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In vitro screening of food peptides toxic for coeliac and other gluten-sensitive patients: a review
Toxicology 132 (1999) 99 – 110
In vitro screening of food peptides toxic for coeliac and other gluten-sensitive patients: a review Marco Silano a, Massimo De Vincenzi b,* a
III Scuola di Specializzazione in Pediatria, IV Clinica Pediatrica, Ospedale S. Paolo, Uni6ersita` di Milano, Via A. Di Rudinı` 8, 20142 Milan, Italy b Laboratorio Alimenti, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy Received 17 April 1998; accepted 10 July 1998
Abstract Experience gained through investigations on coeliac disease makes it possible to propose a screening method based on agglutination of isolated K562(S) cells to evaluate the occurrence in food protein of amino acid sequences that are able to adversely affect coeliac and related gluten-sensitive patients. The method consists of in vitro sequential peptic and tryptic digestion of food protein fractions under optimal pH, temperature and time conditions and in vitro incubation of the digest with K562(S) cells; the toxic potential is detected as an agglutination of K 562 (S) cells after a short incubation. Other in vitro test systems, including atrophic coeliac intestinal mucosa and rat fetal intestine, can be used to confirm the results obtained with the isolated cells. A fractionation step of the proteolytic digest on a sepharose-mannan column before exposure of the in vitro systems to the separated peptide fractions adds to the sensitivity of the method. This screening method is not only very useful to investigate action mechanisms in coeliac disease, but also to assess the safety of genetically-modified plant foods and novel foods for gluten-sensitive patients. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Coeliac disease; Gluten sensitive enteropathies; Gliadin peptides; Prolamin peptides; In vitro toxicology; Toxicity screening; Fetal intestines; Isolated cells; Cereals; Mannan; Oligomers of N-acetyl-glucosamine; Spermidine; Spermine; A-gliadin; Novel foods; Genetically-modified plant foods
1. Introduction
Abbre6iations: PT-digest, peptic-tryptic digest; PTC-digest, peptic-tryptic-cotazym digest. * Corresponding author. Fax: +39-06-49387101.
It is well known that in Europe 1:300 people— and in some European countries possibly even more—are intolerant to bread wheat and some other cereal proteins in the diet, which show
0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 8 ) 0 0 0 9 8 - 5
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Table 1 Cereal proteins and peptides toxic in vivo and in different in vitro systemsa In vitro
Coeliac patients
Atrophic coeliac mucosa
Rat fetal intestine
Chick fetal intestine
K562(S) cells
CaCo-2 cells
n.t. PT-gluten digest (36–39) PT-(20, 40–43) and PTC-gliadin digest (20,43–46) a-gliadin (37), A-gliadin (47), T-a-gliadin digest (44) and PT-a-gliadin digest (20,36,40,41,48)
n.t. n.t.
n.t. n.t.
n.t. n.t.
n.t. n.t.
PT-(51) and P, T, C-gliadin digest (44–46,49) a10-gliadin (45)
PT-gliadin digest (52)
PT- and PTC-gliadin digest (53–54)
PT-gliadin digest (57–58)
T-a-gliadin digest (52)
PT-A-gliadin digest (55)
n.t.
n.t. n.t.
n.t. PT-(51) and PTCprolamin digest (44,50–51)
n.t. n.t.
n.t. PT-prolamin digest (54, 56)
n.t. PT-prolamin digest (57–58)
n.t. n.t.
n.t. PT-(51) and PTCprolamin digest (44,50–51)
n.t. n.t.
n.t. PT-prolamin digest (54,56)
n.t. PT-prolamin digest (57–58)
n.t. n.t.
n.t. PT-(51) and PTCprolamin digest (44,50–51)
n.t. n.t.
n.t. PT-prolamin digest (54,56)
n.t. PT-prolamin digest (57–58)
1. Bread wheat Whole flour (1–5) Gluten (6–13,25) and PTgluten digest (14) Gliadin (13,15–17) and PT-(18) and PTC-gliadin digest (18–19) a-gliadin (20–21) and A-gliadin (22) and T-agliadin digest (23)
2. Rye Whole flour (5,24,28)
3. Barley Whole flour (24–28)
4. Oats Whole flour (25,29–35) Arenin
a n.t., not tested; P, peptic; T, tryptic; C, cotazym. (1) Anderson et al. (1952); (2) Rubin et al. (1960); (3) Rubin et al. (1962a); (4) Sheldon and Lawson (1952); (5) Van De Kamer et al. (1953a,b); (6) Alvey et al. (1957); (7) Bayless et al. (1970); (8) Ross et al. (1955); (9) Schenk and Samloff (1968); (10) Schneider et al. (1960); (11) Sheldon (1955); (12) Shmerling and Shiner (1970); (13) Van De Kamer et al. (1953a,b); (14) Frazer et al. (1959); (15) Kendall et al. (1972); (16) Van De Kamer and Weijers (1955); (17) Sturgess et al. (1994); (18) Bronstein et al. (1966); (19) de Ritis et al. (1982); (20) Jos et al. (1982); (21) Ciclitira et al. (1984); (22) Hekkens et al. (1970); (23) Hekkens et al. (1974); (24) Anand et al. (1978); (25) Baker and Read (1976); (26) Weijers and Van De Kamer (1960); (27) Hansted (1950); (28) Rubin et al. (1962b); (29) Auricchio et al. (1984); (30) Moulton (1958); (31) Weijers (1950); (32) Sheldon (1955); (33) Dissanayake et al. (1974); (34) Janatuinen et al. (1995); (35) Srinivasan et al. (1996); (36) Falchuk et al. (1974a); (37) Fluge and Aksnes (1978); (38) Fluge and Aksnes (1981a); (39) Fluge et al. (1982); (40) Jos et al. (1978); (41) Jos et al. (1980); (42) Jos et al. (1974); (43) Jos et al. (1975); (44) Auricchio et al. (1982); (45) de Ritis et al. (1979); (46) Silano et al. (1981); (47) Falchuk et al. (1974b); (48) Wieser et al. (1982); (49) Auricchio et al. (1979); (50) Auricchio et al. (1984a); (51) Auricchio et al. (1987); (52) Mothes et al. (1985); (53) Auricchio et al. (1984b); (54) De Vincenzi et al. (1998); (55) De Vincenzi et al. (1995); (56) De Vincenzi et al. (1997); (57) Giovannini et al. (1995); (58) Giovannini et al. (1996).
