Oral Enzymatic Supplement: An Effective Approach For Treating Gluten Intolerance

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Shashi Meena, Shivani Nagar & Archana watts
Division of Plant Physiology, ICAR-Indian Agricultural Research Institute, Pusa Campus, New Delhi-110012.
E-mail: meena7shashi@gmail.com

Abstract
Celiac disease (CD) is an autoimmune enteropathy that is triggered by partially hydrolysed gluten proteins. A life‐long adherence to gluten‐free diet is the only effective treatment currently available for CD. Gluten proteins present in wheat, rye and barley cereals are the main stimulating factor for this disease. There are three main causes of celiac disease: the environmental trigger (gluten), genetic susceptibility, and unusual gut permeability. Gluten intolerance affects genetically predisposed individuals carrying the prerequisite genetic markers HLA-DQ2 or -DQ8. Gluten is a heterogeneous mixture of insoluble proteins i.e. gliadins and glutenins. It is rich in proline and glutamine residue content which renders the gluten proteins largely inaccessible to human proteases of the gastrointestinal tract driving the abnormal immune intestinal response. A 33-mer from α-gliadin is currently considered the most immunogenic peptide and are resistant to gastrointestinal digestion. Unfortunately, a majority of patients have difficulty complying with this diet, adversely affects the quality of patient life, and the response to therapy is poor. Therefore, efforts are going on to explore alternative approaches and develop novel therapies. Based on mechanisms of action, these therapies may be classified into five broad categories: Engineering gluten-free grains, decreasing intestinal permeability by blockage of the epithelial zonulin receptor, inducing oral tolerance to gluten with a therapeutic vaccine, microwave thermal treatment of hydrated wheat kernels and degrading immunodominant gliadin peptides using probiotics with endopeptidases or transglutaminase inhibitors. Oral therapy involving exogenous prolylendopeptidases able to detoxify ingested gluten was therefore propounded as an alternative effective treatment to thediet. Developments of new enzymes or enzymatic cocktails offer potentially more potent therapeutic tools and providenew hope for enhanced, lifelong celiac disease management with improved patient compliance and better quality of life.

Keywords: gluten, gliadin, endopeptidases, translutaminases, immunodominant, autoimmune

Introduction
Celiac disease (CD) is a chronic inflammatory immune mediated small intestinal disorder. It is also known as gluten sensitivity, gluten intolerance, celiac sprue, gluten sensitive enteropathy and non-tropical sprue.The name ‘celiac’ is derived from the Greek for ‘suffering in the bowels’. It is mainly triggered by the partially digested gluten proteins in the genetically susceptible individuals, which results in villous atrophy, crypt hyperplasia and mucosal inflammation (Rey et al., 2016). Celiac patients may shows wide range of symptoms range from diarrhea, constipation, vomiting, malnutrition, or failure to thrive, to chronic fatigue, joint pain, anemia, osteoporosis, or migraines (Lammers et al., 2014). CD can occur at any age and can affect a variety of organ systems. Early recognition and treatment of CD are important to prevent complications such as malnutrition, osteoporosis, infertility, and gastrointestinal malignancies (Bakshi et al., 2012).

The sequences convey poor overall digestion kinetics, generating peptides of 30–40 amino acid residues in length that resist further digestion by both intestinal exo- and endoproteases (Fasano et al., 2003) . A fraction of these products, primarily from α and γ-gliadin, have affinity for human leukocyte antigen (HLA) DQ2 and DQ8, which are MHC class II molecules associated with over 90% of CD patients (Kaukinen et al., 2014). The peptides are large enough to span multiple antigenic regions, and present glutamine residues for enzymatic deamidation in the celiac mucosa. The inflammatory response is significantly amplified by this deamidation, as HLA affinity is increased by the conversion of glutamine to glutamate(Sapone et al., 2003).

Epidemiology
Earlier it was thought that CD was most prevalent in those areas where gluten containing grains were staple food. Over time, there is an increasing incidence of CD has been observed in areas that were previously considered as CD-free. This may be occurred due to global changes in the diet, mostly related to higher consumption of wheat-based products (eg, pasta, pizza). Recent studies showed that the overall prevalence of CD in general population is more in the Western countries, as In Europe and the United States, the mean frequency of CD in the general population is approximately 1%. The prevalence of CD is as high as 2% to 3% in Finland and Sweden, whereas it is only 0.2% in Germany, although these areas share a similar distribution of causal factors (level of gluten intake and frequency of HLA-DQ2 and -DQ8).In the Northern part of the India, The frequency of CD seems to be higher, so called “celiac belt,” a finding that is at least partially explained by the wheat-rice shift from the north to the south (Catassi et al., 2014).

