Microbial Transglutaminase Used in Bread Preparation at Standard

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Microbial transglutaminase used in bread preparation at standard bakery concentrations does not increase immuno-detectable amounts of deamidated gliadin Andreas Heil, Jürgen Ohsam, Bernard van Genugten, Oscar Diez, Keiichi Yokoyama, Yoshiyuki Kumazawa, Ralf Pasternack, and Martin Hils J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02414 • Publication Date (Web): 19 Jul 2017 Downloaded from http://pubs.acs.org on July 20, 2017

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Journal of Agricultural and Food Chemistry

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Microbial transglutaminase used in bread preparation at standard bakery

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concentrations does not increase immuno-detectable amounts of deamidated

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gliadin

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Andreas Heil1, Jürgen Ohsam1, Bernard van Genugten2, Oscar Diez2, Keiichi

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Yokoyama3, Yoshiyuki Kumazawa3, Ralf Pasternack1, Martin Hils1,*

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1

Zedira GmbH, Roesslerstrasse 83, Darmstadt, 64293, Germany

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2

AB Enzymes GmbH, Feldbergstrasse 78, Darmstadt, 64293, Germany

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3

Ajinomoto Co., Inc., Institute of Food Sciences & Technologies, 1-1, Suzuki-Cho,

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Kawasaki-Ku, Kawasaki-Shi, 2010-8681, Japan

11 12

* Corresponding author: phone +49 6151 325110; fax +49 6151 325119; e-mail:

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[email protected] (M. Hils)

14 15

Conflict of interest statement

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The authors declare the following competing financial interests: R.P., M.H., A.H., and

17

J.O. are shareholders and/or employees of Zedira. K.Y. and Y.K. are employees of

18

Ajinomoto. B.v.G. and O.D. are employees of AB Enzymes. Zedira received financial

19

support from Ajinomoto to perform this study. Ajinomoto did not participate in the

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analysis and interpretation of the data.

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Abstract

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The effect of standard bakery concentrations of microbial transglutaminase (MTG) in

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wheat bread preparation on the immunoreactivity of celiac disease (CD) patients’

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sera was investigated.

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Immunoblotting using monoclonal

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deamidated gliadin showed no differences between control bread and MTG-bread.

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Deamidation of gliadin could not be detected at standard MTG concentrations.

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CD patients’ sera were characterized using anti-gliadin and anti-DGP ELISA and

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grouped into DGP high and low titer pools. The recognition pattern obtained after

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using both CD sera pools for immunoblotting did not reveal differences between

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control and MTG-treated bread protein extracts.

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Our results indicate that MTG-treatment of wheat bread prepared with typical MTG

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concentrations used in standard bakery processes does not lead to immuno-

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detectable amounts of CD-immunotoxic deamidated gliadins.

antibodies

specific

to unmodified

37 38

Keywords:

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microbial transglutaminase, celiac disease, gliadin, wheat bread

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2 ACS Paragon Plus Environment

and/or

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Introduction

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Celiac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten

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derived from cereals like wheat, rye, or barley in susceptible individuals.1 The major

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symptom of CD is chronic inflammation of the small intestine, combined with severe

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damage of the intestinal mucosa. The wheat grain storage protein gluten is

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composed of alcohol-soluble gliadins and propanol/urea/DTE-soluble glutenins.

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Proteins present in the gliadin fraction, e.g. α- and γ-gliadins, are characterized by a

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high content of proline. This renders gliadin resistant to proteolytic cleavage by

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digestive enzymes of the gastro-intestinal tract. However, gliadins are broken down

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into oligopeptides containing up to several dozen amino acids reaching the intestinal

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mucosa. Another characteristic of these peptides is the high content of glutamine

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residues in close proximity with prolins and hydrophobic residues, resulting in

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multiple copies of the peptide motive QXPF in their sequence. This motif is a

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preferred substrate sequence for human tissue transglutaminase (TG2). Under the

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specific conditions in the inflamed gut TG2 catalyzes deamidation of glutamines

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instead of cross-linking, which is the general function of transglutaminases.2,3

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Deamidation results in a strong increase in affinity of the gliadin peptides towards

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HLA-DQ2/8 receptors present on antigen presenting cells. Binding of deamidated

