Bowman-Birk inhibitors from legumes as colorectal chemopreventive agents

Table 1 Amino acid sequence alignment of Bowman-Birk inhibitor-like proteins from several legumes species

Species	Entry name (accession number)	Amino acid sequence
Dolichos biflorus	IBB_DOLBI (Q9S9E3)	EPSESSKPCCDQCTCTKSIPPQCRCTDVRLNSCHSACSSCVCTFSIPAQCVCVDMKDFCYAPCKSSHDD
Glycine max	IBB1_SOYBN (P01055)	DDESSKPCCDQCACTKSNPPQCRCSDMRLNSCHSACKSCICALSYPAQCFCVDITDFCYEPCKPSEDDKEN
	IBBD2_SOYBN (P1064)	DDEYSKPCCDLCMCTRSMPPQCSCEDIRLNSCHSDCKSCMCTRSQPGQCRCLDTNDFCYKPCKSRDD
	IBBC2_SOYBN (P01063)	DDESSKPCCDLCMCTASMPPQCHCADIRLNSCHSACDRCACTRSMPGQCRCLDTTDFCYKPCKSSDEDDD
Lens culinaris	IBB_LENCU (Q8W4Y8)	GDDVKSACCDTCLCTRSQPPTCRCVDVRESCHSACDKCVCAYSNPPQCQCYDTHKFCYKACHNSEIEE
Lupinus albus	IBB1_LUPAL (P85172)	SLASKPCCDSCLCTRSIPPQCRCTDIGETCHSACKSCICTRSFPPQCRCSDITHFCYKPCTSS
Phaseolus vulgaris	IBB2_PHAVU (P01060)	EPSESSEPCCDICVCTASIPPICQCTDVRLNSCHSACKSCMCTRSMPGKCRCLDTTDYCYKSCKSSGEDDD
	IBB3_PHAVU (P81484)	ASXSSKPCCBHCACTKSIPPQCRCSBLRLNSCHSECKGCICTFSIPAQCICTDTNNFCYEPCKSSHGPBBNN
Pisum sativum	IBBA_PEA (Q41065)	GDDVKSACCDTCLCTKSNPPTCRCVDVRETCHSACDSCICAYSNPPKCQCFDTHKFCYKACHNSEVEEVIKN
	IBB2_PEA (Q41066)	GDDVKSACCDTCLCTKSDPPTCRCVDVGETCHSACDSCICALSYPPQCQCFDTHKFCYKACHNSEVEE VIKN
	IBBB_PEA (P56679)	GDDVKSACCDTCLCTKSNPPTCRCVDVGETCHSACLSCICAYSNPPKCQCFDTQKFCYKACHNSELEEVIKN
Vicia faba	IBB_VICFA (P24661)	GDDVKSACCDTCLCTKSEPPTCRCVDVGERCHSACNSCVCRYSNPPKCQCFDTHKFCYKSCHN
Vigna unguiculata	IBB_VIGUN (P17734)	ZASZSSKPCCRZCACTKSIPPZCRCSZVRLNSCHSACKSCACTFSIPAZCFCGBIBBFCYKPCKSSHSBBBBWN

The primary accession numbers below are reported in UniProtKB database. P₁-P_1’ are the reactive peptide bond sites, in bold text. Either K or R at P₁ position determines specificity for trypsin, whereas L, Y or F determines specificity against chymotrypsin; A determines specificity for elastase.

Table 2 Preclinical studies showing colorectal chemopreventive properties of Bowman-Birk inhibitor-like proteins from several legume species