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In vivo
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Table 2 A-gliadin amino acid sequences toxic or inactive in vivo and in different in vitro systemsa In vivo
In vitro
Coeliac patients
Atrophic mucosa
1. Acti6e amino acid sequences 3–55b (1–2) 1–30 (8) 31–43 (3) 1–55 (8) 31–49 (4) 3–24 (2) 44–55 (3) 3–55 (2) 206–217 (5–7) 25–55 (2) 31–43 (9) 31–55 (8–9) 31–49 (10) 44–55 (9) 1–127 (8) 128–246 (8) 2. Inacti6e amino acid sequences 3–21 (4) 3–21 (10) 56–68 (3) 56–68 (8) 202–220 (4) 202–220 (10) 247–260 (8)
Chick fetal intestine
K562(S) cells
CaCo-2 cells
8–19 (11–12) 9–19 (12) 11–19 (12) 45–56 (11) 75–86 (13) 208–219 (11)
1–30 (14) 1–55 (14) 1–127 (14) 31–55 (14–16) 31–43 (15–16) 31–37 (15–16) 44–55 (15–16) 49–55 (15–16) 128–246 (14)
31–43 (17) 44–55 (18)
13–18 (13) 39–48 (13) 40–45 (13) 46–57 (13) 77–82 (13) 213–227 (12)
56–68 (14) 247–266 (14)
a (1) Wieser et al. (1982); (2) Wieser et al. (1983); (3) Marsh et al. (1995); (4) Sturgess et al. (1994); (5) Karagiannis et al. (1987); (6) Mantzaris et al. (1990); (7) Mantzaris and Jewell (1991); (8) de Ritis et al. (1988); (9) Maiuri et al. (1996); (10) Shidrawi et al. (1995); (11) Kocna et al. (1991); (12) Cornell et al. (1994); (13) Cornell and Mothes (1993); (14) Auricchio et al. (1990a); (15) De Vincenzi et al. (1998); (16) De Vincenzi et al. (1994); (17) Giovannini et al. (1997); (18) Giovannini et al. (1995). b Numbers indicate positions of amino acid residues in the A-gliadin amino acid sequence as determined by Kasarda et al. (1984).
sooner or later small-bowel atrophy with frequent associated malabsorption symptoms (Greco et al., 1992; Grodzinsky et al., 1992; Catassi et al., 1994; McMillian et al., 1996); and several other very serious symptoms may also occur (Ma¨ki and Collins, 1997). Ability to induce a ‘toxic response’ in coeliac patients has been shown (Table 1) by administration of bread wheat to coeliac patients or by in vivo instillation into the proximal ileum of a number of derived protein fractions and low molecular weight digests, including gluten, crude gliadin, a-gliadin, b-gliadin, g-gliadin, and vgliadin fractions, peptic-tryptic (PT-) gluten or gliadin digest, peptic-tryptic-cotazym (PTC-) gliadin digest, and T-a-gliadin digest. The investigations carried out with fragments of A-gliadin (i.e. a highly purified gliadin fraction characterized by Kasarda et al., 1984) or with synthetic
peptides homologous to the A-gliadin amino acid sequences 31–43, 31–49 and 44–55 (Table 2) have shown that toxic peptides consist of relatively small amino acid sequences rich in glutamine and proline (Fig. 1). Toxicity of these proteins and peptides has also been confirmed with the organ culture of human small intestinal biopsies (Table 1). Jejunal specimens obtained from patients with active enteropathy show morphological and biochemical improvement when cultured in a gliadin-free medium but not in the presence of toxic proteins and peptides (Fluge and Aksnes, 1978, 1981a,b; Stroberg, 1978). On the contrary, the jejunal mucosa from normal subjects is not affected by the presence of bread wheat gliadin, whereas the results obtained with jejunal mucosa from coeliac patients in remission (i.e. treated) are controversial (Falchuk and Stro¨ber, 1972, 1974; Fluge and Aksnes, 1981a,b; Howdle
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Fig. 1. Amino acid sequence of A-gliadin as determined by Kasarda et al. (1984). Continuous lines indicate regions of the A-gliadin molecule which have been found ‘toxic’ in all the in vivo and in vitro system tested. Dotted lines indicate regions with controversial results. Bold characters indicate glutamine and proline rich sequences common to the biologically active regions of A-gliadin and absent in non toxic regions.
et al., 1981; Fluge et al., 1982; Auricchio et al., 1985; Cornell et al., 1988; Shidrawi et al., 1995). Several bread wheat gluten-sensitive enteropathies have also been described (Bayless and Swanson, 1964; Hedberg et al., 1966; Levine et al., 1966; Rudman et al., 1971; McNeish et al., 1976; Cooper et al., 1978; Nussle et al., 1978) which, with the exception of coeliac disease that is genet-
ically-determined and permanent, are temporary and dependent on other primary causes including infectious diseases and intestinal surgery. So far the ability to associate proteolytic digestion to biologically-active peptides able to affect the intestinal mucosa of coeliac and related patients has been found to be restricted to some cereals. However, the growing diffusion of
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modified plant foods produced by means of genetic engineering technologies and of novel foods based on parts of plants formerly of very limited food use clearly call for more attention to be paid to possible toxic effects for the above-mentioned susceptible people, deriving from the unexpected emergence of toxic amino acid sequences in new food components. Existing preventive regulations such as the Council Directive 90/220/EEC of 23 April 1990 on the deliberate release into the environment of genetically-modified organisms and the European Parliament and the Council Regulation (EC) N. 258/97 of 27 January 1997 concerning novel foods and novel food ingredients, while providing for extensive safety testing for the general population, do not address the issue of safety for either genetically- or temporary-predisposed people in spite of their large number in Europe and of the severity of possible health problems. The present paper contains a critical appraisal of available data aimed at assessing whether in vitro systems can be reliably used to assess systematically the presence of the toxic peptides.
2. In vitro test systems Because of the many ethical and practical constraints of in vivo studies and of studies with bioptic specimens of intestinal mucosa from coeliac patients available for diagnostic purposes, many investigations aiming at the identification and characterization of cereal peptides deemed to be ‘toxic’ for coeliac and related patients have been performed with in vitro systems including isolated tissues, cells and subcellular fractions from a variety of sources. The digestion of cereal protein fractions under investigation has been mimicked in vitro by means of a preliminary sequential digestion with pepsin and trypsin under optimal pH values (1.8 and 8.0, respectively), temperature (37°C) and time (2 h) conditions. In some studies a third digestion step with a crude hog pancreatic extract (commercial name: cotazym or pancreatin) was also included in order to add a chymotrypsin digestive step before thermal inactivation of added proteolytic enzymes by heating at 100°C for 30 min.
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2.1. Fetal intestine The in vitro developing intestine from rat fetus was proposed by Auricchio et al. (1984a) as a suitable model for the study of peptides toxic in coeliac disease because of the enterocyte changes seen in coeliac disease (e.g. increased cell turnover, increased mitotic index and lengthening of crypts), suggestive of the emergence of less differentiated cells on the villus. Morphological maturation on fetal intestinal mucosa takes place under adequate in vitro conditions in a way comparable to what happens in vivo (de Ritis et al., 1975). Toxic effects in this system were detected as an inhibition of in vitro development and morphogenesis of the small intestine from 17- and 18-day-old rat fetuses and with no effect on the culture of jejunum from 21-day-old fetuses or from newborn rats. These effects were detected after 48 h culture at concentrations of bread wheat peptides as low as 0.1 mg/ml of incubation medium. A fetal chick intestinal assay has also been proposed as a model for the study of the biological effects of gliadin related to those seen in coeliac disease by other authors (Mothes et al., 1985; Kocna et al., 1991; Cornell and Mothes, 1993 Cornell et al., 1994). The assay is based on duodena of 12-day-old chick embryos explanted and cultured for 2 days. The effects of peptides added to the culture medium were evaluated at a concentration of 0.5 mg/ml by determining sucrase activity in the explant and the medium; the decrease in sucrase activity is an indication of test substances able to act on undifferentiated enterocytes.