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gliadin to HLA-DQ2/8 leads to CD4+ T helper 1 cells mediated intestinal

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inflammation.4 In addition it leads to the production of antibodies against deamidated

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gliadin peptides (DGP) as well as auto-antibodies against the endogenous TG2.2,5,6

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These (auto)antibodies are used as standard serological markers in CD

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diagnostics 6,7, whereas anti-gliadin antibodies can also be related to other diseases.8

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The vicious circle of inflammation, transglutaminase release, deamidation of gliadin,

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and further immune stimulation currently can only be stopped by adhering to a

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completely gluten-free diet.9 3 ACS Paragon Plus Environment


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Microbial transglutaminase (MTG) is an enzyme derived from Streptomyces

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mobaraensis and catalyzes cross-linking of protein-bound glutamine to protein-bound

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lysine. Although it has a similar catalytic mechanism, it is not related to eukaryotic

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transglutaminases in structure or sequence but is a result of convergent evolution.

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Most importantly, and in contrast to human transglutaminases, the microbial enzyme

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does not require Ca2+ for catalytic activity, thus enabling its industrial application.

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Under certain conditions, transglutaminases may also catalyze deamidation of

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protein-bound glutamine residues forming the respective glutamic acid. While TG2

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shows equal cross-linking and deamidation activities under the slightly acidic

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conditions as given in the duodenum this is not the case for MTG. Over a broad pH-

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range from 4 – 9 the deamidation activity of MTG constantly is more than a

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magnitude lower than its cross-linking tendency.10

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The idea of a potential generation of CD-specific deamidated gliadin epitopes

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resulting from MTG-treatment of gluten-containing food was described in 2005.11 It is

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based on the hypothesis that MTG may mimic TG2 due to the similar mode of action.

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Since then this topic was further investigated by other research groups who found

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that MTG is able to deamidate gluten-related peptides which are immunotoxic in CD

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at least under certain experimental conditions.10,12,13 In contrast, other studies

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suggest that the use of MTG in bread preparations has no effect on the

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immunogenicity of gluten, but may even be used to detoxify gluten by covalent

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incorporation of primary amines.14,15 Despite of this, some research groups

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postulated the hypothesis that MTG used for production of baked products could lead

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to an increased incidence in celiac disease in Western countries.16–18 So far, to our

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knowledge no clinical nor epidemiologic data is available whether there is a


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relationship between the apparent increase in CD and the use of MTG in baked

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goods.

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Commercial use of MTG

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Since its discovery in 1989, the use of MTG has spread into many different

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biotechnological applications, most importantly in food processing.19,20 The cross-

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linking function is used to improve properties of protein containing food like meat,

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sausage, fish, dairy products, pasta

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preparation was described using many different flour types, e.g. wheat, rye

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barley, soybean 24,25, oats 26, and mixtures of wheat and millet.27

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and bread. The use of MTG in bread

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22,23

,

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The use of MTG in bakery products was patented by Röhm GmbH in 1990

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was marketed by this company (nowadays known as AB Enzymes GmbH) for bakery

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related applications since approximately the mid 1990’s. First products were just

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enzyme preparations combining MTG with other enzyme classes. At a later stage a

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single activity MTG named VERON® TG (a product with 100 TG Units/g) was put on

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the market and happens to be the subject of this investigation. Since this product (like

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the majority of bakery enzymes available in the market) is based on a wheat flour

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carrier it is not suitable for the use in gluten-free bakery products. Therefore only a

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normal standard wheat bread application was considered for the purpose of this

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study.

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Various articles and studies have been published showing the potential technological

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benefits of MTG in bakery applications.22,23,26,27,29–37 Generally, MTG has a positive

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effect on loaf volume when used at low concentrations, and at high concentrations

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the loaf volume is reduced due to formation of a strong meshwork of cross-linked

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proteins and therefore reduced expansion of gas bubbles.30–32


and


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However, typically MTG is never used as a single enzyme in a given bakery

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application but always in combination with other technologically required enzyme

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classes. Due to the limited field of benefits it can be considered a niche enzyme

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within the group of typical baking enzymes such as amylases, xylanases, lipases,

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oxidases, hemicellulases, cellulases, proteases, etc. and the global market share in

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terms of volume and value of MTG used for baking is estimated by industry experts

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to be lower than 1% of the total baking enzymes market.

