The complementary roles of molecules in nature

In nature, there are many examples of molecules that play complementary roles resulting in benefits to humans. The common saying “nature knows best” gives credit to “mother nature” as someone who is all-knowing and nurturing. Another perspective is that throughout evolution, humans selected for healthy foods simply because consuming such foods is healthy — a self-serving motive. For centuries, Native Americans in North and South America consume beans (or legumes) and corn, a cereal. This is similar to eating mungo and rice in our diet. Later on, biochemists and nutritionists established that the consumption of beans and cereals together is healthy because their essential amino acid profiles are complementary. We all need essential amino acids from proteins in our diet in the right proportion because we are unable to synthesize them in the right amount. When a protein provides all the essential amino acids we need, it is called “complete,” otherwise, it is “incomplete.” Plant proteins are incomplete while animal proteins are complete. Beans have low methionine (a sulfur essential amino acid) and high lysine (another essential amino acid) while cereals are low in lysine and high in methionine. Thus, beans and cereals when consumed separately provide incomplete proteins but when consumed together make a complete protein. This is called nutritional complementation and is the way by which plant proteins are made complete. Practicing vegetarians know this by heart.

Another example is in the field of cancer prevention — the complementary roles of lunasin, a cancer preventive peptide (small protein) and the so-called Bowman-Birk Bowman Protease Inhibitor (BBI). BBI is the pure form and BBIC (BBI Concentrate) is not pure and in fact shows 5 protein components when analyzed by Western Blot — a biochemical analysis that involves separation of a mixture of proteins based on size and charge followed by detection of specific proteins by an antibody that selectively reacts with a particular protein.

Lunasin was originally discovered in soybean as a cancer preventive agent in my laboratory at University of California at Berkeley, California. I wrote an article about lunasin in this Star Science series six years ago. We gave it the name lunasin from the Tagalog word “lunas” for cure. The usual convention in naming a protein is that it ends in “in,” thus “lunas-in”. Later we showed that lunasin is also present in many other seeds such as barley, wheat, garbanzo, amaranth (the Golden Grain of the Aztecs of South America), etc. We expect lunasin to be present in all kinds of seeds, including mungo, based on our hypothesis that lunasin plays a crucial role in seed development.

An initial major criticism of our lunasin work is that being a peptide (a small protein), lunasin would be subject to digestion when we eat seeds and once digested it would lose its cancer preventive property — a very valid criticism. So we proceeded to demonstrate that lunasin survives digestion to some extent and ends up intact in the tissues of the body. Our first experiment involved labeling synthetic lunasin with a radioactive label of carbon-14 (as opposed to the non-radioactive, carbon-12) so that it can be tracked by a radioactivity counter. Indeed, when we fed labeled synthetic lunasin to mice orally together with soy protein, after 24 hours, the radioactivity label can be found in all the tissues we collected including liver, pancreas, kidney, brain, etc suggesting that lunasin is present in these tissues. However, the radioactivity alone did not prove that lunasin in the tissues was intact. We then proceeded to prove that lunasin in the tissues is indeed intact. In collaboration with our Korean colleagues, we fed animals with lunasin-enriched soy (LES) protein for several weeks and collected the liver. We extracted the protein from the liver and ran a Western blot. Unexpectedly, two bands showed up for lunasin, one corresponding to the molecular weight (MW) of lunasin and another band corresponding to a dimer, twice the size of lunasin. Evidently, lunasin formed a dimer in the tissues, an observation that we are unable to explain. Nevertheless, at this point, we had proven that lunasin ended up intact in the liver of animals fed lunasin-enriched soy. The liver of the control group of animals that were not fed LES showed no lunasin, as expected. We then proceeded to demonstrate that the lunasin in the liver was bioactive using an in vitro assay. In this assay, lunasin was purified from the liver extract, and added to mammalian cells in the presence of a chemical carcinogen (a cancer causing chemical) and the number of cancerous cells was counted. The results showed clearly that the lunasin extracted from the liver of mice fed LES significantly prevented cancer formation. For the control, the liver extract from mice not fed LES showed no cancer preventive effect.

The next question was how is lunasin protected from digestion in the gastrointestinal (GI) tract of the animals? Note that in the previous experiments, we always used LES (lunasin-enriched soy), meaning that lunasin is not pure but always present with some other soy proteins. And that is the key! The “other proteins” in soy protected lunasin from digestion. One of these “other proteins” is the protease inhibitor BBI. BBI inhibits the digestion of proteins by blocking the action of proteases, enzymes that digest proteins. It should be noted that BBI is just one of many protease inhibitors in seeds. Interestingly, BBI is a well-known cancer preventive protein administered in the form of BBIC (BBI Concentrate) which contains not only BBI but other proteins as well. BBIC is now in human clinical trials against mouth cancer and has been studied for almost 30 years, longer than lunasin which has been around for 11 years.

At this point, the story becomes extremely interesting! We suspected that BBIC, already a well-known cancer preventive protein, contains lunasin. This is quite simple to demonstrate by doing a Western blot — and guess what we found — lunasin is present in BBIC! The next question is what happens to the cancer preventive property of BBIC when lunasin is removed. Indeed, removal of lunasin makes BBIC ineffective as a cancer preventive agent while the lunasin removed from BBIC is bioactive using an in vitro assay! Our conclusion is that lunasin is in fact the bioactive component in BBIC while BBI simply protects lunasin from digestion. In another set of experiments, we clearly showed that synthetic lunasin (99 percent pure) is digested by pancreatin (a mixture of digestive enzymes) but is protected by adding BBI or another soy protease inhibitor called Kunitz Trypsin Inhibitor (KTI).

So here is a beautiful story of complementation: when we eat beans, including mungo, naturally occurring protease inhibitors such as BBI protects lunasin from digestion, lunasin ends up intact in our tissues and protect us from cancer. Lunasin and BBI play distinct complementary roles. “Nature knows best” and it is up to science to discover how nature works.

Epidemiology, the study of human populations, show that consumption of plant foods such as cereals and legumes is associated with reduced risks of cancer. Our studies give one explanation: lunasin and protease inhibitors play complementary roles to prevent cancer.

The data quoted here are mostly from a publication that appeared in the scientific journal PLoS ONE, January 2010, Volume 5, page e8890. There are five authors, signifying the team effort of our work and I want to acknowledge the contributions of each member of our team: CC Hsieh, B Hernandez-Ledesma, HJ Park and HJ Jeong. There are also many hardworking Berkeley undergraduates who helped in many aspects of the experiments. For a complete list of publications on lunasin, including ours, please go to the PubMed website maintained by the National Library of Medicine, National Institute of Health, USA and put in the search word “lunasin.”

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Ben O. de Lumen, Ph.D., is a professor at the Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA where he has been on the faculty for 33 years. He is also president and CEO of Filgen Biosciences Inc., which he co-founded to commercialize lunasin technology which is protected by patents owned by the University of California Berkeley. E-mail at [email protected].