LEGUMBRES Y SALUD GASTROINTESTINAL

ALFONSO CLEMENTE

Departamento de Fisiología y Bioquímica de la Nutrición Animal

Estación Experimental del Zaidín (EEZ-CSIC)

alfonso.clemente@eez.csic.es

INIA-CSIC-AEL. XXVIII Foro de Colaboración Público-Privada. LEGUMINOSAS. Producción y consumo. Una revolución pendiente

Madrid, 15 de marzo 2016

LEGUMBRES Y SALUD GASTROINTESTINAL

Investigadores

Dr. Luis A. Rubio

Dr. Alfonso Clemente

Investigadores contratados

Dra. Maria del Carmen Marín Manzano

Dra. Raquel Ruiz Arroyo

Dra. Isabel Aranda Olmedo

GRUPO DE INVESTIGACION: SALUD GASTROINTESTINAL

1. Metabolismo y ecología de la microbiota intestinal. Efectos beneficiosos de prebióticos y

probióticos.

2. Mecanismo de acción y efectos biológicos de las proteínas de legumbres

LEGUMBRES Y SALUD GASTROINTESTINAL

1. Metabolismo y ecología de la microbiota intestinal. Efectos beneficiosos de prebióticos y

probióticos.

Colaboraciones

Financiación

Redes de investigación

LEGUMBRES Y SALUD GASTROINTESTINAL

2. Mecanismo de acción y efectos biológicos de las proteínas de legumbres

Colaboraciones

Financiación

Redes de investigación

Protease inhibitor TI1 from pea (Pisum sativum)

T

- Serine protease inhibitors

- Leguminosae, Gramineae

- Seeds and vegetative organs

- Low-molecular-weight (6-9 kDa)

- Form stable stoichiometric complexes

- Inhibit digestive proteases

- Double headed (T/T, T/C, T/E)

- 7 disulphide bridges

C

Clemente & Domoney (2006) Curr Prot & Pept Sci 7, 201-216

NUTRITIONAL SIGNIFICANCE OF BOWMAN-BIRK INHIBITORS FROM

LEGUMES IN FOOD AND FEED

* *

Amino acid sequence alignment of BBI proteins deduced from three gene (TI1, TI2 and TI9) and two cDNA (f69-2 and

f69-4) sequences from garden pea (Pisum sativum)

Functional and physiological role of

BBI-like proteins•Physiological role in plants

- Stores of sulphur-containing amino acids

- Regulators of endogenous proteases

- Defensive agents

- Abiotic stress

•Anti-nutritional properties

-Hyperactive pancreas

-Negative effect on digestibility of dietary proteins

• Health-promotion properties

- Cancer chemoprevention

- Anti-inflammatory

- Radioprotection

- Skeletal muscle atrophy

Clemente & Domoney (2006) Curr Prot & Pept Sci 7, 201-216; Clemente et al. (2011). Curr Prot & Pept Sci 12, 358-373

FUNCTIONAL AND PHYSIOLOGICAL ROLE OF BOWMAN-BIRK INHIBITORS

TILLING (Targeting Induced Local Lesions IN Genomes) mutants for TI1,

a major BBI in pea seeds

C77Y

S85F

E109K

N

C

Clemente et al. (2015) PLOS ONE 10: E0134634

GDDVKSACCDTCLCTKSNPPTCRCVDVRETCHSACDSCICAYSNPPKCQCFDTHKFCYKACHNSEVEEVIKN* *

77 85 109

TI1

Impact of mutations on TI1 structure and inhibitory activity

C77K - Abolished both TIA and CIA

- Overall reduction of 60% TIA and CIA in pea seeds

- Requirement for the disulphide bond C77-C92 for function

S85F - Abolished CIA

E109K - TIA and CIA was not affected

- Oligomerization pattern was affected

- Involvement in protein stability?

