Papaya latex enzymes capable of detoxification of gliadin.

Assay of fractions obtained from ion exchange chromatography of papaya latex on CM Sephadex-C50, size exclusion chromatography on Sephacryl S-300 and size exclusion HPLC have provided an insight into the relative contributions of the gluten-detoxifying enzymes present. This outcome has been achieved by the use of the above chromatographic techniques, coupled with assays of lysosomal activity, protease activity using benzylarginine ethyl ester (BAEE) as substrate, prolyl endopeptidase (PEP) using glycylprolylnitroanilide and a prolidase assay using acetylprolylglycine. These procedures have shown that the activity in papaya latex is due largely to caricain and to a lesser extent, chymopapain and glutamine cyclotransferase. The presence of caricain and these other enzymes was confirmed by mass spectrometry of trypsin digests of the most active fraction obtained by CM Sephadex-C50 chromatography and size exclusion HPLC. Fractions rich in caricain would be suitable for enzyme therapy in gluten intolerance and appear to have synergistic action with porcine intestinal extracts.

A unified hypothesis of coeliac disease with implications for management of patients.

This mini-review presents the research carried out within the context of two of the main hypotheses of the aetiology of coeliac disease. The enzymopathic hypothesis of the disease has been placed clearly as the underlying deficiency causing increased levels of toxic peptides, while the immunological hypothesis has been implicated in the pathogenesis of the disorder as the result of the action of undigested peptides in the small intestine. As a consequence, we are proposing a unified hypothesis of coeliac disease, which takes into account the actions of these undigested peptides through their direct cytotoxicity and their immunoactivity. At the same time, work aimed at defining some of these biologically active peptides, which could be said to be involved in the aetiopathogenesis of coeliac disease, will be reported. The review also focusses on the use of enzyme therapy for management of the disease, which when used in conjunction with the gluten-free diet, offers a safeguard against damage to the small intestine caused by small amounts of gluten.

The use of extended amino acid motifs for focussing on toxic peptides in coeliac disease.

Cereal prolamins of wheat, rye and barley are the major proteins that have been implicated in toxicity in patients with coeliac disease. The gliadins of wheat are the best characterised with the identification of toxic peptides from rye and barley not as well advanced. This study has employed extended motifs, based on the known toxic motifs are derived from the sequence of A-gliadin, to search protein databases for matches with coeliac-toxic cereals. The results obtained have provided pointers to specific regions in rye and barley prolamins, which have received little attention in in vitro and in vivo studies of toxicity in coeliac disease. The results obtained in this study indicate that the size of the extended motif is critical when searching for coeliac-toxic cereals using protein databases. Extended motifs that are common to all three coeliac-toxic cereals and found in active wheat gliadin peptides are QQPYP, PQQPY and QQQPFP.

Extraction of cereal prolamins and their toxicity in coeliac disease.

Simple extraction of prolamins from cereal flour using 70% aqueous ethanol leads to co-extraction of lipids and other secondary products. Treatment of the crude extract with an excess of pure ethanol resulted in the removal of the majority of these compounds. Prolamin extracts obtained following ethanol precipitation showed little difference to products of more complex, multi stage, selective extractions, when compared using MALDI-TOF mass spectrometry. Toxicity tests on enzymic digests of ethanol-precipitated prolamins from coeliac-toxic cereals, coeliac-non-toxic cereals and oats were carried out using a rat liver lysosome assay. The prolamins from the ethanol-precipitated extracts showed greater activity than those extracted using only 70% ethanol for extraction. As the ethanol precipitation method is simple and provides a prolamin extract of sufficient purity for further evaluation, this procedure has been adopted as an alternative to more tedious procedures for preparation of cereal prolamins.

Structure-activity relationships in coeliac-toxic gliadin peptides.

