Maize Prolamin Research Articles

Breeding strategies for quality protein maize: A Review

Zein proteins in maize seeds regulate food and feed quality and belong to several subfamilies. Most maize prolamin is α-zeins, which are rich in glutamine, leucine, and proline but lacking in critical amino acids (lysine and tryptophan). RNAi experiments show that 22kD zeins downregulate the quality protein maize (QPM) phenotype, not 19kD. The opaque-2 mutation caused a chalky, soft endosperm and many defects. QPM genotypes with opaque-2 alleles and firm kernels were improved. As opaque-2 is recessive and biochemical lysine and tryptophan analysis is costly, conventional backcrossing alone is not very useful. Markerassisted selection (MAS) allowed top inbreds to be homozygous o2o2 utilizing opaque-2 gene-specific markers. Vivek QPM-9, a mixture of two QPM introgression lines, benefits various clients and customers. This review highlights the breeding strategies for QPM.

Large and stable genome edits at the sorghum alpha kafirin locus result in changes in chromatin accessibility and globally increased expression of genes encoding lysine enrichment.

Sorghum is a resilient and widely cultivated grain crop used for feed and food. However, it's grain is deficient in lysine, an essential amino acid. This is due to the primary seed storage proteins, the alpha-kafirins, lacking lysine. It has been observed that reductions in alpha-kafirin protein results in rebalancing of the seed proteome and a corresponding increase in non-kafirin proteins which leads to an increased lysine content. However, the mechanisms underlying proteome rebalancing are unclear. This study characterizes a previously developed gene edited sorghum line, with deletions at the alpha kafirin locus. A single consensus guide RNA leads to tandem deletion of multiple members of the gene family in addition to the small target site mutations in remaining genes. RNA-seq and ATAC-seq were utilized to identify changes in gene expression and chromatin accessibility in developing kernels in the absence of most alpha-kafirin expression. Several differentially accessible chromatin regions and differentially expressed genes were identified. Additionally, several genes upregulated in the edited sorghum line were common with their syntenic orthologues differentially expressed in maize prolamin mutants. ATAC-seq showed enrichment of the binding motif for ZmOPAQUE 11, perhaps indicating the transcription factor's involvement in the kernel response to reduced prolamins. Overall, this study provides a resource of genes and chromosomal regions which may be involved in sorghum's response to reduced seed storage proteins and the process of proteome rebalancing.

Progressive Aggregation of 16 kDa Gamma-Zein during Seed Maturation in Transgenic Arabidopsis thaliana.

Prolamins constitute a unique class of seed storage proteins, present only in grasses. In the lumen of the endoplasmic reticulum (ER), prolamins form large, insoluble heteropolymers termed protein bodies (PB). In transgenic Arabidopsis (Arabidopsis thaliana) leaves, the major maize (Zea mays) prolamin, 27 kDa γ-zein (27γz), assembles into insoluble disulfide-linked polymers, as in maize endosperm, forming homotypic PB. The 16 kDa γ-zein (16γz), evolved from 27γz, instead forms disulfide-bonded dispersed electron-dense threads that enlarge the ER lumen without assembling into PB. We have investigated whether the peculiar features of 16γz are also maintained during transgenic seed development. We show that 16γz progressively changes its electron microscopy appearance during transgenic Arabidopsis embryo maturation, from dispersed threads to PB-like, compact structures. In mature seeds, 16γz and 27γz PBs appear very similar. However, when mature embryos are treated with a reducing agent, 27γz is fully solubilized, as expected, whereas 16γz remains largely insoluble also in reducing conditions and drives insolubilization of the ER chaperone BiP. These results indicate that 16γz expressed in the absence of the other zein partners forms aggregates in a storage tissue, strongly supporting the view that 16γz behaves as the unassembled subunit of a large heteropolymer, the PB, and could have evolved successfully only following the emergence of the much more structurally self-sufficient 27γz.

Two γ-zeins induce the unfolded protein response.