C77

S85

E109

GTTTTCCTCTTGAGCTTCGCCGCAAA--------------GTTTTGATTCAACTTCCTTC

ATAACTCAAGTGCTCTCCAATGGTGATGATGTCAAATCAGCATGTTGTGATACCTGCCTT

TGCACAAAATCAAACCCTCCTACCTGTCGCTGTGTTGATGTTGGAGAAACTTGTCACTCA

GCATGCAACAGTTGCATTTGTGCACTATCTTATCCTCCCCAGTGTCAGTG

JI262_TI2 MELMNKKAMMKLALMVFLLSFAAKF*---FDSTSFITQVLSNGDDVKSACCDTCLCTKSN 56

TI2_X83210 MELMNKKVMMKLALMVFLLSFAANVVNARFDSTSFITQVLSNGDDVKSACCDTCLCTKSD 60

JI262_TI1 MELMNKKAMMKLALMVFLLSFAAKF*---FDSTSFITQVLSNGDDVKSACCDTCLCTKSN 56

TI1_AJ276900 MELMNKKAMMKLALMVFLLSFAANVVNARFDSTSFITQVLSNGDDVKSACCDTCLCTKSN 60

*******.*************** ******************************:

JI262_TI2 PPTCRCVDVGETCHSACNSCICALSYPPKCQCFDTQKFCYKACHNSELEEVIKN86

TI2_X83210 PPTCRCVDVGETCHSACDSCICALSYPPQCQCFDTHKFCYKACHNSEVEEVIKN 114

JI262_TI1 PPTCRCVDVRETCHSACDSCICAYSNPPKCQCFDTHKFCYKACHNSEVEEVIKN86

TI1_AJ276900 PPTCRCVDVRETCHSACDSCICAYSNPPKCQCFDTHKFCYKACHNSEVEEVIKN 114

********* *******:***** * **:******:***********:******

A

C

B

D

Identification of a TI1/TI2 double null mutant in a pea germplasm collection

Clemente et al. (2015) PLOS ONE 10: E0134634

COLORECTAL CHEMOPREVENTION

• Colorectal cancer is one of the major causes of morbidity and mortality in

western countries

• Colorectal cancer is a multi-year process providing opportunities for intervention

prior to accumulation of mutations or phenotypic changes

• Colon cancer is preventable. One of the most effective means of reducing risk is

through appropriate diet

• Diets rich in fruits and vegetables are associated with a reduced risk of many

types of cancer, including colorectal cancer, and are believed to affect different

stages of carcinogenesis (initiation/promotion/progression/invasion)

•More than 300 plant compounds can prevent cancer (Source: Chemoprevention

of Colorectal Cancer Database. http://www.inra.fr/internet/Projets/reseau-

nacre/sci-memb/corpet/indexan.html)

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Functional Bowman-Birk inhibitors survive gastrointestinal

digestion at the terminal ileum of pigs fed chickpea-based diets

5-8 % of ingested BBI reach the large intestine in active form (anti-T and -C)

0.5

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0NaCl (M)

5ACCDSCVCTKSIPPQCRCNDM25

TIA CIA

Diet (U/100mg) 863 ± 120 886 ± 99

Ileal contents (U/100 mg) 222 ± 7 120 ± 13

Apparent ileal digestibility (%) 92.7 ± 0.2 95.6 ± 1.3

Clemente et al. (2008) J Sci Food Agric 8, 513-521

SDS-PAGE

1. Ileal extract

2. Partially purified BBI

Elution profile of proteins of ileal samples from pigs fed chickpea-based diets

Faecal samples of pigs

Masticator

Fermentation under anaerobiosis

37ºC 24 h

Exogenous soybean BBI

(93µM)

14

MW (kDa) 1 2 3 4 5 6 7 8 9

Effect of faecal fermentation on SDS-PAGE pattern of soybean BBI.