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This low share is a result of its relatively low dosage (VERON® TG is used at 1-3 g /

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100 kg of flour, corresponding to 1-3 U/kg flour), that is a result of a relatively high

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cost in use and very specific functionality. Regardless of the low usage volumes MTG

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is sold and used for bakery applications on all continents without any significant focus

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countries. MTG is only required for solving explicit technical cases such as industrial

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bakers using wheat dough with short proofing times, where optimization of dough

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properties directly after mixing is crucial and MTG can complement the enzymes

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such as xylanase and amylase that are already used. This is the main reason why

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only low dosages of MTG (1-3U/kg flour) are required.

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Countries with traditionally high protein quantity and quality flours (such as derived

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from e.g. US or Canadian wheat) tend to have lower need for MTG use as compared

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to countries with predominant medium to poor quality flours, where MTG is used to

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improve dough elasticity to more closely resemble properties of high protein flour.38

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Currently in scientific journals as well as in the public, a discussion on a postulated

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celiac disease triggering effect by MTG takes place – controversial, due to lacking

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scientific data. Therefore, in order to provide a more solid base to assess this topic,

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the aim of this study was to investigate the effect of common industrially applied MTG


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concentrations in wheat bread production on the formation of deamidated gluten

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epitopes.

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Materials and Methods

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Materials

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Microbial transglutaminase was applied as formulation VERON® TG (AB Enzymes,

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Darmstadt, Germany) containing MTG from Ajinomoto (Tokyo, Japan). Deamidated

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gliadin peptide DGPx2, 26mer gliadin peptide G051, as well as the monoclonal

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antibodies A011, A054, A064, A067 and A062 were from Zedira (Darmstadt,

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Germany). Celiac disease patients’ sera (n=26) were anonymized residual samples

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purchased from in.vent Diagnostica (Hennigsdorf, Germany).

151 152

ELISA

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Celiac disease patients’ sera were analyzed for the following antibody titers (IgA and

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IgG) using commercial ELISA test kits according to the manufacturer’s instructions:

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gliadin (Steffens Biotechnische Analysen, Ebringen, Germany), DGP (Zedira,

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Darmstadt, Germany).

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Bread preparation

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Bread was prepared according to an internal recipe by AB Enzymes (Darmstadt,

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Germany). Each bread contained 550 g wheat flour, 33 mg ascorbic acid, 11 g salt, 7 ACS Paragon Plus Environment


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11 g butter, 11 g sugar, and 27.5 g yeast. Three bread preparations contained

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different concentrations of VERON® TG (100 U/g): 20, 40, and 80 ppm,

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corresponding to a total of 2, 4 and 8 U MTG/kg flour. 2 U/kg flour is within the

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recommended dose range of 1 – 3 U/kg, 4 U/kg and 8 U/kg exceed the upper dose

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limit by up to 170%. The doughs were mixed in Diosna Spiral mixers type SP12

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applying standard mixing times of 2 minutes slow speed and 8 minutes fast speed to

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achieve an optimum dough development. The breads were fermented at 32°C and

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85% relative humidity for 80 minutes and then baked at 235°C for 35 minutes in

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unlidded baking tins.

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As positive control, bread was prepared according to Cabrera-Chávez et al.16 but

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with 2,000 U MTG/kg flour. For this MTG-concentration preliminary experiments

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revealed increased immuno-detectable deamidated gliadin peptides.

173 174

Preparation of albumin/globulin, gliadin and glutenin extracts

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Extraction was essentially performed as previously described.39 Briefly, 0.5 g bread

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sample were added to 10 mL buffer consisting of 67 mM HKNaPO4, 400 mM NaCl,

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pH 7.6. Albumins and globulins were extracted at room temperature through vigorous

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mixing of the sample for 2 min, followed by gentle stirring for further 10 min and

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subsequent centrifugation at 6,000 x g for 15 min. Extraction of gliadins was

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performed using 60% ethanol (v/v). Glutenins were extracted at 60°C using 50 mM

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Tris-HCl pH 7.5, 50% (v/v) 1-propanol, 2 M urea, 1% (w/v) DTE.