Computer modelling studies of two groups of biologically-active peptides derived from A-gliadin indicated that the most likely structures were a-helical ones, in the case of serine-containing peptides, and random peptides coil types featuring beta-turns, in the case of proline-rich, tyrosine-containing peptides. The serine-containing group of peptides appear to be essentially cytotoxic in animal models of coeliac disease, whilst the tyrosine-containing group have the capacity to initiate damaging immunological reactions in patients with coeliac disease. Both types of activity in coeliac disease are only possible if there is defective digestion of the active peptides, as mucosal digestion studies indicate. In the case of the serine-containing peptides, activity of the peptides is linked to the presence of PSQQ and also probably QQQP motifs. With the tyrosine-containing peptides, sequences such as QQPY and/or QPYP are associated with immunological activity and hence toxicity.

Partial in vitro digestion of active gliadin-related peptides in celiac disease.

Peptides corresponding to residues 75-86 (RPQQPYPQPQPQ) and 75-85 of the A-gliadin structure, which were shown to be active in an animal model of celiac disease, were digested in vitro with small intestinal mucosa from children with celiac disease in remission and with mucosa from normal children. The products of digestion were separated into two fractions by gel permeation chromatography. Undigested residues (Mr > 400 fraction) from both peptides contained mainly glutamine, proline, and tyrosine, while the digested materials (Mr < 400 fraction) contained mainly proline, glutamine and arginine. Much larger amounts of undigested peptides were obtained from digestion with celiac mucosa than from normal mucosa. The results with peptide 75-86 indicated that the octapeptide 77-84 (QQPYPQPQ) was the main residual component and this peptide was shown to be active in the assay. Peptide 77-84 was also obtained as a residue from digestion of peptide 75-85, together with heptapeptide 77-83. The results lend further support for a primary mucosal defect in celiac disease and indicate that residual peptides in the small intestine of patients with the disease still retain appreciable toxicity.

Coeliac disease: A review of the causative agents and their possible mechanisms of action.

This review outlines the main theories for the aetiology of coeliac disease and presents in more detail the work carried out in an attempt to define the nature of the toxins in wheat gluten. This includes the results of work with synthetic peptides and a discussion of the various assays used.Evidence is presented for an enzyme deficiency in coeliac disease which leads to abnormally high concentrations of certain peptides in the small bowel. These peptides can bring about damage by direct toxic action and by immunological mechanisms.Investigations of activity of synthetic peptides based on the structure of Agliadin appear to be making good progress and point to certain regions of that molecule as being responsible for toxicity. Certain key sequences of amino acids appear to be of fundamental importance to these studies.

In vitro mucosal digestion of synthetic gliadin-derived peptides in celiac disease.

Two celiac-active synthetic peptides derived from the A-gliadin structure corresponding to residues 8-19 (LQPQNPSQQQPQ) and to 11-19 were digested in vitro with small intestinal mucosa from children with celiac disease in remission and from normal children. The products of digestion were separated into two fractions on the basis of M(r) < 400 and M(r) > 400 by gel permeation chromatography and subjected to amino acid analysis. After digestion of the dodecapeptide with celiac mucosa, 71 +/- 14% (molar) of the total digestion products remained in the M(r) > 400 fraction. Glutamine, proline, serine, and asparagine were the major amino acids present. Glutamine, proline, and leucine were the major amino acids in the M(r) < 400 fraction. The M(r) > 400 fraction from the celiac mucosal digestion of the nonapeptide was of similar composition to the corresponding fraction from the dodecapeptide and represented 78 +/- 15% of the total products. Digestion of the two peptides with normal mucosa gave lower amounts of products in the M(r) > 400 fraction, but they were of similar composition to the corresponding fractions from the celiac mucosal digestion. Peptides such as NPSQQP and QNPSQQQ may be present in the M(r) > 400 fractions since glutamine and proline are present in the approximate ratio of 2:1, respectively. The results indicate a defect in the mucosal digestion of peptides which are active in an animal model of celiac disease.

Studies of in vitro gamma-interferon production in coeliac disease as a response to gliadin peptides.