Gluten protein
Gluten is a heterogeneous mixture of insoluble storage proteins of wheat, barley and rye, which are deposited in the endosperm of the developing cereal grain. It is comprised mostly of glutenin and prolamines, and when mixed with water it generates molecular networks that impart useful viscoelastic properties to flour dough (Green, 2005). Of these, prolamines are the gluten componentsthat are implicated in CD; they are found variously in different grains as gliadins in wheat, secalins in rye, a mix of both in triticale, hordeins in barley, and avenins in oats. In wheat, gliadins are in turn composed of sub-fractions–α/β, γ, ω1, ω2 and ω5. The α-gliadin sub-fraction has the maximum immunogenicity in CD, contributing the most to toxic epitopes upon digestion (Yoosuf and Makharia, 2019).It is rich in proline (15%) and glutamine (35%) residue content which renders the gluten proteins largely resistant/ sterically inaccessible to human proteolytic enzymes of the gastrointestinal tract driving the abnormal immune intestinal response (Ludvigsson et al., 2013).

Immune response to gluten
Digestion of gluten protein by luminal proteases results in larger oligopeptides which are immunogenic in CD and some of them include, the most immunotoxic, 33- mer peptide 57–89 (with the amino acid sequence LQLQPFPQPQLPYPQPLPYPQPQLPYPQPQPF (Yoosuf and Makharia, 2019). The partially digested gliadin peptides enter the lamina propria of the small intestine by a transporter protein called zonulin. Gliadin uptake by zonulin is known as paracellular pathway. Itis structurally similar to the zona occludens toxin associated with Vibrio cholera and has been observed to be a controller of epithelial permeability. Through this pathway, gliadin products bind to the chemokine receptor CXCR3 on the luminal side of the intestinal epithelium. This interaction in turn enhances the formation of zonulin, which ultimately relaxes the interepithelial tight junctions via PAR2/EGFR (Protease activated receptor 2/Epithelial Growth Factor Receptor) pathway. This increased permeability leads to influx of gliadin. An alternative pathway implicated in gliadin uptake is the transcellular pathway. This involves secretory Immunoglobulin A (IgA) that co-localizes with another molecule, the CD71 to promote transcellular uptake of gliadin products into the lamina propria (Fasano et al., 2011). CD71 is the transferrin receptor, but is found to be expressed in higher amounts on the luminal aspect of intestinal epithelial cells in CD. The tissue transglutaminase-2 (tTG-2) enzyme modifies the digested gluten immunogenic peptides that have entered the mucosa, by deamidating their glutamine residues to glutamate. These negatively charged glutamate side chains have a higher potential to be recognized as immunogenic. Also, by virtue of the relatively large size of these partially digested proline containing fragments, and the negative charge of glutamate, they tend to settle and form bonds with the neighboring extracellular matrix, resulting in immobilized neoepitopes. The formation of these bonds may be directly catalyzed by the tTG

Ultimately, all the gluten- derived antigens are recognized and processed by the HLA-DQ2 and -DQ8 bearing antigen presenting cells (APCs), which activate CD4+ helper T cells, setting off an inflammatory cascade. Activated CD4+cells release cytokines like Interferon- γ (IFN-γ) and Tumor Necrosis Factor- a (TNF-α), thereby further enhancing the permeability and facilitating a self-propagating mechanism of gliadin uptake. T-cells also activate B-cells which mature to produce antibodies against gluten and tissue transglutaminase-2 (celiac antibodies). These antibodies further contribute to the ensuing immune-mediated enteropathy. Aswell, the immunotoxicity is mediated through the increased production of interleukin-15 (IL-15) by the intestinal epithelial cells and the intraepithelial lymphocytes (IEL). This interaction ligand-receptor pairs activates the IELs and triggers them to kill epithelial cells through perforins, and other mechanisms (Yoosuf and Makharia, 2019).

Oral enzymatic therapy for celiac disease
A gluten-free diet (GFD) is the primary and obvious treatment option for CD patients, as it improves gastrointestinal symptoms within a few weeks. If patients strictly follow this diet the risk of concomitant autoimmune disease symptoms is reduced. However, many patients fail to comply with this lifelong adherence to this restrictive diet, as gluten is a common ingredient in diets throughout the world, and gluten-free foods are not widely available. Therefore, maintaining a truly gluten-free status is both difficult and costly (See et al., 2015). Gluten-free foods are also more expensive than their gluten-containing counterparts. Compliance issues and hidden gluten contamination produce a constant low-level stimulation that remains the norm for a very large fraction of CD patients.Supplemental or even alternative treatment options are desirable but the bar is necessarily high (Mahadev et al.,2015). Alternative therapies must be safe, and at least as effective as a GFD in reducing both inflammation and pain. A variety ofstrategies are being considered based on our improved understanding of disease mechanism (McCarville et al., 2015), but successful alternatives to a GFD have yet to emerge (Janssen et al., 2015).Therefore, efforts are going on to explore alternative approaches and develop novel therapies. Based on mechanisms of action, these therapies may be classified into five broad categories: genetically modified wheat, decreasing intestinal permeability by blockage of the epithelial zonulin receptor, inducing oral tolerance to gluten with a therapeutic vaccine, microwave thermal treatment of hydrated wheat kernels, enzymatically modified wheat gluten, use of translutaminase and cathepsin inhibitors, HLA blocker, polymeric binding and degrading immunodominant gliadin peptides using probiotics with endopeptidases or transglutaminase inhibitors (Yasoof and Makhari, 2019).