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All extraction steps were performed three times with the same sample and the

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supernatants from each step were pooled.

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Gel electrophoresis and immunoblotting

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SDS-PAGE was performed according to Laemmli.40 Briefly, samples were mixed with

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5x SDS-PAGE loading buffer (128 mM Tris-HCl pH 6.8, 10% β-mercaptoethanol,

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10% glycerol, 4% SDS, 13% bromophenol blue), boiled for 10 min and loaded on a

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10% SDS-PAGE gel. Separation was performed at 200 V for 40 min. Gels were

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stained with silver nitrate according to Blum et al.41 Electro-blotting was performed in

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a Trans Blot SD Semi Dry Transfer Cell (Bio-Rad, Hercules, U.S.A.) at 20 V for 80

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min. After blotting, the nitrocellulose membranes were presoaked in 48 mM Tris, 39

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mM glycine, 1.3 mM SDS, 20% (v/v) methanol. Residual binding sites were blocked

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in 5% skimmed milk powder in TBS-T (10 mM Tris, 150 mM NaCl, 0.05% (v/v)

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Tween 20, pH 8.0) for 60 min at RT. The membrane was washed in TBS-T wash

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buffer and incubated for 1 h at room temperature with primary antibody/serum

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(diluted 1,000 fold in TBS-T). After washing for 3 × 5 min in TBS-T, the secondary

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antibodies (anti-human IgA (Thermo Scientific, Karlsruhe, Germany), anti-human

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IgG, anti-mouse IgG (both Sigma, Schnelldorf, Germany), each conjugated to

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alkaline phosphatase and diluted 10,000 fold in TBS-T buffer) were added to the

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membrane followed by a 1 h incubation at room temperature. After intensive

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washing, the membrane was placed in detection reagent (100 fold dilution of AP

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color reagent in color development buffer, BioRad, Hercules, U.S.A.) and incubated

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for 30 to 60 s at room temperature. Excess detection reagent was drained off and the

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staining reaction was stopped with 20 mM Tris-HCl, 5 mM EDTA, pH 8.0. All washing

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steps were performed at room temperature on a shaker.

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Dot blots

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To analyze the specific binding epitopes of the antibodies used in this study, dot blot

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analysis was performed using unmodified or deamidated γ-gliadin 26mer (aa 59-84;

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FLQPQQPFPQQPQQPYPQQPQQPFPQ

214

PEQPFPQ

215

LQLQPFPQPQLPYPQPQLPYPQPQLPYPQPQPF

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PELPYPQPELPY-PQPQPF [33mer DGP], respectively) fused to a carrier protein

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(Zedira, Darmstadt, Germany). DGPx2 is a recombinant protein composed of both

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33mer DGP and 26mer DGP fused to a carrier protein. Different amounts (1 and 10

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µg) of either wheat gliadin (extracted from commercially available standard wheat

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flour) or recombinant protein were spotted on a nitrocellulose membrane and dried.

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The membrane was cut into small strips and each of them incubated with an

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individual antibody (A011, A054, A064, A057, A062). Further steps were identical to

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the immunoblotting procedure already described.

[26mer

DGP],

and

respectively)

or

FLQPEQPFPEQPEQPYPEQα-gliadin and


33mer

(aa

57-89;

LQLQPFPQPELPYPQ-

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Results and Discussion

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Determination of the specificity of monoclonal antibodies against wheat gliadin

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The monoclonal antibodies used in this study were evaluated for their specific binding

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epitope using dot blotting with different antigens containing either unmodified or

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deamidated immunotoxic CD epitopes, as well as wheat gliadin extracted from wheat

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flour. Figure 1 shows, that the antibodies strongly differed in their recognition pattern.

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A011 and A054 showed strong reactivity with wheat gliadin and recombinant

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unmodified 26mer γ-gliadin. A064 was able to recognize both unmodified and

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deamidated gliadin independent of the peptide sequence, which indicates that the

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binding epitope does not cover a deamidation site. Monoclonal antibodies A057 and

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A062

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(KLQPFPQPELPYPQPQ).13 Whereas A057 is very specific for the deamidated

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peptide, A062 detects both unmodified and deamidated 33mer gliadin to a similar

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extent. Therefore, A057 is one of the most important tools for identifying deamidated

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33mer gliadin peptide at the moment.