The effects of certain fractions of a peptic-tryptic-pancreatinic (PTP) digest of wheat gliadin and of synthetic peptides on the production of gamma interferon (gamma-IFN) in cultures of whole blood from adult patients with coeliac disease (CD) have been studied using a sandwich enzyme immunoassay. The most active peptides were fraction 9, its two principal sub-fractions (sub-fractions 1 and 2) and a synthetic peptide of sequence RPQQPYPQPQPQ (peptide V) corresponding to the principal peptide obtained from reversed-phase HPLC of fraction 9. Results with blood from the control group of subjects also indicated some response to these antigens, in most cases at similar levels to those observed with the coeliacs. Fraction 1 of the PTP digest and the other nine synthetic peptides tested were inactive with both coeliacs and controls. These results are in agreement with the results of in vivo and in vitro toxicity tests. They provide evidence of a link between toxicity and cell-mediated immune response in CD, and suggest that peptide V represents one of the active parts of the gliadin molecule.

Effects of insulin-like growth factor binding protein complexes on human fibroblast growth.

Insulin-like growth factor binding protein (IGF-BP) complexes containing either IGF-I or IGF-II were purified from human plasma and coincubated with human foreskin fibroblasts. Incorporation of [3H]thymidine into fibroblasts was measured to determine the mitogenic effects of the IGF-BP complexes in comparison with purified recombinant IGF-I or IGF-II. The IGF-I-BP complex was shown to have no stimulatory activity when added to cultures at a comparable Non Suppressible Insulin-Like Activity (NSILA) dose level to that of purified recombinant IGF-I which produced maximal stimulation. In contrast, the IGF-II-BP complex produced a similar stimulatory activity to that of purified recombinant IGF-II when added at comparable NSILA doses. NSILA-p, a contaminating factor in the purified preparations of IGF-BP complexes, was shown to have no effect on fibroblast growth. This study suggests that plasma IGF binding proteins may have a differential effect on the stimulatory activities of IGFs I and II on human fibroblasts.

The activity of wheat gliadin peptides in in vitro assays for coeliac disease.

A fraction of a peptic-tryptic-pancreatinic digest of wheat gliadin (fraction 9), known to be toxic to individuals with coeliac disease, together with synthetic peptides containing key gliadin sequences, were tested for their effects on foetal chick intestine and on rat liver lysosomes. Fraction 9 and a dodecapeptide corresponding to residues 75-86 of A-gliadin (RPQQPYPQPQPQ) were the only peptides to display appreciable activity in both assays. A synthetic hexapeptide corresponding to residues 77-82 was only weakly toxic to lysosomes and non-toxic to chick duodenum. A decapeptide corresponding to residues 76-85 was non-toxic in both assays. Two serine-containing peptides containing the key sequence PSQQ were also tested but were found to be non-toxic, as was the hexapeptide PSQQQP. The results suggest that the key sequences QQQP and PSQQ are not sufficient by themselves to cause activity. Further tests on synthetic peptides will be necessary to define the sequences of highest toxicity.

Effects of free and bound insulin-like growth factors on proteoglycan metabolism in articular cartilage explants.

This article describes the effects of bound forms of insulin-like growth factors (IGFs) on proteoglycan metabolism by bovine articular cartilage in explant culture. When these growth factors were added to articular cartilage explants complexed with their native serum binding proteins (BPs), both IGF-I-BP complex and IGF-II-BP complex stimulated proteoglycan synthesis to different degrees over a 3-day period. When added to the medium of cultures of articular cartilage over 5 days, IGF-II-BP complex induced high rates of synthesis and low rates of catabolism of proteoglycans, giving rise to tissue levels of proteoglycan similar to those observed in fresh tissue. When articular cartilage was maintained in culture with the same concentration of IGF-I-BP complex, tissue levels of proteoglycans fell over the culture period because of lower rates of proteoglycan synthesis. Analysis of the proteoglycans synthesized by articular cartilage in the presence of free or bound IGF-I or IGF-II showed that these growth factors stimulated the rate of synthesis of the large proteoglycan species present in cartilage but did not affect the synthesis of the small proteoglycans.

Mucosal digestion studies of whole gliadin fractions in coeliac disease.