One promising approach involves enzyme supplementation of the gastrointestinal tract, to avoid the induction of immune responses by partially digested gluten peptide is the key antigene that binds to HLA and stimulate inflammatory cascade (Bethune et al., 2012).A small number of candidates have been tested for such purposes, mostly involving prolyl endoproteases (PEPs)or prolyl oligopeptidases (POPs). Two options in advanced testing are AN-PEP19–21, a prolyl endoprotease from Aspergillus niger, and ALV00322, a combination of a POP from Sphingomonas capsulate and a glutamine-targeting cysteine endoprotease (Gass et al., 2007). It has been shown that they are well tolerated and work perfectly at low pH. Based on clinical studies it has been demonstrated that these enzymes can attenuate intestinal injury (Seigel et al., 2012).

The glutenase EP-B2 (endoprotease B, isoform 2) is a glutamine specific peptidase and their active sites contain Cys-His-Asn catalytic triad.They serve to digest hordein, the analog of gliadin and secreted naturally in the acidic endosperm of germinating barley seeds (Hordeumvulgare). Glutenase EP-B2 shows maximum active at low pH and it is resistantto pepsin but it lysed at physiological concentrations of trypsin.This enzyme recognizes sequence QXP, which is highly abundant in the 33-mer as well as other immunotoxic gluten compounds.These factors make it a good option for therapy in CD as a gastricactive enzyme (Bethune et al., 2006).

Proline specific endoproteases (PEP) from the microbes Flavobacterium meningosepticum (FM-PEP), Sphingomonas capsulata (SC-PEP), and Myxococcus xanthus (MX-PEP) have also been investigated as potential glutenases. They are serine proteases and each has a larger b-propeller domain and a smaller,N-terminal catalytic domain that breaks the peptide bond of proline residues at the carboxy end of the gluten protein (Gass et al., 2007). Activity of SC-PEP extends into the acidic range of pH, and is by and large, unaltered in the presence of pepsin. However,the other two PEPs are lysed by pepsin. Furthermore, FM-PEP is inactivated by the small intestinal enzyme trypsin in the presence of bile acids. MX-PEP too is unstable in the presence of bile salts (Gass et al., 2007). Considering these limitations, SC-PEP has been explored as a more favorable candidate for therapy in CD. In order to improve its action further, mutant variants (variant 10,224 or 10,230) of SC-PEP have been developed which have 200-fold higher resistance to pepsin and 20% higher turnover at acidic pH (Ehren et al., 2007).

While SC-PEP has high specificity for gluten immunogenic epitopes, it has relatively low specificity for long peptide sequences. This is because the larger b-propeller domain preferentially allows smaller gliadin fragments into the active site, and therefore, is unable to completely eliminate the immunogenic gliadin peptides. This limitation could be overcome by combining it with other enzymes with complementary specificity. Combination of EP-B2 with SC-PEP, for instance has been explored for application in CD. The EP-B2 efficiently digests the 33 mer peptides into smaller, not necessarily non-toxic proline containing fragments. The PEP complements its action by digesting the proline- glutaminelinks in these smaller oligopeptides, thereby reducing their immunotoxicity (Gass et al., 2007).

Several clinical trials have been conducted to assess the effectiveness of this enzyme mixture in making dietary gluten safe for patients with CD. One of the most prominent of these has been using the enzyme cocktail ALV 003, now known as latiglutenase. Latiglutenase is a 1:1 combination of different types of enzymes such as EP-B2, or ALV 001 plus PEP, or ALV 002.It has been seen that ALV003group showed significantly lower immunological activation, as found in peripheral T cell IFN-γ responses to gliadin (TyeDin et al., 2010).