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Importantly, none of the antibodies showed cross-reactivity with the carrier protein.

were

raised

against

the

same

immunogenic

peptide

240 241

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Analysis of wheat gluten extracts from MTG-treated breads

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As expected baked breads prepared with increasing MTG-concentrations (2, 4 and 8

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U MTG/kg flour) did not show visible differences, while high-dose positive control

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MTG-bread (2,000 U/kg) was characterized by a small dough volume and reduced air

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bubble size. Albumin/globulin, gliadin and glutenin extracts from these breads were 11 ACS Paragon Plus Environment


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initially analyzed using SDS-PAGE followed by silver staining and Western blotting

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with an antibody detecting unmodified gliadin (A011) (Figure 2). Similar protein

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amounts were extractable for albumin/globulin, gliadin, and glutenin fractions of the

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control (0), 2, 4 and 8 U MTG/kg flour extracts. Western blot analysis revealed only

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little gliadin detectable in the albumin/globulin extracts. As expected, more intensive

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staining was observed for gliadin and glutenin extracts from the samples. The cross

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reactivity of anti-gliadin antibody A011 (as well as A064 and A054, see Figure 3) to

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glutelins is in agreement with the observation by Martínez-Esteso et al. for anti-

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gliadin antibodies used in gluten diagnostics.42

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However, neither in silver stained gels nor in western blotting, differences in the band

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pattern between the 0, 2, 4 and 8 U MTG/kg samples were observed. An influence of

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MTG on the protein composition of the bread samples prepared with increasing

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amounts of MTG could not be detected. This is in line with earlier data by Bauer et al.

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who, in a detailed analysis, did observe polymerization and decreased extractability

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of wheat flour proteins starting with 891 U MTG/kg flour. In contrast, with up to

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133 U MTG/kg no polymerization was detectable.43 Further the results are in

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agreement with the observation for bread or pasta prepared with MTG (both about

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300 U MTG/kg flour).16,39

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In the positive control extract prepared with the extraordinarily high MTG-

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concentration of 2,000 U MTG/kg, high molecular weight bands are present

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predominantly for gliadins and glutenins.

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Further analysis (Figure 3) of the extracts was performed using additional monoclonal

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antibodies recognizing unmodified gliadin (A054) or both unmodified and deamidated

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gliadin peptides (A064) (for specificity, see Figure 1). All samples showed intensive

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staining with both antibodies. The staining in the albumin/globulin-fraction may be 12 ACS Paragon Plus Environment

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explained by residual amounts of gliadin. Again, and in agreement with the silver

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stained gels (Figure 2 A, B, C), an influence of MTG on the protein composition of the

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extracts prepared with increasing amounts of MTG could not be observed. We

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already reported analogous results for pasta treated with 300 U MTG/kg.39

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Taken together, MTG-treatment of wheat dough only results in detectable amounts of

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high molecular weight proteins, if MTG concentration is extraordinarily high (>300

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U/kg) which is not the case in standard bakery products.

279 280

Analysis of MTG-treated bread for gliadin deamidation

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Antibodies raised against deamidated α2-gliadin 33mer epitopes, either specific for

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the deamidated epitope (A057) or recognizing both unmodified and deamidated

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33mer (A062) were used to detect possible deamidation of gluten by MTG.13

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Western blot analysis showed that in the industrially prepared bread samples (0, 2, 4

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and 8 U MTG/kg flour), basically no staining was observed using deamidation-

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specific antibody A057 (Figure 4). However, for the 2,000 U MTG/kg controls,

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staining of multiple bands with a molecular size of 36-40 kDa was observed in every

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extract. Also the positive control DGPx2 was intensively stained. A062 finally

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showed, an increase in staining intensity of bands with a molecular size of approx. 37

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kDa in the albumin/globulin and gliadin extracts for samples with 4 U and 8 U

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MTG/kg flour. As for the more sensitive antibody A057 (Figure 1) no signal increase

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was observed, the growing intensity with A062 (the antibody shows some selectivity

293

for the unmodified gliadin, Figure 1) should be due to unmodified gliadin. It can be

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speculated that MTG-treatment increases accessibility of the (unmodified) epitope.