2.2. Isolated cells To investigate agglutinating activity of gliadin peptides, K562 (S) undifferentiated cells, a subline isolated from an outgrowth of a clone from a patient with chronic myelogenouss leukemia that responds to hemin or butyrate induction with a differential expression of the embryonal globin genes, have been extensively used (Auricchio et al., 1984a,b, 1990a,b; De Vincenzi et al., 1995, 1997, 1998). Control undifferentiated K 562 (S)
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cells exhibited a typical spherical shape with an average diameter of about 5 m and no significant evidence of cell clustering. After 30 min incubation in the presence of active cereal peptides at concentrations varying (according to the purity and activity of the peptides) between a few and 300 micrograms/ml of incubation medium, the cells were agglutinated with a peculiar appearance, and a tendency toward the formation of a continuous cell layer, which showed a high resistance to shearing and whirling forces. Giovannini et al. (1995, 1996, 1997) have also extensively investigated the effects of a number of peptides derived from cereal proteins on CaCo-2 cells at different days of culture in order to test undifferentiated cells (initial culture phase), actively undifferentiated proliferating cells (day 5) and more differentiated cells (day 19). In fact, within 19 days the CaCo-2 cell line, derived from a human colon adenocarcinoma, spontaneously undergoes some differentiation in vitro with enterocyte-like features both structurally (microvilli, tight junctions) and functionally (brush border-associated enzymes, transport across the surface membrane) (Pinto et al., 1983; Rousset, 1986). As far as undifferentiated CaCo-2 cells are concerned, toxicity has been evaluated mainly through growth inhibition of cells, whereas toxicity on more differentiated CaCo-2 cells has been assessed through colony forming ability, measurements of alkaline phosphatase activity (one of the specific enzymatic activities of the intestinal brush border cells) and a series of metabolic parameters such as DNA and RNA synthesis and (glyco)protein synthesis. Effects of active cereal peptides were detected at concentrations varying between 1 and 2 mg of peptides/ml of incubation medium.
3. Cereal peptides Tables 1–3 show the responsiveness of several in vitro systems to peptide fractions from different cereals.
3.1. Prolamin peptides from toxic cereals Digests of prolamines from cereals known to be non tolerated in coeliac disease (i.e. bread wheat, rye and barley) (Table 1) all induced in vitro inhibition of fetal differentiation, agglutination of K 562 (S) cells and toxic response in CaCo-2 cells. However, in vivo data suggesting toxicity of barley and rye in coelaic disease are very old, based only on a small number of patients and performed with diagnostic methods (i.e. fat absorption test, xylase test and biochemical indicators) that are often difficult to interpret. Data concerning toxicity of oats in coeliac patients are controversial in vivo but not in vitro. Weijers (1950) reported oats to be harmful for coeliac patients on the basis of stool fat studies on two patients, but Sheldon (1955) failed to confirm these results. Dissanayake et al. (1974) fed 4 patients 40–60 g oats per day for 4 wk and failed to demonstrate histologic changes, whereas Baker and Read (1976) challenged 12 patients with 60 g oat flour/day over 2–14 wk and demonstrated toxicity in 3 patients. Fifty-two adults with coeliac disease in remission followed for 6 months and 40 with newly diagnosed disease for 12 months were studied by Janatuinen et al. (1995); the group were treated with 50–70 g of oats/day and patients did not differ significantly in nutritional status, symptoms, or laboratory measures from the control group. Srinivasan et al. (1996) treated 10 coeliac patients in remission with 50 g oats cereal daily for 12 wk and reported no induction of histologic changes or changes in serologic immune markers. The absence of effects seen in some studies might be due to the fact that the prolamin content of oats is approximately five times less than that of wheat, rye, and barley; such a standpoint is also supported by the fact that toxicity of oat prolamin does not significantly differ from that of bread wheat gliadin when tested on rat fetal intestine, K 562 (S) and CaCo-2 cells (Table 1). Moreover, no difference has been detected between oats and bread wheat prolamins in their abilities to induce lymphocytic infiltration upon instillation in the coeliac rectal mucosa, and in vitro studies using organ culture of treated coeliac mucosa have shown (Mayer et al., 1997)
Table 3 Cereal proteins and peptides non toxic in vivo and in different in vitro test systemsa In vitro
Coeliac patients
Atrophic coeliac mucosa
Rat fetal intestine
K562(S) cells
CaCo-2 cells
n.t. n.t.
n.t. n.t.
n.t. n.t.
PT-and PTC-gliadin digest+mannan (5) PT-gliadin digest +N, N%, N¦-triacetylchitotriose or spermidine (5–6)
PTC-glutelin digest (7–8) PTC-albumin and globulin digest (7–9) PT- and PTC-gliadin digest+mannan (10) PT-gliadin digest +N, N%, N¦-triacetylchitotriose or spermidine (10–11)
PT-gliadin digest+mannan (10) PT-gliadin digest +N, N%, N¦-triacetylchitotrioseor spermidine or spermine (10, 13)
n.t. n.t.
2. Rice Whole flour (3–4) n.t.
n.t. n.t.
n.t. PTC-prolamin digest (12)
n.t. PT-prolamin digest (5)
n.t. n.t.
3. Maize Whole flour (3–4) n.t.
n.t. n.t.
n.t. PTC-prolamin digest (12)
n.t. PT-prolamin digest (5)
n.t. PT-prolamin digest (14)
1. Bread wheat Glutelins (1) Albumins (1, 2) and albumins+globulins (1) n.t. n.t.
a n.t., not tested; P, peptic; T, tryptic; C, cotazym. (1) Van De Kamer et al. (1953a); (2) Rubin et al. (1962b); (3) Van De Kamer et al. (1953b); (4) Anand et al. (1978); (5) Auricchio et al. (1990a); Auricchio et al. (1990b); (7) Auricchio et al. (1982); (8) Silano et al. (1981); (9) de Ritis et al. (1979); (10) Auricchio et al. (1987); (11) Auricchio et al. (1990b); (12) Auricchio et al. (1984a); (13) Auricchio et al. (1984b); (14) Giovannini et al. (1995).