The rapid, massive synthesis of storage proteins that occurs during seed development stresses endoplasmic reticulum (ER) homeostasis, which activates the ER unfolded protein response (UPR). However, how different storage proteins contribute to UPR is not clear. We analyzed vegetative tissues of transgenic Arabidopsis (Arabidopsis thaliana) plants constitutively expressing the common bean (Phaseolus vulgaris) soluble vacuolar storage protein PHASEOLIN (PHSL) or maize (Zea mays) prolamins (27-kDa γ-zein or 16-kDa γ-zein) that participate in forming insoluble protein bodies in the ER. We show that 16-kDa γ-zein significantly activates the INOSITOL REQUIRING ENZYME1/BASIC LEUCINE ZIPPER 60 (bZIP60) UPR branch-but not the bZIP28 branch or autophagy-leading to induction of major UPR-controlled genes that encode folding helpers that function inside the ER. Protein blot analysis of IMMUNOGLOBULIN-BINDING PROTEIN (BIP) 1 and 2, BIP3, GLUCOSE REGULATED PROTEIN 94 (GRP94), and ER-localized DNAJ family 3A (ERDJ3A) polypeptides confirmed their higher accumulation in the plant expressing 16-kDa γ-zein. Expression of 27-kDa γ-zein significantly induced only BIP3 and ERDJ3A transcription even though an increase in GRP94 and BIP1/2 polypeptides also occurred in this plant. These results indicate a significant but weaker effect of 27-kDa γ-zein compared to 16-kDa γ-zein, which corresponds with the higher availability of 16-kDa γ-zein for BIP binding, and indicates subtle protein-specific modulations of plant UPR. None of the analyzed genes was significantly induced by PHSL or by a mutated, soluble form of 27-kDa γ-zein that traffics along the secretory pathway. Such variability in UPR induction may have influenced the evolution of storage proteins with different tissue and subcellular localization.

Identification of peptides in the terminal ileum of broiler chickens fed diets based on maize and soybean meal using proteomics

A total of 160 Ross PM3 birds were used in a two treatment feeding study in order to explore the usefulness of proteomics to identify the origin of peptides in ileal digesta. Two diets were fed, one conventional maize/soy-based diet acted as a reference whereas a second diet, formulated to be nutritionally equivalent to the reference diet in protein and energy provision, contained 20% raw soy meal in order to (putatively) elicit changes in intestinal protein flow. Each diet was fed to 10 replicate cages of eight birds per cage from Day 1 to 21. Feed and water were available ad libitum and an indigestible marker was included for assessment of ileal digestibility. Weight gain and feed intake were monitored and at the end of the trial period birds were killed, pancreatic mass was measured and the ileum was excised and the contents were collected, immediately frozen in liquid nitrogen and were subsequently lyophilised. Protein from the ileal digesta was extracted and exposed to proteomic analysis with peptide fragments identified and compared with an amalgamated database containing protein sequences from chicken, soy and maize. Addition of 20% raw soy meal to the maize/soy-based diet resulted in a reduction in weight gain, feed intake and an increase in feed conversion ratio (P < 0.001). Pancreatic mass was significantly increased and the apparent ileal digestibility of protein was significantly decreased by raw soy meal inclusion. Overall, a total of 248 proteins were identified from endogenous origin, 336 from soy and 411 from maize. However, the relative abundance of these proteins were ~20–30% for endogenous protein, 65–75% for soy protein and ~2–4% for maize protein. The addition of 20% raw soy meal resulted in an increase in the relative abundance of endogenous protein and a reduction in the relative abundance of protein from soy with no measurable effect on the presence of protein from maize. Specifically, in the endogenous protein fraction, there was a significant reduction in the relative abundance of metalloendopeptidase, aminopeptidase and alkaline phosphatase and a significant increase in the relative abundance of colipase and trypsin, in response to raw soy meal inclusion. For proteins originating from soybean, the addition of raw soy meal to the diet resulted in a significant increase in the relative abundance of protein from the 2S albumin fraction, Kunitz and Bowman–Birk trypsin inhibitors and soybean agglutinin whereas there was a reduction in the relative abundance of globulin and glycinin. Addition of raw soy meal to the diet also resulted in a significant increase in the presence of maize prolamin in the lumen and a significant decrease in the presence of globulin-2, β-1–3-glucanase and cystatin. These results demonstrate considerable potential of proteomics technology to identify changes in the digestion and secretion of protein in the intestine of chickens. Although these data are preliminary and based on an animal model that included diets that were formulated to have a chronic effect on intestinal physiology it is evident that changes in diet composition can have a profound effect on the origin of protein that leaves the ileum.