Species (common name)	Model system (carcinogen)	Effect and/or mechanisms of action	Ref.
Glycine max (soy)	Rodent colon carcinogenesis (DMH)	Soybean BBI is effective at concentrations as low as 10 mg/100 g diet in reducing the incidence and frequency of colorectal tumors. Its ability to inhibit serine proteases is required for their chemopreventive properties. No adverse effects are observed in treated animals	[52,53]
	Mouse colorectal carcinogenesis (DMH)	Soybean BBI, when simultaneously treated with DMH, prevent the development of neoplastic lesions and protect against the onset of severe inflammatory processes	[79]
	Mouse colon inflammation (DSS)	A soybean Bomwan-Birk inhibitor concentrate reduces colon inflammation in mice with induced ulcerative colitis. Lower mortality rates and delayed onset of mortality are observed	[58]
	Colon cancer cell proliferation	The antiproliferative properties of BBI isoinhibitors, IBB1 and IBBD2, reveal that both trypsin- and chymotrypsin–like proteases involved in carcinogenesis should be considered as potential targets
			[26]
Lens culinaris (lentil)	Colon cancer cell proliferation	Lentil BBI is able to inhibit the growth of HT29 colon cancer cells at concentrations as low as 19 μmol/L, in a concentration-dependent manner; by contrast, colonic fibroblast CCD-18Co cells are unaffected	[30]
Pisum sativum (pea)	Colon cancer cell proliferation	TI1B, a major pea protease inhibitor, affect in a dose-dependent manner the growth of HT29 colon cancer cells whereas an inactive mutant did not show any significant effect	[55]
Vicia faba (field bean)	Mouse stomach carcinogenesis (benzopyrene)	BBI proves to be biologically active, under acidic conditions, in suppressing benzopyrene-induced forestomach carcinogenesis in mice following oral treatment; the oncopreventive properties are related to its protease inhibitory activity	[93]

DMH: Dimethylhydrazine; DSS: Dextran sulphate sodium; BBI: Bowman-Birk inhibitor.

Table 3 Serine proteases as potential therapeutic targets of Bowman-Birk inhibitor-like proteins in pathological processes

Serine protease	Function	Pathological processes	Evidence of interaction with BBI-like proteins
Proteasome	Control of the turn-over of regulatory proteins involved in critical cellular processes including cell cycle progression, cell development and differentiation, apoptosis, angiogenesis and signaling pathways	Cancer, inflammatory processes, autoimmune diseases and aging	Soybean BBI specifically and potently inhibits the proteasomal chymotrypsin-like activity in vitro and in vivo in MCF7 cancer breast cells[76]
			Mice treated simultaneously with BBI and DMH show a significant decrease in the chymotrypsin- and trypsin-like proteasomal activity in comparison with those treated with DMH only[79]
			Soybean BBI suppress proteasomal chymotrypsin-like activity in U2OS human osteosarcoma cells in vitro[80]
Matriptase	Differentiation and function of epithelial tissues	Activator of critical molecules associated with tumor invasion and metastasis	SFTI-1, a cyclic peptide from sunflower having similar features to the trypsin inhibitory binding domain of BBI, is a very potent inhibitor (Ki: 0.92 nmol/L)[85]
			BBI from soybean and lima bean have been reported to inhibit matriptase activity in vitro[86]
Chymase	Key mediator in inflammatory cell signaling pathways	Inflammatory processes, allergic reactions and pulmonary fibrosis	Soybean BBI strongly inhibits chymase from rat mast cells (Ki: 13.2 nmol/L)[94]
			Soybean BBI is a highly effective inhibitor of human mast cell chymase, being not effective against human tryptase[88]
Cathepsin G	Degradation of extracellular matrix components, regulates inflammatory response and promotes apoptosis	Inflammatory processes, cancer and aging	Soybean BBI inhibits strongly cathepsin G (Ki: 1.2 nmol/L)[86]
Duodenase	Morphogenesis and tissue repair; inflammatory and mitogenic role; participation in activation cascade of digestive proteases	Inflammatory processes	Duodenase interacts specifically with the chymotrypsin inhibitory domain of soybean BBI (Ki: 4 nmol/L)[87]
Elastase	Degradation of extracellular matrix components	Pulmonary emphysema, cystic fibrosis, infections, inflammation and atherosclerosis	Soybean BBI inhibits hydrolysis of extracellular matrix components by leukocyte enzymes[95]
			Soybean BBI inhibit human leukocyte elastase (Ki: 2.3 nmol/L)[96]

BBI: Bowman-Birk inhibitor; DMH: Dimethylhydrazine; Ki: Constant of inhibition; SFTI-1: Sunflower trypsin inhibitor.