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that oat prolamins are able to activate a T-cell mediated mucosal immune response in the coeliac patients. Auricchio et al. (1982) showed that the PTC-digest of durum wheat gliadins, as compared to the corresponding digest of bread wheat gliadins, was much less active in inhibiting recovery of and in damaging the in vitro cultured small intestinal mucosa from patients with active coeliac disease. At concentrations of 0.5 and 1.0 mg/ml of culture medium, the PTC-digest of gliadin fraction from durum wheat, did not inhibit in vitro occurring epithelial recovery in a number of flat biopsies and caused no change in several other cases. However, at a concentration of 2.5 mg/ml, this digest induced some mucosal damage in all three biopsies tested. The PTC-digest of prolamins from durum wheat were completely inactive when tested on the rat fetal intestine (Auricchio et al., 1979) and on CaCo-2 cells (Giovannini et al., 1996). Although the whole gliadin digests from 12 durum wheat varieties were unable to agglutinate K 562 (S) cells even when tested at a concentration of 14 mg/ml, all these digests were found to contain an actively agglutinating Fraction C also present in bread wheat gliadin digest (De Vincenzi et al., 1995). The lack of agglutinating activity of the whole durum wheat gliadin digests has been shown to depend on the presence in these digests of another peptide fraction (coded as Fraction B) that is eluted much earlier from the sepharose 6-B-mannan column and is able to inhibit the cell agglutinating activity of Fraction C. Such an active Fraction B is not present in bread wheat gliadin peptides (De Vincenzi et al., 1995). The cell agglutination inhibiting activity of Fraction B was shown to be associated with a peptide with molecular weight 1157.5 Da, which was synthesized in a high degree of purity with the solid phase method (De Vincenzi et al., 1997). The 1157.5 Da peptide, consisting of 10 amino acid residues with sequence H2N – gln – gln – pro – gln–asp–ala–val – gln – pro – phe – COOH, was able to prevent the agglutination induced by PT-digests of gliadins from bread wheat and of prolamins from rye, barley and oats as well as by the synthetic peptides homologous to A-gliadin amino acid sequences 31 – 43 and 44 – 55 (De Vincenzi et al., 1997). The ability to protect K 562 (S)
cells from agglutination was also exhibited to the full extent by all the peptides derived by the ‘1157.5 Da’ peptide by progressive deletions of five times the terminal carboxylic residue, whereas the sixth consecutive deletion yielded an inactive peptide (De Vincenzi et al., 1998). The smallest active peptide has molecular weight 614.6 Da and amino acid sequence H2N–gln–gln–pro–gln–asp– COOH. The activity of the 1157.5 Da and related peptides is likely due to an effective competition for the binding sites of toxic gliadin peptides on the cell surface.
3.2. Prolamin peptides from non toxic cereals Not only is there a high correlation between in vivo and in vitro results for toxic components, but also for the non toxic ones. Table 3 shows that all cereals and cereal fractions which are well tolerated in coeliac disease do not affect in vitro differentiation of rat fetal intestine and do not induce agglutination of K 562 (S) cells. Another important aspect which has been detected by means of in vitro systems is the protective action of several substances including mannan, N, N%, N¦-triacetylchitotriose and spermidine on intestinal atrophic mucosa, rat fetal intestine and K 562 (S) cells resulting in the complete removal of in vitro toxicity of bread wheat PT- and PTC-gliadin digest (Table 3). These substances were even able to reverse the expression of biological activities of toxic prolamin peptides on immature intestines and undifferentiated cells. The affinity chromatography studies of the whole gliadin digest on sepharose 6-B-mannan or sepharose 6-B-oligomers of Nacetyl-glucosamine, suggest that the agglutination–inhibition effect of these carbohydrates is due to a binding (sequestrating) activity of the toxic peptides (De Vincenzi et al., 1995), whereas the protection exerted by the polycationic polyamines can be more likely attributed to the effective competition for the binding sites of toxic gliadin peptides on the cell surface.
3.3. A-gliadin fragments As far as the amino acid sequence responsible for toxicity of the bread wheat A-gliadin, the
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fragments 31–43 and 44 – 55 are the only ones which have been tested and found to be toxic in coeliac patients and active on atrophic and treated coeliac mucosa as well as on fetal intestines and undifferentiated cells (Table 2). In vitro toxicity of synthetic peptides homologous to A-gliadin amino acid sequences 31 – 55, 31 – 43 or 44 – 55 was completely prevented by mannan or N,N%-diacetylchitotriose or N, N%, N¦-triacetylchitotriose (De Vincenzi et al., 1994). As already pointed out by de Ritis et al., (1988), peptides ‘31 – 43’ and ‘44 – 55’ contain the amino acid sequence – gln – gln – gln–pro– and/or that – pro – ser – gln – gln – (Fig. 1), which are believed to be essential for the expression of toxicity. It is striking the similarity of these two sequences with that of H2N – gln – gln – pro–gln–asp–COOH, which is able to prevent the agglutination of K562(s) cells induced by the toxic peptides. The amino acid sequence – gln – gln–gln–pro– is also present in the peptide ‘31 – 49’ which is toxic in coeliac disease and active on the in vitro mucosa, but has not been tested for cell agglutination so far. De Vincenzi et al., (1998) have recently shown that the agglutinating activity on K 562 (S) cells is also significantly exhibited, although to a lower extent than for peptides 31–43 and 44 – 55, by the very small peptides ‘31–37’ (H2N – leu – gly – gln – gln – gln – pro – phe–COOH) and ‘49 – 55’ (H2N – phe – pro – ser – gln–gln–pro–tyr – COOH). On the other hand, the results on the toxicity of the amino acid sequences concerning the Agliadin fragments ‘1 – 30’ and ‘206 – 217’ are more controversial (Table 2).They may depend on the different ways in which these peptides are split into smaller peptides by peptidases operating in the test systems chosen by the different authors (De Vincenzi et al., 1997, 1998; Silano and De Vincenzi, 1998), but further investigation is needed to clarify this issue.
4. Conclusion The data available in the literature clearly show that in vitro studies on fetal intestinal mucosa as well as on undifferentiated K 562 (S) cells and CaCo-2 cells have proven to be extremely useful
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in identifying toxic and non toxic cereals and cereal components as well as elucidating the fragments of bread wheat A-gliadin which are toxic in the so called gluten-sensitive enteropathies. Studies with these in vitro systems have a key role in clarifying the molecular mechanisms of glutensensitive enteropathies. The very high correlation existing between data obtained with the different in vitro test systems and those available in vivo (Tables 1–3) strongly indicate that a screening method based on the ability of peptic–tryptic protein digests to induce agglutination of K 562 (S) cells provides a very sensitive and reliable system to assess the potential risk for susceptible individuals of geneticallymodified plant foods and novel foods. This cell agglutination test is very fast, simple to perform and inexpensive; moreover, it shows high sensitivity to a few micrograms of active peptides/ml of culture medium. Not only is the test able to easily identify cereal peptides toxic in coeliac disease (i.e. those from bread wheat, barley, rye and oats prolamins) and those which are well tolerated (e.g. from maize and rice), but it is also able to identify geneticallymodified bread wheat cereals which have a lower content of toxic peptides (De Vincenzi et al., 1996). An additional degree of confidence for both positive and negative findings would be added by a preliminary fractionation step of the PT-protein digest on a sepharose 6B-mannan column and separate testing with K 562 (S) cells of the obtained fractions; in fact, through such an approach it is possible to identify those cereals such as durum and Monococcum wheats that contain both toxic and protective peptides. When needed, confirmation can be achieved by testing fractions of interest in vitro with atrophic coeliac intestinal mucosa.