Wheat gliadin was chromatographed on carboxymethyl cellulose and the major fractions (alpha-, beta- and gamma-gliadin) digested with pepsin, trypsin and pancreatin. The digests from each fraction were chromatographed on SP Sephadex and the fractions corresponding to the one previously shown to be toxic by oral feeding tests (fraction 9) were incubated with homogenates of small intestinal mucosa from children with coeliac disease in remission and from normal children. Electrophoretic patterns of the coeliac mucosal digests of fraction 9 from the alpha-, beta- and gamma-gliadin fractions all showed that appreciable amounts of undigested peptides were present in each of the three digests. In contrast, patterns of all digests prepared using normal mucosa showed only small amounts of undigested peptides. The results suggest that all three of these whole gliadin fractions contain substances which may be causative agents in coeliac disease.

Amino acid composition of peptides remaining after in vitro digestion of a gliadin sub-fraction with duodenal mucosa from patients with coeliac disease.

Sub-fraction 9-2, derived from fraction 9 of a peptic-tryptic-pancreatinic digest of wheat gliadin and previously shown to be toxic to individuals with coeliac disease, was digested in vitro with duodenal mucosa from patients with coeliac disease in remission and with mucosa from normals. Digestion products from coeliac mucosa caused damage to rat liver lysosomes in contrast to digestion products from normal mucosa which had practically no effects on the lysosomes. Ion exchange chromatography of the digestion products, followed by gel permeation chromatography to remove tissue proteins and amino acids allowed the separation of small peptides. Purification of the peptide residues by reversed-phase HPLC on a C18 column resulted in four subfractions, two of which were cytotoxic to rat liver lysosomes. Amino acid analysis of these latter peptide fractions showed that they were both rich in glutamine/glutamic acid, proline, serine and tyrosine. The results support the hypothesis of defective mucosal digestion as being the aetiology of coeliac disease and suggest that the causative agents are small peptides of apparent Mr congruent to 700 Da, with amino acid analysis corresponding to (Glx)3, (Pro)2, Ser and (Glx)3, (Pro)2, Tyr. These hexapeptides are likely to be H-Pro-Ser-Glx-Glx-Glx-Pro-OH and H-Glx-Glx-Pro-Tyr-Pro-Glx-OH.

Intestinal mucosa of celiacs in remission is unable to abolish toxicity of gliadin peptides on in vitro developing fetal rat intestine and cultured atrophic celiac mucosa.

Subfraction 2R of fraction 9 from a peptic-tryptic-pancreatic digest of wheat gliadin is known to be toxic in vivo to celiac patients. We have found that fractions 9 and 2R inhibit the in vitro development of fetal rat intestine and the increase of enterocyte height occurring in organ culture of atrophic celiac mucosa (0.1-0.5 mg/ml medium). Other peptide fractions of the gliadin digest are devoid of such in vitro effects. Subfraction 2R, after incubation with morphologically normal small intestinal mucosa of celiacs in remission and ultrafiltration, was still very active in both culture systems at low concentration (0.1 mg/ml); on the contrary, subfraction 2R was inactivated after incubation with normal mucosa. These results are compatible with the hypothesis that there is a mucosal defect in handling gliadin peptides in celiac disease, and suggest that there is either a primary (or secondary) enzyme deficiency or some other mechanism operating in the intestinal mucosa of celiac patients in remission.

Application of preparative reversed-phase high-performance liquid chromatography to the isolation of insulin-like growth factor II from human serum.

Substantially purified insulin-like growth factor II (IGF-II) was prepared from human serum. Initial enrichment using ion-exchange chromatography on DEAE Sephadex A50, followed by gel permeation chromatography on Sephadex G-75 in 1% formic acid produced material suitable for application to a preparative reversed-phase high-performance liquid chromatographic (HPLC) column containing LiChroprep RP-18. The latter step gave about 90-fold purification with a recovery of about 70% IGF-II bio-activity. Finally, a small reversed-phase HPLC column achieved a 17-fold purification with similar yield of activity. Overall, the four steps gave IGF-II of about 90% purity in yield of 12%.

Preferential association of the insulin-like growth factors I and II with metabolically inactive and active carrier-bound complexes in serum.