(1) fermentation control (without BBI) after 24 h; lanes 2-7,

fermentation samples with soybean BBI (93μM) at 0, 2, 4, 6, 8 y 24 h,

respectively; lane 8, soybean BBI incubated in fermentation media

without faeces; lane 9, soybean BBI

Effect of faecal microbiota on trypsin and chymotrypsin inhibitory

activity of soybean BBI

Soybean Bowman-Birk inhibitors survive faecal fermentation in their active

form retainaing significance as a bioactive compound in the human GIT

Marín-Manzano et al., (2009) Br J Nutr 101, 967-971

Bacterial enumeration by colony-

counting assays

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TIA CIA

No modulation of intestinal microbiota was observed

Natural and induced variation has been exploited to dissect the potential

role of BBI in human health-promoting programmes. We have successfully

used two eukaryotic expression systems (Aspergillus niger and Pichia

pastoris), alternatives to E. coli, capable of efficient re-folding of extensively

disulphide-bonded proteins

Aspergillus niger Pichia pastoris

Clemente et al. (2004) Protein Expression Purif 36, 106-114 Clemente et al. (2012) Br J Nutr 108, S135-S138

2014

Internalization of the anti-carcinogenic IBB1, a major Bowman-Birk isoinhibitor

from soybean (Glycine max), in colon cancer cells

PROTEIN ENGINEERING IN ORDER TO OBTAIN BBI PROTEINS WITH

MODIFIED POTENCY AND SPECIFICITY AGAINST TARGET SERINE

PROTEASES

rTI1B (template) Trypsin inhibitory domain Chymotrypsin inhibitory domain Inactive domain

P1 determines especificity against serine proteases: K / Y determines inhibition against trypsin/ chymmotrypsin, respectively

1---------10----------20---------30---------40----------50----------60---------70--

rTI1B mature GDDVKSACCDTCLCTK-SNPPTCRCVDVRETCHSACDSCICAY-SNPPKCQCFDTHKFCYKACHNSEVEEVIKN

rTI1B T-T GDDVKSACCDTCLCTK-SNPPTCRCVDVRETCHSACDSCICTK-SNPPTCQCFDTHKFCYKACHNSEVEEVIKN

rTI1B C-C GDDVKSACCDTCLCAY-SNPPKCRCVDVRETCHSACDSCICAY-SNPPKCQCFDTHKFCYKACHNSEVEEVIKN

rTI1B inactive GDDVKSACCDTCLCTG-SNPPTCRCVDVRETCHSACDSCICAG-SNPPKCQCFDTHKFCYKACHNSEVEEVIKN

rTI1B inactive GDDVKSACCDTCLCTKGSNPPTCRCVDVRETCHSACDSCICAYGSNPPKCQCFDTHKFCYKACHNSEVEEVIKN

Pea TI1B

Clemente et al. (2012) Br J Nutr 108, S135-S138; Marín-Manzano et al. (2015) (in preparation)

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THE ANTI-PROLIFERATIVE EFFECT OF TI1B, A MAJOR BOWMAN-BIRK ISOINHIBITOR FROM PEA (Pisum sativum L.),

ON HT29 COLON CANCER CELLS ARE MEDIATED THROUGH PROTEASE INHIBITION

Clemente et al., (2012) Br J Nutr 108, S135-S138

rTI1B Inactive rTI1B

HT29 cells

IC50 = 31 ± 7 μM

Serine protease Function Pathological processes Evidence of interaction with BBI-like proteins[refs.]

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[78]

Soybean BBI suppress proteasomal chymotrypsin-like

activity in U2OS human osteosarcoma cells in vitro[79]

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 nM) [84]

BBI from soybean and lima bean have been reported

to inhibit matriptase activity in vitro[85]

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 nM)[93]

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

nM)[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 nM)[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[94]

Soybean BBI inhibit human leukocyte elastase (Ki:2.3

nM)[95]

Serine proteases as potential therapeutic targets of BBI-like proteins in pathological processes

Clemente and Arques (2014). World J Gastroenterol 20: 10305-10315

Pea (Pisum sativum L.) seed albumin extracts show anti-inflammatory effect in the DSS

model of mouse colitis

• All treated groups showed anti-

inflammatory effect, evidenced by

reduced microscopic histological

damage in comparison to untreated

colitic mice.

• The treatments ameliorated the colonic

mRNA expression of different pro-

inflammatory markers.

• All treated groups showed an

improvement in the intestinal barrier

function. As a result, restoration of

bacterial populations to values in

healthy mice was observed.

Utrilla et al. (2015). Mol Nutr Food Res 59, 807-819