Kuma030 is an engineered glutenase developed by the Institute for Protein Design, University of Washington. Before its development, the researchers identified Kumamolisin-As (KumaWT), a naturally occurring enzyme from the acidophilic microbe Alicyclobacillus sendaiensis. It is a serine endoprotease, with optimal activity over the pH range of 2–4/37◦C and therefore adaptable for use in the gastric environment (Gordon et al., 2012). Based on the structure of KumaWT, an enzyme was designed, with specificity toward known gliadinpeptides. The new enzyme called KumaMax or Kuma010, had 116 times higher proteolytic activity, and 877 times higher specificity for the target gliadin oligopeptides. Further modification was done in KumaMaxor Kuma010 which results in several-fold higher activity against immunotoxic 33-mer and 26-mer peptides. This modified version was called Kuma030. It has been reported that Kuma030 is more efficient than SC PEP- EP B2.It works efficiently at a lesser concentration of 1:40 weight/weight (w/w) ratio, achieved >99.9% gliadin degradation thereby reducing the gluten content to 3 ppm, well-below the 20 ppm threshold for“gluten-free” labeling but the mixture of PEP-EP B2 effectively works at higher concentration i.e. 1:10 w/w ratio and achieved 84.4% gluten degradation (Wolf et al., 2015). When gluten sensitive T cells isolated from patients with CD were incubated with gliadin pre-treated with Kuma030, a dose dependent reduction in IFN-γ production and T cell proliferation was observed (Wolf et al., 2015). Based on these findings Kuma030 may hold promise in the near future.

Dipeptidyl peptidase- IV (DPP-IV) is an exopeptidase that acts on the amino-terminal side to liberate X-Pro dipeptides in gliadin. It occurs naturally in small amounts in the small intestinal brush border. It has been obtained commercially from the fungus Aspergillus oryzae and its potential as a glutenase has been investigated. On its own, DPP-IV has modest efficiency as it can only act on peptides starting with X-Pro. Additionally, DPP-IV has a neutral pH optimum and hence it starts action only in the intestine. It has been investigated that DPP-IV executes higher efficiency in the presence of AN-PEP . The combination when administered as an oral mixture has been found to successfully degrade small amounts of gluten (Ehren et al., 2009). Because of non-specificity of AN-PEP and the very limited proteolytic effect of DPP-IV, the effect of this combination appears to be the best. This combination was studied as a part of STAN1, a cocktail of microbial enzymes commonly used in food supplements. However, in presence of trypsin/Ph 8.0/37◦C, the enzyme is susceptible to destruction. Hence it may be effective only in the gastric digestion of wheat gluten prior to the food bolus reaching the intestine. Further invitro testing to study its efficiency as detoxifying enzyme in celiac disease is required.

Endopeptidase 40 is a novel glutenase isolated from the soil actinomycete Actinoallomurus A8 as a secreted protein. It is active at low pH ranges 3 to 6 and shows resistance against pepsin and trypsin. The most immunogenic 33-mer as well as the whole gliadin proteins are efficiently degraded and no toxic peptides are produced during gluten digestion by E40 into the stomach, thus preventing the intestinal inflammatory reactions to occur in CD patients. Hence, E40 is proposed as a novel candidate in oral enzymatic therapy for the dietary management of gluten intolerance (Cavaletti et al., 2019).

Although all the enzymes described above have been proven effective glutenase activity under appropriate thermochemical conditions, whether these enzymes completely eliminate all immunogenic epitopes and prevent any possible immune-activation by dietary gluten is the most pertinent question. Moreover, most of the glutenases that are now in advanced stages of clinical trials, have been studied in the context of small amounts of gluten challenge, in patients who are already on the GFD. Such small challenges simulate inadvertent gluten exposure in patients that adhere to GFD, and are useful to study patients that are not responsive despite adherence to GFD. Whether these oral enzymatic mixture would help them remains to be proven. Clinical trials with higher doses of gluten challenge are required.

Conclusions
The high incidence of CD in the worldwide population is a challenging task, imposed the negative impact of a strict gluten free diet on the perceived quality of life of celiac patients for several reasons. A life-long adherence to gluten-free diet is not easy since gluten is the most common ingredient in the human diet. Multidirectional research efforts are currently going on to find out new treatment strategies in order to reduce sensitivity to gluten. The use of oral proteases capable of detoxifying ingested gluten and bacterial-derived endopeptidases currently represent the most advanced and promising strategies to manage CD.GFD is likely to remain the mainstay of therapy of CD in the near future, since all other treatment modalities are only in the preliminary stages of research. An ideal therapeutic agent would be one that permits a CD patient to consume gluten in usual amounts, without compromising her/his quality of life. So, Glutenases have been extensively explored as therapeutic agents and their list is continuously growing with newer discoveries. It has been showed that among these, latiglutenase had reached far away in terms of clinical trials. Based on the results of preliminary studies, Kuma030 (Type of glutenase), suggested that this enzyme may provide more promising results in future, compared to other glutenases studied so far. Finally it has been concluded that although most trials on novel therapeutics are currently in phase 2 or earlier stages, ongoing research in areas targeting various molecular pathways in CD is robust. This provides much scope to find definitive alternatives to GFD in the coming years, in order to improve the quality of life of patients with CD. Celiac patients abide by a gluten-free diet, and the supplementation of exogenous gluten-digestive enzymes (glutenases) is expected as a promising way to reduce the risk of dietary gluten boost.

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