References Alvey, C., Anderson, C.M., Freeman, M., 1957. Wheat gluten and coeliac disease. Arch. Dis. Child. 32, 434 – 437. Anand, B.S., Piris, J., Truelove, S.C., 1978. The role of various cereals in coeliac disease. Quart. J. Med. N.S. 47, 101 – 110.
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In vitro screening of food peptides toxic for coeliac and other gluten-sensitive patients: a review Marco Silano a, Massimo De Vincenzi b,* a
III Scuola di Specializzazione in Pediatria, IV Clinica Pediatrica, Ospedale S. Paolo, Uni6ersita` di Milano, Via A. Di Rudinı` 8, 20142 Milan, Italy b Laboratorio Alimenti, Istituto Superiore di Sanita`, Viale Regina Elena 299, 00161 Rome, Italy Received 17 April 1998; accepted 10 July 1998
Abstract Experience gained through investigations on coeliac disease makes it possible to propose a screening method based on agglutination of isolated K562(S) cells to evaluate the occurrence in food protein of amino acid sequences that are able to adversely affect coeliac and related gluten-sensitive patients. The method consists of in vitro sequential peptic and tryptic digestion of food protein fractions under optimal pH, temperature and time conditions and in vitro incubation of the digest with K562(S) cells; the toxic potential is detected as an agglutination of K 562 (S) cells after a short incubation. Other in vitro test systems, including atrophic coeliac intestinal mucosa and rat fetal intestine, can be used to confirm the results obtained with the isolated cells. A fractionation step of the proteolytic digest on a sepharose-mannan column before exposure of the in vitro systems to the separated peptide fractions adds to the sensitivity of the method. This screening method is not only very useful to investigate action mechanisms in coeliac disease, but also to assess the safety of genetically-modified plant foods and novel foods for gluten-sensitive patients. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Coeliac disease; Gluten sensitive enteropathies; Gliadin peptides; Prolamin peptides; In vitro toxicology; Toxicity screening; Fetal intestines; Isolated cells; Cereals; Mannan; Oligomers of N-acetyl-glucosamine; Spermidine; Spermine; A-gliadin; Novel foods; Genetically-modified plant foods
1. Introduction
Abbre6iations: PT-digest, peptic-tryptic digest; PTC-digest, peptic-tryptic-cotazym digest. * Corresponding author. Fax: +39-06-49387101.
It is well known that in Europe 1:300 people— and in some European countries possibly even more—are intolerant to bread wheat and some other cereal proteins in the diet, which show
0300-483X/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 0 0 - 4 8 3 X ( 9 8 ) 0 0 0 9 8 - 5
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Table 1 Cereal proteins and peptides toxic in vivo and in different in vitro systemsa In vitro
Coeliac patients
Atrophic coeliac mucosa
Rat fetal intestine
Chick fetal intestine
K562(S) cells
CaCo-2 cells
n.t. PT-gluten digest (36–39) PT-(20, 40–43) and PTC-gliadin digest (20,43–46) a-gliadin (37), A-gliadin (47), T-a-gliadin digest (44) and PT-a-gliadin digest (20,36,40,41,48)
n.t. n.t.
n.t. n.t.
n.t. n.t.
n.t. n.t.
PT-(51) and P, T, C-gliadin digest (44–46,49) a10-gliadin (45)
PT-gliadin digest (52)
PT- and PTC-gliadin digest (53–54)
PT-gliadin digest (57–58)
T-a-gliadin digest (52)
PT-A-gliadin digest (55)
n.t.
n.t. n.t.
n.t. PT-(51) and PTCprolamin digest (44,50–51)
n.t. n.t.
n.t. PT-prolamin digest (54, 56)
n.t. PT-prolamin digest (57–58)
n.t. n.t.
n.t. PT-(51) and PTCprolamin digest (44,50–51)
n.t. n.t.
n.t. PT-prolamin digest (54,56)
n.t. PT-prolamin digest (57–58)
n.t. n.t.
n.t. PT-(51) and PTCprolamin digest (44,50–51)
n.t. n.t.
n.t. PT-prolamin digest (54,56)
n.t. PT-prolamin digest (57–58)
1. Bread wheat Whole flour (1–5) Gluten (6–13,25) and PTgluten digest (14) Gliadin (13,15–17) and PT-(18) and PTC-gliadin digest (18–19) a-gliadin (20–21) and A-gliadin (22) and T-agliadin digest (23)
2. Rye Whole flour (5,24,28)
3. Barley Whole flour (24–28)
4. Oats Whole flour (25,29–35) Arenin
a n.t., not tested; P, peptic; T, tryptic; C, cotazym. (1) Anderson et al. (1952); (2) Rubin et al. (1960); (3) Rubin et al. (1962a); (4) Sheldon and Lawson (1952); (5) Van De Kamer et al. (1953a,b); (6) Alvey et al. (1957); (7) Bayless et al. (1970); (8) Ross et al. (1955); (9) Schenk and Samloff (1968); (10) Schneider et al. (1960); (11) Sheldon (1955); (12) Shmerling and Shiner (1970); (13) Van De Kamer et al. (1953a,b); (14) Frazer et al. (1959); (15) Kendall et al. (1972); (16) Van De Kamer and Weijers (1955); (17) Sturgess et al. (1994); (18) Bronstein et al. (1966); (19) de Ritis et al. (1982); (20) Jos et al. (1982); (21) Ciclitira et al. (1984); (22) Hekkens et al. (1970); (23) Hekkens et al. (1974); (24) Anand et al. (1978); (25) Baker and Read (1976); (26) Weijers and Van De Kamer (1960); (27) Hansted (1950); (28) Rubin et al. (1962b); (29) Auricchio et al. (1984); (30) Moulton (1958); (31) Weijers (1950); (32) Sheldon (1955); (33) Dissanayake et al. (1974); (34) Janatuinen et al. (1995); (35) Srinivasan et al. (1996); (36) Falchuk et al. (1974a); (37) Fluge and Aksnes (1978); (38) Fluge and Aksnes (1981a); (39) Fluge et al. (1982); (40) Jos et al. (1978); (41) Jos et al. (1980); (42) Jos et al. (1974); (43) Jos et al. (1975); (44) Auricchio et al. (1982); (45) de Ritis et al. (1979); (46) Silano et al. (1981); (47) Falchuk et al. (1974b); (48) Wieser et al. (1982); (49) Auricchio et al. (1979); (50) Auricchio et al. (1984a); (51) Auricchio et al. (1987); (52) Mothes et al. (1985); (53) Auricchio et al. (1984b); (54) De Vincenzi et al. (1998); (55) De Vincenzi et al. (1995); (56) De Vincenzi et al. (1997); (57) Giovannini et al. (1995); (58) Giovannini et al. (1996).