A Fusion between Domains of the Human Bone Morphogenetic Protein-2 and Maize 27 kD γ-Zein Accumulates to High Levels in the Endoplasmic Reticulum without Forming Protein Bodies in Transgenic Tobacco.

Human Bone Morphogenetic Protein-2 (hBMP2) is an osteoinductive agent physiologically involved in bone remodeling processes. A commercialized recombinant hBMP2 produced in mammalian cell lines is available in different clinical applications where bone regeneration is needed, but widespread use has been hindered due to an unfavorable cost/effective ratio. Protein bodies are very large insoluble protein polymers that originate within the endoplasmic reticulum by prolamine accumulation during the cereal seed development. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body biogenesis when fused to other proteins. To produce high yield of recombinant hBMP2 active domain (ad) in stably transformed tobacco plants we have fused it to the γ-zein domain. We show that this zein-hBMP2ad fusion is retained in the endoplasmic reticulum without forming insoluble protein bodies. The accumulation levels are above 1% of total soluble leaf proteins, indicating that it could be a rapid and suitable strategy to produce hBMP2ad at affordable costs.

Effect of Maize Prolamins on Peripheral Blood Mononuclear Cells from Celiac Disease Patients

Celiac disease (CD) is an enteropathy induced by wheat prolamins (gliadins) and in some rare cases by maize prolamins (zeins) possibly due to a similar immune response. The aim was to study the cellular immune response to zeins in comparison to gliadins of peripheral blood mononuclear cells (PBMC) from CD patients. Isolated PBMC from two treated CD patients and three non-CD controls were challenged in vitro with gliadins or zeins and released gamma-interferon (IFN-γ) in culture medium was measured. PBMC were stimulated with gliadin or zein immunogenic peptides or their digested fractions (3-5 kDa).The gliadin peptide G33-mer induced an expected IFN-γ releasing of PBMC from both patients 1 and 2, with a higher level between days 0 and 6 for patients 1 and no differences para patient 2. The zein peptide Z34-mer induced a higher increase of IFN-γ in both CD patients at 0 day, even higher that it of G33-mer for patient 2, and both of them were highly decreased at day 6. Finally, the zein digested fraction induce an IFN-γ release similar to that of gliadin digested fraction in both cases, although negligible for patient 1 and significant for patient 2. In conclusion, the cellular response to zeins was partially similar to it of gliadins after an in vitro challenge.

Prolamins of maize and wheat differentially affect intestinal cells both in biopsies of celiac patients and CACO-2 cell line

ABSTRACTWe study the effect of maize zeins on cellular immune response as compared to that of wheat gliadins for exacerbating celiac disease due to a hypothetical similar response in some patients. In vitro activation of celiac duodenal mucosa with gliadin or zein immunoreactive peptides and pro-inflammatory response of CACO-2 cells to digested gliadin or zein fractions were evaluated as indicators of adaptive and innate response, respectively. In 3/5 biopsies, zein increased production of IFN-γ, whereas gliadin has done it in all the patient biopsies. In CACO-2 cells, two zein fractions (3–5 and 1–3 kDa), similar to gliadin fractions, stimulated the production of IL-8, p38 MAPK, COX2, and release of ZO-1 as compared to medium alone. The 3–5 kDa zein fraction increased permeability in cell monolayers, although less than gliadin. Zein peptides are immunogenic for some patients and induce a similar innate response, but to a lesser extent than gliadin peptides.

Identification of the region of rice 13 kDa prolamin essential for the formation of ER-derived protein bodies using a heterologous expression system.