Ion-exchange chromatography of serum on DEAE-Sephadex A-50 using a stepwise NaCl gradient showed that complexes enriched with insulin-like growth factors I and II (IGF-I and IGF-II) could be preferentially eluted. A fraction eluted with 0.075 M-NaCl preferentially contained immunoreactive IGF-I with peak levels appearing in fractions of Mr approx. 110,000. The IGF-I-binding protein complex itself had low bioactivity as measured in a non-suppressible insulin-like (NSILA) bioassay. On conversion to free IGF-I by gel-permeation chromatography on Sephadex G-75 in 1% formic acid, however, the IGF-I did express its intrinsic NSILA bioactivity. In contrast, an IGF-II-enriched complex was eluted from the DEAE-Sephadex with 0.15 M-NaCl. Practically all of the recovered NSILA of the original serum was present in this fraction, in the Mr range 70,000-300,000 with a peak of 150,000. Chromatography on Sephadex G-75 in 1% formic acid separated this high-Mr NSILA into low-Mr (less than 15000) IGF-II and high-Mr acid-stable NSILA-P. The high-Mr IGF-II complex bound to concanavalin A-Sepharose, suggesting that it was a glycoprotein. The results confirm previous reports that a large portion of the NSILA of whole serum can be accounted for by a biologically active acid-dissociable complex. These data show for the first time that this active complex consists of an IGF-II-preferring binding protein. In direct contrast, the IGF-I-preferring complex does not express NSILA bioactivity until the IGF-I is liberated through acidification. The presence of a metabolically active IGF-II complex in serum raises questions as to its possible biological role in the adult.

Differentiation between carrier-bound forms of non-suppressible insulin-like activity (NSILA) in serum.

Gel-permeation chromatography of serum on Sephacryl S-300 at pH 7.4 has shown that NSILA was detected over a range of MW 50,000-400,000 with a peak at about MW 200,000. When fractions from the above chromatography were rechromatographed on Sephadex G-75 at pH 2.4 major amounts of acid-stable NSILA were found in a fraction of MW 200,000-600,000 (77% of the fraction NSILA or 28% of total serum NSILA). Further evidence was obtained for the presence of an active acid-dissociable complex in serum. This was present in both the MW 100,000-200,000 and 35,000-100,000 fractions and corresponded to 37% of total serum NSILA. Con-A Sepharose affinity chromatography of the serum fractions from Sephacryl S-300 chromatography, followed by Sephadex G-75 chromatography under acid conditions, showed that the acid-stable complex was consistently found in weakly bound materials. The active acid-dissociable complex was found in the bound fractions, especially in the Sephacryl S-300 pool of MW 35,000-100,000. Low MW NSILA (less than 15,000) was also released on acid treatment from an otherwise inactive high MW complex(es) of MW 35,000-600,000. This complex was not bound by Con-A Sepharose.

Isolation of insulin-like growth Factors I and II from human plasma.

Insulin-like growth factors I and II have been isolated from Cohn fraction IV-1 of human plasma using gel permeation chromatography, ion exchange chromatography, reversed phase chromatography, isoelectric focusing (IEF) and high performance liquid chromatography(HPLC). IGF I of specific activity 89 U/g, as measured by the isolated rat adipocyte assay, and IGF II, of specific activity 78 U/g, were obtained in yields of 16 micrograms and 34 micrograms respectively per 100g of Cohn fraction. Although this process yields IGF I which is contaminated with IGF II (due to the relatively large amount of the latter present in the original plasma), the IGF II preparations appear to be relatively free from IGF I. This separation was mainly achieved with IEF since the two factors elute close together on HPLC. Nevertheless, HPLC is important for their subsequent purification. The process is thus especially suitable for the preparation of IGF II and appears to give better yields than those obtained by earlier methods which used acid-ethanol extraction, gel permeation chromatography and polyacrylamide gel electrophoresis.

Gliadin subfraction skin test for the diagnosis of coeliac disease.

Skin hypersensitivity to a gliadin subfraction (Fraction 3) was tested in patients with coeliac disease, patients with inflammatory bowel disease, and controls. Those with coeliac disease showed substantially greater hypersensitivity than controls, but there was no significant difference between the frequency of hypersensitivity in patients with coeliac disease and in those with inflammatory bowel disease. Fraction 3 did not provide a sufficiently specific skin reaction to be of use in the diagnosis of coeliac disease.