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Table 2 A-gliadin amino acid sequences toxic or inactive in vivo and in different in vitro systemsa In vivo
In vitro
Coeliac patients
Atrophic mucosa
1. Acti6e amino acid sequences 3–55b (1–2) 1–30 (8) 31–43 (3) 1–55 (8) 31–49 (4) 3–24 (2) 44–55 (3) 3–55 (2) 206–217 (5–7) 25–55 (2) 31–43 (9) 31–55 (8–9) 31–49 (10) 44–55 (9) 1–127 (8) 128–246 (8) 2. Inacti6e amino acid sequences 3–21 (4) 3–21 (10) 56–68 (3) 56–68 (8) 202–220 (4) 202–220 (10) 247–260 (8)
Chick fetal intestine
K562(S) cells
CaCo-2 cells
8–19 (11–12) 9–19 (12) 11–19 (12) 45–56 (11) 75–86 (13) 208–219 (11)
1–30 (14) 1–55 (14) 1–127 (14) 31–55 (14–16) 31–43 (15–16) 31–37 (15–16) 44–55 (15–16) 49–55 (15–16) 128–246 (14)
31–43 (17) 44–55 (18)
13–18 (13) 39–48 (13) 40–45 (13) 46–57 (13) 77–82 (13) 213–227 (12)
56–68 (14) 247–266 (14)
a (1) Wieser et al. (1982); (2) Wieser et al. (1983); (3) Marsh et al. (1995); (4) Sturgess et al. (1994); (5) Karagiannis et al. (1987); (6) Mantzaris et al. (1990); (7) Mantzaris and Jewell (1991); (8) de Ritis et al. (1988); (9) Maiuri et al. (1996); (10) Shidrawi et al. (1995); (11) Kocna et al. (1991); (12) Cornell et al. (1994); (13) Cornell and Mothes (1993); (14) Auricchio et al. (1990a); (15) De Vincenzi et al. (1998); (16) De Vincenzi et al. (1994); (17) Giovannini et al. (1997); (18) Giovannini et al. (1995). b Numbers indicate positions of amino acid residues in the A-gliadin amino acid sequence as determined by Kasarda et al. (1984).
sooner or later small-bowel atrophy with frequent associated malabsorption symptoms (Greco et al., 1992; Grodzinsky et al., 1992; Catassi et al., 1994; McMillian et al., 1996); and several other very serious symptoms may also occur (Ma¨ki and Collins, 1997). Ability to induce a ‘toxic response’ in coeliac patients has been shown (Table 1) by administration of bread wheat to coeliac patients or by in vivo instillation into the proximal ileum of a number of derived protein fractions and low molecular weight digests, including gluten, crude gliadin, a-gliadin, b-gliadin, g-gliadin, and vgliadin fractions, peptic-tryptic (PT-) gluten or gliadin digest, peptic-tryptic-cotazym (PTC-) gliadin digest, and T-a-gliadin digest. The investigations carried out with fragments of A-gliadin (i.e. a highly purified gliadin fraction characterized by Kasarda et al., 1984) or with synthetic
peptides homologous to the A-gliadin amino acid sequences 31–43, 31–49 and 44–55 (Table 2) have shown that toxic peptides consist of relatively small amino acid sequences rich in glutamine and proline (Fig. 1). Toxicity of these proteins and peptides has also been confirmed with the organ culture of human small intestinal biopsies (Table 1). Jejunal specimens obtained from patients with active enteropathy show morphological and biochemical improvement when cultured in a gliadin-free medium but not in the presence of toxic proteins and peptides (Fluge and Aksnes, 1978, 1981a,b; Stroberg, 1978). On the contrary, the jejunal mucosa from normal subjects is not affected by the presence of bread wheat gliadin, whereas the results obtained with jejunal mucosa from coeliac patients in remission (i.e. treated) are controversial (Falchuk and Stro¨ber, 1972, 1974; Fluge and Aksnes, 1981a,b; Howdle
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Fig. 1. Amino acid sequence of A-gliadin as determined by Kasarda et al. (1984). Continuous lines indicate regions of the A-gliadin molecule which have been found ‘toxic’ in all the in vivo and in vitro system tested. Dotted lines indicate regions with controversial results. Bold characters indicate glutamine and proline rich sequences common to the biologically active regions of A-gliadin and absent in non toxic regions.
et al., 1981; Fluge et al., 1982; Auricchio et al., 1985; Cornell et al., 1988; Shidrawi et al., 1995). Several bread wheat gluten-sensitive enteropathies have also been described (Bayless and Swanson, 1964; Hedberg et al., 1966; Levine et al., 1966; Rudman et al., 1971; McNeish et al., 1976; Cooper et al., 1978; Nussle et al., 1978) which, with the exception of coeliac disease that is genet-
ically-determined and permanent, are temporary and dependent on other primary causes including infectious diseases and intestinal surgery. So far the ability to associate proteolytic digestion to biologically-active peptides able to affect the intestinal mucosa of coeliac and related patients has been found to be restricted to some cereals. However, the growing diffusion of
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modified plant foods produced by means of genetic engineering technologies and of novel foods based on parts of plants formerly of very limited food use clearly call for more attention to be paid to possible toxic effects for the above-mentioned susceptible people, deriving from the unexpected emergence of toxic amino acid sequences in new food components. Existing preventive regulations such as the Council Directive 90/220/EEC of 23 April 1990 on the deliberate release into the environment of genetically-modified organisms and the European Parliament and the Council Regulation (EC) N. 258/97 of 27 January 1997 concerning novel foods and novel food ingredients, while providing for extensive safety testing for the general population, do not address the issue of safety for either genetically- or temporary-predisposed people in spite of their large number in Europe and of the severity of possible health problems. The present paper contains a critical appraisal of available data aimed at assessing whether in vitro systems can be reliably used to assess systematically the presence of the toxic peptides.
2. In vitro test systems Because of the many ethical and practical constraints of in vivo studies and of studies with bioptic specimens of intestinal mucosa from coeliac patients available for diagnostic purposes, many investigations aiming at the identification and characterization of cereal peptides deemed to be ‘toxic’ for coeliac and related patients have been performed with in vitro systems including isolated tissues, cells and subcellular fractions from a variety of sources. The digestion of cereal protein fractions under investigation has been mimicked in vitro by means of a preliminary sequential digestion with pepsin and trypsin under optimal pH values (1.8 and 8.0, respectively), temperature (37°C) and time (2 h) conditions. In some studies a third digestion step with a crude hog pancreatic extract (commercial name: cotazym or pancreatin) was also included in order to add a chymotrypsin digestive step before thermal inactivation of added proteolytic enzymes by heating at 100°C for 30 min.
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2.1. Fetal intestine The in vitro developing intestine from rat fetus was proposed by Auricchio et al. (1984a) as a suitable model for the study of peptides toxic in coeliac disease because of the enterocyte changes seen in coeliac disease (e.g. increased cell turnover, increased mitotic index and lengthening of crypts), suggestive of the emergence of less differentiated cells on the villus. Morphological maturation on fetal intestinal mucosa takes place under adequate in vitro conditions in a way comparable to what happens in vivo (de Ritis et al., 1975). Toxic effects in this system were detected as an inhibition of in vitro development and morphogenesis of the small intestine from 17- and 18-day-old rat fetuses and with no effect on the culture of jejunum from 21-day-old fetuses or from newborn rats. These effects were detected after 48 h culture at concentrations of bread wheat peptides as low as 0.1 mg/ml of incubation medium. A fetal chick intestinal assay has also been proposed as a model for the study of the biological effects of gliadin related to those seen in coeliac disease by other authors (Mothes et al., 1985; Kocna et al., 1991; Cornell and Mothes, 1993 Cornell et al., 1994). The assay is based on duodena of 12-day-old chick embryos explanted and cultured for 2 days. The effects of peptides added to the culture medium were evaluated at a concentration of 0.5 mg/ml by determining sucrase activity in the explant and the medium; the decrease in sucrase activity is an indication of test substances able to act on undifferentiated enterocytes.
