control and Prevention. (2015). Normal iron absorption and storage. Retrieved December 15, 2015, from http://www.cdc.gov/ncbddd/hemochromatosis/training/pathophysiology/iron_cycle_popup.htm

Kanwar, J. R., Roy, K., Patel, Y., Shu-Feng, Z., Rawat Singh, M., Singh, D., & ... Kanwar, R. K. (2015). Multifunctional Iron Bound Lactoferrin and Nanomedicinal Approaches to Enhance Its Bioactive Functions. Molecules, 20(6), 9703-9731. doi:10.3390/molecules20069703

McCord, J. M. (2004). Iron, free radicals, and oxidative Injury1. The Journal of Nutrition, 134(11), 3171S-3172S. Retrieved from http://search.proquest.com.ac.ezproxy.switchinc.org/docview/197451869?accountid=8278

Moore, S. A., Andersona, B. F., Groom, C. R., Haridasa, M., & Baker, E. N. (1997). Three-dimensional structure of diferric bovine lactoferrin at 2.8 Å resolution. Journal of Molecular Biology, 274(2), 222-236.http://dx.doi.org/10.1006/jmbi.1997.1386

Sujata Sharma, Mau Sinha, Sanket Kaushik, Punit Kaur, and Tej P. Singh, 'C-Lobe of Lactoferrin: The Whole Story of the Half-Molecule,' Biochemistry Research International, vol. 2013, Article ID 271641, 8 pages, 2013. doi:10.1155/2013/271641

Rastogi, N., Singh, A., Pandey, S. N., Sinha, M., Bhushan, A., Kaur, P., & ... Singh, T. P. (2014). Structure of the iron-free true C-terminal half of bovine lactoferrin produced by tryptic digestion and its functional significance in the gut. FEBS Journal, 281(12), 2871-2882. doi:10.1111/febs.12827