295

At least using monoclonal antibody A057 specific to deamidated α2-gliadin 33mer

296

neither increase nor presence of any deamidated gliadin could be detected. To

297

further examine this finding, celiac disease patients’ sera were used in Western blot

298

analysis of the bread extracts.

299 300

Analysis of MTG-treated bread using celiac disease patients’ sera

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In total, 26 celiac disease patients’ sera were characterized for their antibody-titers

302

against unmodified gliadin and deamidated gliadin peptide (DGP). All sera were

303

positive for DGP antibodies (either IgA or IgG) with the exception of two sera (225224

304

and 232559), which were in addition negative for both types of anti-gliadin antibodies.

305

Further, half of the sera were negative for anti-gliadin antibodies (IgA and IgG), 5 of

306

them with a high anti-DGP-titer. The sera were grouped in high (>50 U/mL, Table 1)

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and low titer anti-DGP (500 U/kg. While we were able to generate deamidated gliadin peptides in

338

high-dose MTG-treated bread (2,000 U/kg) no deamidated gliadin could be detected

339

in bread produced with standard bakery amounts of MTG. In addition, MTG has a

340

more than 10-fold transamidation preference. In the presence of sufficient amounts of

341

primary amine substrates deamidation can be blocked completely.10 Taking these

342

observations together, deamidation probably may take place only at high dose MTG 15 ACS Paragon Plus Environment


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when primary amines are not available in sufficient amounts anymore or already

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depleted by the MTG-reaction. This is supported by the finding from Mazzeo et al.,

345

who did not detect deamidated gliadin by nano-HPLC-ESI-MS/MS upon addition of

346

even 8,000 U MTG/kg flour in the presence of 20 mM Lysine-ethylester.45

347

To our knowledge so far one example for a food based trigger for celiac disease is

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described. In the 1980s the Swedish national recommendation to postpone

349

introduction of gluten to 6 months old children resulted in an immediate increase of

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celiac disease in Sweden. Within 3 years celiac disease incidence almost

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quadrupled.46 MTG has been introduced in bakery products in the mid-1990s.

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However, no evidence for an immediate increase in celiac disease incidence at the

353

same time has been reported. Thus, epidemiological data do not support a celiac

354

disease triggering impact by MTG.

355

It is a fact that MTG is able to deamidate gliadin rendering the peptides immunotoxic,

356

as shown by ourselves and other groups. Therefore, it cannot be excluded that MTG

357

treatment of bread has an impact on celiac disease. However, in the light of our

358

results and the published data mentioned above, citing Philippus Theophrastus

359

Aureolus Bombastus von Hohenheim (1493 – 1541) - also known as Paracelsus -

360

seems appropriate: “sola dosis facit venenum – the dose makes the poison”.

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In summary, standard bakery doses of MTG are magnitudes lower than used in

362

academic examinations. They do not lead to detectable amounts of deamidated

363

gliadin using a deamidated gliadin specific monoclonal antibody and celiac disease

364

patients’ sera in western blotting. Especially no increase in deamidated gliadin was

365

found.

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Abbreviations used:

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CD, celiac disease; DGP, deamidated gliadin peptide; ELISA, enzyme linked immune

370

sorbent assay; MTG, microbial transglutaminase; DGP, deamidated gliadin peptide;

371

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; DTE,

372

dithioerythritol.

373 374 375

Acknowledgement:

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Preparation of MTG-containing bread by Norman Burkhardt (AB Enzymes) and

377

preparation of graphics by Katrin Bott-Fischer (Zedira) is gratefully acknowledged.



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References

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(1)

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Dieterich, W.; Esslinger, B.; Schuppan, D. Pathomechanisms in Celiac Disease. Int. Arch. Allergy Immunol. 2003, 132 (2), 98–108.

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Molberg, Ø.; Mcadam, S. N.; Körner, R.; Quarsten, H.; Kristiansen, C.; Madsen, L.; Fugger, L.;

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Scott, H.; Norén, O.; Roepstorff, P.; et al. Tissue transglutaminase selectively modifies gliadin

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peptides that are recognized by gut-derived T cells in celiac disease. Nat. Med. 1998, 4 (6),

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713–717.