2.2. Isolated cells To investigate agglutinating activity of gliadin peptides, K562 (S) undifferentiated cells, a subline isolated from an outgrowth of a clone from a patient with chronic myelogenouss leukemia that responds to hemin or butyrate induction with a differential expression of the embryonal globin genes, have been extensively used (Auricchio et al., 1984a,b, 1990a,b; De Vincenzi et al., 1995, 1997, 1998). Control undifferentiated K 562 (S)
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cells exhibited a typical spherical shape with an average diameter of about 5 m and no significant evidence of cell clustering. After 30 min incubation in the presence of active cereal peptides at concentrations varying (according to the purity and activity of the peptides) between a few and 300 micrograms/ml of incubation medium, the cells were agglutinated with a peculiar appearance, and a tendency toward the formation of a continuous cell layer, which showed a high resistance to shearing and whirling forces. Giovannini et al. (1995, 1996, 1997) have also extensively investigated the effects of a number of peptides derived from cereal proteins on CaCo-2 cells at different days of culture in order to test undifferentiated cells (initial culture phase), actively undifferentiated proliferating cells (day 5) and more differentiated cells (day 19). In fact, within 19 days the CaCo-2 cell line, derived from a human colon adenocarcinoma, spontaneously undergoes some differentiation in vitro with enterocyte-like features both structurally (microvilli, tight junctions) and functionally (brush border-associated enzymes, transport across the surface membrane) (Pinto et al., 1983; Rousset, 1986). As far as undifferentiated CaCo-2 cells are concerned, toxicity has been evaluated mainly through growth inhibition of cells, whereas toxicity on more differentiated CaCo-2 cells has been assessed through colony forming ability, measurements of alkaline phosphatase activity (one of the specific enzymatic activities of the intestinal brush border cells) and a series of metabolic parameters such as DNA and RNA synthesis and (glyco)protein synthesis. Effects of active cereal peptides were detected at concentrations varying between 1 and 2 mg of peptides/ml of incubation medium.
3. Cereal peptides Tables 1–3 show the responsiveness of several in vitro systems to peptide fractions from different cereals.
3.1. Prolamin peptides from toxic cereals Digests of prolamines from cereals known to be non tolerated in coeliac disease (i.e. bread wheat, rye and barley) (Table 1) all induced in vitro inhibition of fetal differentiation, agglutination of K 562 (S) cells and toxic response in CaCo-2 cells. However, in vivo data suggesting toxicity of barley and rye in coelaic disease are very old, based only on a small number of patients and performed with diagnostic methods (i.e. fat absorption test, xylase test and biochemical indicators) that are often difficult to interpret. Data concerning toxicity of oats in coeliac patients are controversial in vivo but not in vitro. Weijers (1950) reported oats to be harmful for coeliac patients on the basis of stool fat studies on two patients, but Sheldon (1955) failed to confirm these results. Dissanayake et al. (1974) fed 4 patients 40–60 g oats per day for 4 wk and failed to demonstrate histologic changes, whereas Baker and Read (1976) challenged 12 patients with 60 g oat flour/day over 2–14 wk and demonstrated toxicity in 3 patients. Fifty-two adults with coeliac disease in remission followed for 6 months and 40 with newly diagnosed disease for 12 months were studied by Janatuinen et al. (1995); the group were treated with 50–70 g of oats/day and patients did not differ significantly in nutritional status, symptoms, or laboratory measures from the control group. Srinivasan et al. (1996) treated 10 coeliac patients in remission with 50 g oats cereal daily for 12 wk and reported no induction of histologic changes or changes in serologic immune markers. The absence of effects seen in some studies might be due to the fact that the prolamin content of oats is approximately five times less than that of wheat, rye, and barley; such a standpoint is also supported by the fact that toxicity of oat prolamin does not significantly differ from that of bread wheat gliadin when tested on rat fetal intestine, K 562 (S) and CaCo-2 cells (Table 1). Moreover, no difference has been detected between oats and bread wheat prolamins in their abilities to induce lymphocytic infiltration upon instillation in the coeliac rectal mucosa, and in vitro studies using organ culture of treated coeliac mucosa have shown (Mayer et al., 1997)
Table 3 Cereal proteins and peptides non toxic in vivo and in different in vitro test systemsa In vitro
Coeliac patients
Atrophic coeliac mucosa
Rat fetal intestine
K562(S) cells
CaCo-2 cells
n.t. n.t.
n.t. n.t.
n.t. n.t.
PT-and PTC-gliadin digest+mannan (5) PT-gliadin digest +N, N%, N¦-triacetylchitotriose or spermidine (5–6)
PTC-glutelin digest (7–8) PTC-albumin and globulin digest (7–9) PT- and PTC-gliadin digest+mannan (10) PT-gliadin digest +N, N%, N¦-triacetylchitotriose or spermidine (10–11)
PT-gliadin digest+mannan (10) PT-gliadin digest +N, N%, N¦-triacetylchitotrioseor spermidine or spermine (10, 13)
n.t. n.t.
2. Rice Whole flour (3–4) n.t.
n.t. n.t.
n.t. PTC-prolamin digest (12)
n.t. PT-prolamin digest (5)
n.t. n.t.
3. Maize Whole flour (3–4) n.t.
n.t. n.t.
n.t. PTC-prolamin digest (12)
n.t. PT-prolamin digest (5)
n.t. PT-prolamin digest (14)
1. Bread wheat Glutelins (1) Albumins (1, 2) and albumins+globulins (1) n.t. n.t.
a n.t., not tested; P, peptic; T, tryptic; C, cotazym. (1) Van De Kamer et al. (1953a); (2) Rubin et al. (1962b); (3) Van De Kamer et al. (1953b); (4) Anand et al. (1978); (5) Auricchio et al. (1990a); Auricchio et al. (1990b); (7) Auricchio et al. (1982); (8) Silano et al. (1981); (9) de Ritis et al. (1979); (10) Auricchio et al. (1987); (11) Auricchio et al. (1990b); (12) Auricchio et al. (1984a); (13) Auricchio et al. (1984b); (14) Giovannini et al. (1995).