Cereal prolamins, which are alcohol-soluble seed storage proteins, can induce ER-derived protein bodies (PBs) in heterologous tissue. Like maize and wheat prolamins, rice prolamins can form ER-derived PBs, but the region of mature polypeptides that is essential for PB formation has not been identified. In this study, we examined the formation mechanisms of ER-derived PB-like structures by expressing rice 13 kDa prolamin-deletion mutants fused to green fluorescent protein (GFP) in heterologous tissues such as yeast. The 13 kDa prolamin-GFP fusion protein was stably accumulated in transgenic yeast and formed an ER-derived PB-like structure. In contrast, rice α-globulin-GFP fusion protein was transported to vacuoles. In addition, the middle and COOH-terminal regions of 13 kDa prolamin formed ER-derived PB-like structures, whereas the NH2-terminal region of 13 kDa prolamin did not form such structures. These results suggest that the middle and COOH-terminal regions of 13 kDa prolamin can be retained and thus can induce ER-derived PB in yeast.

Protein body formation in the endoplasmic reticulum as an evolution of storage protein sorting to vacuoles: insights from maize γ-zein.

The albumin and globulin seed storage proteins present in all plants accumulate in storage vacuoles. Prolamins, which are the major proteins in cereal seeds and are present only there, instead accumulate within the endoplasmic reticulum (ER) lumen as very large insoluble polymers termed protein bodies. Inter-chain disulfide bonds play a major role in polymerization and insolubility of many prolamins. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body formation when fused to other proteins and contains seven cysteine residues involved in inter-chain bonds. We show that progressive substitution of these amino acids with serine residues in full length γ-zein leads to similarly progressive increase in solubility and availability to traffic from the ER along the secretory pathway. Total substitution results in very efficient secretion, whereas the presence of a single cysteine is sufficient to promote partial sorting to the vacuole via a wortmannin-sensitive pathway, similar to the traffic pathway of vacuolar storage proteins. We propose that the mechanism leading to accumulation of prolamins in the ER is a further evolutionary step of the one responsible for accumulation in storage vacuoles.

Gluten‐like cellular response of maize prolamins in intestinal biopsies and Caco‐2 cells (916.6)

Celiac disease (CD) is an autoimmune‐mediated enteropathy triggered by wheat gliadins in genetically prone individuals. Some celiac patients are affected by maize zeins, possibly by a gliadin‐like immune response. The immunogenic peptides of gliadin (G33mer) and zein (Z34mer) were used to evaluate activation of cultured intestinal biopsies of patients with different gastrointestinal diseases. Additionally, changes in the permeability of a monolayer of Caco‐2 cells challenged with peptides of digested gliadin (GdDig) and zeins (ZeDig), were evaluated. Pathological report indicated inflamed duodenal mucosa (7), mucosal atrophy (2) and normal mucosa (1) in 10 patient biopsies. Interferon‐gamma (IFN‐γ) and interleucin (IL‐2) specifically increased in atrophic mucosa by effect of G33mer, while the Z34mer induce a smaller increase in cases of atrophic and inflamed mucosa compared to control. ZeDig peptides and G33mer induced a higher permeability of the monolayer of Caco‐2 cells, as compared to that induced by GdDig or Z34mer. Zeins could have an effect on disrupted mucosa similar to gliadins but not necessarily by induction of the initial damage.

Maize Prolamins Could Induce a Gluten-Like Cellular Immune Response in Some Celiac Disease Patients

Celiac disease (CD) is an autoimmune-mediated enteropathy triggered by dietary gluten in genetically prone individuals. The current treatment for CD is a strict lifelong gluten-free diet. However, in some CD patients following a strict gluten-free diet, the symptoms do not remit. These cases may be refractory CD or due to gluten contamination; however, the lack of response could be related to other dietary ingredients, such as maize, which is one of the most common alternatives to wheat used in the gluten-free diet. In some CD patients, as a rare event, peptides from maize prolamins could induce a celiac-like immune response by similar or alternative pathogenic mechanisms to those used by wheat gluten peptides. This is supported by several shared features between wheat and maize prolamins and by some experimental results. Given that gluten peptides induce an immune response of the intestinal mucosa both in vivo and in vitro, peptides from maize prolamins could also be tested to determine whether they also induce a cellular immune response. Hypothetically, maize prolamins could be harmful for a very limited subgroup of CD patients, especially those that are non-responsive, and if it is confirmed, they should follow, in addition to a gluten-free, a maize-free diet.