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(3)

Stamnaes, J.; Fleckenstein, B.; Sollid, L. M. The propensity for deamidation and transamidation

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of peptides by transglutaminase 2 is dependent on substrate affinity and reaction conditions.

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Biochim. Biophys. Acta 2008, 1784 (11), 1804–1811.

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Figure captions

505 506

Figure 1. Specificity of monoclonal antibodies used for the analysis of MTG-treated

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bread. Recombinant proteins carrying different unmodified and deamidated

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immunotoxic peptide sequences isolated from wheat gliadin were analyzed using dot

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blot to identify specific binding patterns of the antibodies.

510 511

Figure 2. SDS-PAGE and Western blot analysis of wheat protein fractions extracted

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from bread prepared without (0), 2, 4 and 8 U MTG/kg flour. A, B, C: silver-stained

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gels; D, E, F: Western blots using monoclonal antibody A011 specific to gliadin as

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detection antibody. Gliadin extract from 2,000 U MTG/kg flour and DGPx2 were

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deamidated gliadin positive controls.

516 517

Figure 3. Western blot analysis of MTG treated bread extracts using antibodies

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against unmodified gliadin. Protein extracts as described in figure 2. A, B, C: Western

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blots with gliadin specific antibody A054; D, E, F: Western blots with gliadin specific

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antibody A064. DGPx2: deamidated gliadin control. 26mer GP: non-deamidated

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gliadin control (the multiple bands visible in the blots are essentially due to multimers

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or degradation products of recombinant 26mer GP visible at high concentrations in

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combination with sensitive detection).

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Figure 4. Western blot analysis of extracts from MTG treated bread samples using

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antibodies against deamidated gliadin. Protein extracts as described in figure 2. A, B, 24 ACS Paragon Plus Environment

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C: Western blots with gliadin specific antibody A057; D, E, F: Western blots with

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gliadin specific antibody A062. DGPx2: deamidated gliadin control.

529 530

Figure 5. Western blot analysis of MTG treated bread samples using high titer celiac

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disease patients’ sera. Protein extracts as described in figure 2. A, B, C: Western

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blots with high titer sera pool of IgG type; D, E, F: Western blots with high titer sera

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pool of IgA type. DGPx2: deamidated gliadin control. 26mer GP: non-deamidated

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gliadin control.

535 536

Figure 6. Western blot analysis of MTG treated bread samples using low titer celiac

537

disease patients’ sera. Protein extracts as described in figure 2. A, B, C: Western

538

blots with low titer sera pool of IgG type; D, E, F: Western blots with low titer sera

539

pool of IgA type. DGPx2: deamidated gliadin control. 26mer GP: non-deamidated

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gliadin control.



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Tables Table 1: Celiac disease patients’ sera with high titers of anti-DGP and anti-gliadin antibodies (IgA and IgG; c/o = cut off).

Serum No. 221096 223261 224184 224187 224234 225254 225380 225833 225872 227542 232554 240820 240993 232394

High titer sera, either IgA or IgG > 50 U/mL (n = 14) DGPx1 IgA DGPx1 IgG Gliadin IgA c/o = 8 c/o = 7 c/o = 14 31 65 8 100 33 >100 96 99 68 6 50 20 70 12 >100 23 >100 4 100 3 41 >100 24 100 0 >100 >100 40 27 >100 11 >100 87 >100 >100 >100 55 27 60 16

Gliadin IgG c/o = 14 7 4 9 3 3 80 16 >100 8 14 2 12 10 2

Table 2: Celiac disease patients’ sera with low titers of anti-DGP and anti-gliadin antibodies (IgA and IgG; c/o = cut off), respectively.

Serum No. 221057 223253 223260 223333 224360 225224 225292 225387 232553 232559 225938 223332

Low titer sera, IgA and IgG < 50 U/mL (n = 12) DGPx1 IgA DGPx1 IgG Gliadin IgA c/o = 8 c/o = 7 c/o = 14 33

Microbial Transglutaminase Used in Bread Preparation at Standard

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