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that oat prolamins are able to activate a T-cell mediated mucosal immune response in the coeliac patients. Auricchio et al. (1982) showed that the PTC-digest of durum wheat gliadins, as compared to the corresponding digest of bread wheat gliadins, was much less active in inhibiting recovery of and in damaging the in vitro cultured small intestinal mucosa from patients with active coeliac disease. At concentrations of 0.5 and 1.0 mg/ml of culture medium, the PTC-digest of gliadin fraction from durum wheat, did not inhibit in vitro occurring epithelial recovery in a number of flat biopsies and caused no change in several other cases. However, at a concentration of 2.5 mg/ml, this digest induced some mucosal damage in all three biopsies tested. The PTC-digest of prolamins from durum wheat were completely inactive when tested on the rat fetal intestine (Auricchio et al., 1979) and on CaCo-2 cells (Giovannini et al., 1996). Although the whole gliadin digests from 12 durum wheat varieties were unable to agglutinate K 562 (S) cells even when tested at a concentration of 14 mg/ml, all these digests were found to contain an actively agglutinating Fraction C also present in bread wheat gliadin digest (De Vincenzi et al., 1995). The lack of agglutinating activity of the whole durum wheat gliadin digests has been shown to depend on the presence in these digests of another peptide fraction (coded as Fraction B) that is eluted much earlier from the sepharose 6-B-mannan column and is able to inhibit the cell agglutinating activity of Fraction C. Such an active Fraction B is not present in bread wheat gliadin peptides (De Vincenzi et al., 1995). The cell agglutination inhibiting activity of Fraction B was shown to be associated with a peptide with molecular weight 1157.5 Da, which was synthesized in a high degree of purity with the solid phase method (De Vincenzi et al., 1997). The 1157.5 Da peptide, consisting of 10 amino acid residues with sequence H2N – gln – gln – pro – gln–asp–ala–val – gln – pro – phe – COOH, was able to prevent the agglutination induced by PT-digests of gliadins from bread wheat and of prolamins from rye, barley and oats as well as by the synthetic peptides homologous to A-gliadin amino acid sequences 31 – 43 and 44 – 55 (De Vincenzi et al., 1997). The ability to protect K 562 (S)
cells from agglutination was also exhibited to the full extent by all the peptides derived by the ‘1157.5 Da’ peptide by progressive deletions of five times the terminal carboxylic residue, whereas the sixth consecutive deletion yielded an inactive peptide (De Vincenzi et al., 1998). The smallest active peptide has molecular weight 614.6 Da and amino acid sequence H2N–gln–gln–pro–gln–asp– COOH. The activity of the 1157.5 Da and related peptides is likely due to an effective competition for the binding sites of toxic gliadin peptides on the cell surface.
3.2. Prolamin peptides from non toxic cereals Not only is there a high correlation between in vivo and in vitro results for toxic components, but also for the non toxic ones. Table 3 shows that all cereals and cereal fractions which are well tolerated in coeliac disease do not affect in vitro differentiation of rat fetal intestine and do not induce agglutination of K 562 (S) cells. Another important aspect which has been detected by means of in vitro systems is the protective action of several substances including mannan, N, N%, N¦-triacetylchitotriose and spermidine on intestinal atrophic mucosa, rat fetal intestine and K 562 (S) cells resulting in the complete removal of in vitro toxicity of bread wheat PT- and PTC-gliadin digest (Table 3). These substances were even able to reverse the expression of biological activities of toxic prolamin peptides on immature intestines and undifferentiated cells. The affinity chromatography studies of the whole gliadin digest on sepharose 6-B-mannan or sepharose 6-B-oligomers of Nacetyl-glucosamine, suggest that the agglutination–inhibition effect of these carbohydrates is due to a binding (sequestrating) activity of the toxic peptides (De Vincenzi et al., 1995), whereas the protection exerted by the polycationic polyamines can be more likely attributed to the effective competition for the binding sites of toxic gliadin peptides on the cell surface.
3.3. A-gliadin fragments As far as the amino acid sequence responsible for toxicity of the bread wheat A-gliadin, the
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fragments 31–43 and 44 – 55 are the only ones which have been tested and found to be toxic in coeliac patients and active on atrophic and treated coeliac mucosa as well as on fetal intestines and undifferentiated cells (Table 2). In vitro toxicity of synthetic peptides homologous to A-gliadin amino acid sequences 31 – 55, 31 – 43 or 44 – 55 was completely prevented by mannan or N,N%-diacetylchitotriose or N, N%, N¦-triacetylchitotriose (De Vincenzi et al., 1994). As already pointed out by de Ritis et al., (1988), peptides ‘31 – 43’ and ‘44 – 55’ contain the amino acid sequence – gln – gln – gln–pro– and/or that – pro – ser – gln – gln – (Fig. 1), which are believed to be essential for the expression of toxicity. It is striking the similarity of these two sequences with that of H2N – gln – gln – pro–gln–asp–COOH, which is able to prevent the agglutination of K562(s) cells induced by the toxic peptides. The amino acid sequence – gln – gln–gln–pro– is also present in the peptide ‘31 – 49’ which is toxic in coeliac disease and active on the in vitro mucosa, but has not been tested for cell agglutination so far. De Vincenzi et al., (1998) have recently shown that the agglutinating activity on K 562 (S) cells is also significantly exhibited, although to a lower extent than for peptides 31–43 and 44 – 55, by the very small peptides ‘31–37’ (H2N – leu – gly – gln – gln – gln – pro – phe–COOH) and ‘49 – 55’ (H2N – phe – pro – ser – gln–gln–pro–tyr – COOH). On the other hand, the results on the toxicity of the amino acid sequences concerning the Agliadin fragments ‘1 – 30’ and ‘206 – 217’ are more controversial (Table 2).They may depend on the different ways in which these peptides are split into smaller peptides by peptidases operating in the test systems chosen by the different authors (De Vincenzi et al., 1997, 1998; Silano and De Vincenzi, 1998), but further investigation is needed to clarify this issue.
4. Conclusion The data available in the literature clearly show that in vitro studies on fetal intestinal mucosa as well as on undifferentiated K 562 (S) cells and CaCo-2 cells have proven to be extremely useful
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in identifying toxic and non toxic cereals and cereal components as well as elucidating the fragments of bread wheat A-gliadin which are toxic in the so called gluten-sensitive enteropathies. Studies with these in vitro systems have a key role in clarifying the molecular mechanisms of glutensensitive enteropathies. The very high correlation existing between data obtained with the different in vitro test systems and those available in vivo (Tables 1–3) strongly indicate that a screening method based on the ability of peptic–tryptic protein digests to induce agglutination of K 562 (S) cells provides a very sensitive and reliable system to assess the potential risk for susceptible individuals of geneticallymodified plant foods and novel foods. This cell agglutination test is very fast, simple to perform and inexpensive; moreover, it shows high sensitivity to a few micrograms of active peptides/ml of culture medium. Not only is the test able to easily identify cereal peptides toxic in coeliac disease (i.e. those from bread wheat, barley, rye and oats prolamins) and those which are well tolerated (e.g. from maize and rice), but it is also able to identify geneticallymodified bread wheat cereals which have a lower content of toxic peptides (De Vincenzi et al., 1996). An additional degree of confidence for both positive and negative findings would be added by a preliminary fractionation step of the PT-protein digest on a sepharose 6B-mannan column and separate testing with K 562 (S) cells of the obtained fractions; in fact, through such an approach it is possible to identify those cereals such as durum and Monococcum wheats that contain both toxic and protective peptides. When needed, confirmation can be achieved by testing fractions of interest in vitro with atrophic coeliac intestinal mucosa.
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