Nonredundant Function of Zeins and Their Correct Stoichiometric Ratio Drive Protein Body Formation in Maize Endosperm

Zeins, the maize (Zea mays) prolamin storage proteins, accumulate at very high levels in developing endosperm in endoplasmic reticulum membrane-bound protein bodies. Products of the multigene α-zein families and the single-gene γ-zein family are arranged in the central hydrophobic core and the cross-linked protein body periphery, respectively, but little is known of the specific roles of family members in protein body formation. Here, we used RNA interference suppression of different zein subclasses to abolish vitreous endosperm formation through a variety of effects on protein body density, size, and morphology. We showed that the 27-kilodalton (kD) γ-zein controls protein body initiation but is not involved in protein body filling. Conversely, other γ-zein family members function more in protein body expansion and not in protein body initiation. Reduction in both 19- and 22-kD α-zein subfamilies severely restricted protein body expansion but did not induce morphological abnormalities, which result from reduction of only the 22-kD α-zein class. Concomitant reduction of all zein classes resulted in severe reduction in protein body number but normal protein body size and morphology.

Divergent properties of prolamins in wheat and maize

Cereal grains are an important nutritional source of amino acids for humans and livestock worldwide. Wheat, barley, and oats belong to a different subfamily of the grasses than rice and in addition to maize, millets, sugarcane, and sorghum. All their seeds, however, are largely devoid of free amino acids because they are stored during dormancy in specialized storage proteins. Prolamins, the major class of storage proteins in cereals with preponderance of proline and glutamine, are synthesized at the endoplasmic reticulum during seed development and deposited into subcellular structures of the immature endosperm, the protein bodies. Prolamins have diverged during the evolution of the grass family in their structure and their properties. Here, we used the expression of wheat glutenin-Dx5 in maize to examine its interaction with maize prolamins during endosperm development. Ectopic expression of Dx5 alters protein body morphology in a way that resembles non-vitreous kernel phenotypes, although Dx5 alone does not cause an opaque phenotype. However, if we lower the amount of γ-zeins in Dx5 maize through RNAi, a non-vitreous phenotype emerges and the deformation on the surface of protein bodies is enhanced, indicating that Dx5 requires γ-zeins for its proper subcellular organization in maize.

Accumulation of rice prolamin–GFP fusion proteins induces ER-derived protein bodies in transgenic rice calli

KEY MESSAGE : We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Rice prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin-GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin-GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation.

Maize Prolamins Resistant to Peptic-tryptic Digestion Maintain Immune-recognition by IgA from Some Celiac Disease Patients

Maize is used as an alternative to wheat to elaborate food stuffs for celiac patients in a gluten-free diet.However, some maize prolamins (zeins) contain amino acid sequences that resemble the wheat gluten immunodominant peptides and their integrity after gastrointestinal proteolysisis unknown. In this study, the celiac IgA-immunoreactivity to zeins from raw or nixtamalized grains, before and after peptic/tryptic digestion was evaluated and their possible immunogenicity was investigated by in silico methods.IgA from some celiac patients with HLA-DQ2 or DQ8 haplotypes recognized two alpha-zeins even after peptic/ tryptic proteolysis. However, digestion affected zeins after denaturation, reduction, and alkylation, used for identification of prolamins as alpha-zein A20 and A30 by MS/MS sequencing. An in silico analysis indicated that other zeins contain similar sequences, or sequences that may bind even better to the HLA-DQ2/DQ8 molecules compared to the already identified ones. Results concur to indicate that relative abundance of these zeins, along with factors affecting their resistance to proteolysis, may be of paramount clinical relevance, and the use of maize in the formulation and preparation of gluten-free foods must be reevaluated in some cases of celiac disease.

Removal of surface lipids improves the functionality of commercial zein in viscoelastic zein-starch dough for gluten-free breadmaking

Maize prolamin (zein), together with starch, hydroxypropyl methylcellulose, sugar, salt, yeast and water can form wheat-like cohesive, extensible, viscoelastic dough when mixed above room temperature (e.g. 40 °C). This dough is capable of holding gas. However, it is excessively extensible, and when used for hearth-type rolls, it tends to become flat. Bench-scale defatting of zein with chloroform at room temperature significantly ( P < 0.05) improved specific volume (4.5 ml/g vs. 3.3 ml/g) and shape of the rolls (width-to-height 2.0 vs. 3.9). The total lipid content determined by accelerated solvent extraction (100 °C, 69 bar, chloroform), however, only decreased from 8.0 to 6.6% due to this bench-scale defatting. Staining experiments with Naphthol Blue Black suggested that bench-scale defatting removed surface lipids from the zein particles, and thus facilitated their aggregation. Aggregation experiments with zein and water at 40 °C, and laser scanning confocal microscopy with zein-starch dough confirmed that zein particles aggregated more easily when surface-defatted. Dynamic oscillatory temperature sweeps demonstrated that surface-defatting lowered the temperature at which protein cross-linking occurred by 2 °C. This research can help to produce superior gluten-free bread and could also possibly contribute to the better understanding of wheat dough.

Bovine Milk Caseins and Transglutaminase-Treated Cereal Prolamins Are Differentially Recognized by IgA of Celiac Disease Patients According to Their Age

The prevalence of celiac disease (CD) has increased worldwide, which could be related to some dietary proteins in infant regimens and/or new food processes, affecting CD-predisposed infants and older children or adults differentially. IgA reactivity to human and bovine caseins, as well as yogurt caseins and prolamins from wheat or maize breads, microbial transglutaminase (mTG)-treated or not, was evaluated in three patient groups: G1, <2 years old; G2, approximately 3 years old; and G3 >8 years old. Human caseins were not recognized by IgA, whereas IgA reactivity of G2 and G3 was higher to bovine milk caseins. Immunoreactivity of G1 to yogurt caseins was lower and comparable to controls, with no effects due to mTG treatment. However, mTG treatment increased reactivity of G3 to wheat and maize prolamins. IgA immunoreactivity of CD patients to caseins and mTG-treated or not prolamins was age-dependent, which could reflect a differential manifestation of the effects of such proteins on the intestinal barrier.

Studies on glass transition temperature of mono and bilayer protein films plasticized by glycerol and olive oil

AbstractThermomechanical and thermal properties of whey protein, maize prolamin protein (zein), and the laminated whey protein–zein films were studied. The dynamic mechanical (thermal) analysis (DMTA) results showed that the single zein film had higher Tg than single whey protein and zein–whey laminated films. The shift in the Tg values of films from 31.2°C in whey protein film and 88.5°C in the zein film to 82.8°C in the laminated whey protein–zein films may be implied some interaction formation between the two polymers. The small tan δ peaks were observed at −50°C in zein–glycerol films and at −22.37°C in the whey protein films and can be related to β‐relaxation phenomena or presence of glycerol rich region in polymer matrix. Zein‐olive oil and zein–whey protein–olive oil films showed tan δ peaks corresponded the Tg values at 113.8, and 92.4°C, respectively. Thus, replacing of glycerol with olive oil in film composition increased Tg. A good correspondence was obtained when DSC results were compared with the tan δ peaks in DMTA measurements. DSC thermograms suggested that plasticizers and biopolymers remained a homogeneous material throughout the cooling and heating cycle. The results showed that Tg of zein–glycerol films predicted by Couchman and Karasz equation is very close to value obtained by DSC experiments. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

Transglutaminase Treatment of Wheat and Maize Prolamins of Bread Increases the Serum IgA Reactivity of Celiac Disease Patients

Celiac disease (CD) is mediated by IgA antibodies to wheat gliadins and tissue transglutaminase (tTG). As tTG is homologous to microbial transglutaminase (mTG) used to improve foodstuff quality, it could elicit the immune response of celiac patients. This study evaluated the reactivity of IgA of celiac patients to prolamins of wheat and gluten-free (maize and rice flours) breads mTG-treated or not. Prolamins extracted from wheat and gluten-free breads were analyzed by ELISA and immunodetected on membranes with individual or pooled sera from nine celiac patients recently diagnosed. Sera pool IgA titers were higher against prolamins of mTG-treated wheat or gluten-free breads than against mTG-untreated, mainly due to two individual patients' sera. The electrophoretic pattern of gluten-free bread prolamins was changed by the mTG treatment, and a new 31000 band originated in maize was recognized by three CD patients' IgA.