Transglutaminase 3 crosslinks secreted MUC2 and stabilizes the colonic mucus layer

12 The colonic mucus layer is organized as a two-layered system providing a physical barrier 13 against pathogens and simultaneously harboring the commensal flora. The factors 14 contributing to the organization of this gel network are not well understood. In this study, the 15 impact of transglutaminase activity on this architecture was analyzed. Here, we show that 16 transglutaminase TGM3 is the major TGM isoform expressed and synthesized in the colon. 17 Furthermore, intrinsic extracellular TGM activity in the secreted mucus was demonstrated in 18 vitro and ex vivo . Absence of this acyl-transferase activity resulted in faster degradation of 19 the major mucus component the MUC2 mucin and changed the biochemical properties of 20 mucus. Finally, TGM3-deficient mice showed an early increased susceptibility to DSS- 21 induced colitis. Thus, these observations suggest that natural isopeptide cross-linking by 22 TGM3 is important for mucus homeostasis and protection of the colon from inflammation, a 23 suggested pre-stage of colon carcinoma.


Introduction
The epithelium in the intestinal tract is covered by mucus that provides protection from 27 luminal challenges and bacterial infiltration 1 . Despite the similar proteome composition, the 28 organization of the mucus gel network differs considerably in the small and large intestine 2 . 29 Whereas small intestinal mucus is non-attached, the colonic mucus is a two-layered system 30 with an attached, bacteria-free inner layer and an outer layer harboring the commensal flora 1,  Transglutaminases (R:protein-glutamine γ-glutamyltransferases; E. C. 2.3.2.13) 51 comprise a family of Ca 2+ -dependent acyl-transferases that can catalyze the transamidation or 52 deamidation of protein-bound glutamine residues that can lead to natural cross-links through 53 the formation of an isopeptide bond between the side chains of glutamine and lysine. This 54 PTM is known to limit protein degradation by conformational changes and modification of 55 protease-labile Lys residues 13, 14 . There are nine mammalian TGMs where TGM2 is the most 56 ubiquitously expressed isoform 13,15 . This isoform is predominantly localized in the cell 57 cytosol, but can also be found associated with the plasma membrane. Furthermore, it can be 58 secreted by unknown mechanisms after P2X7 receptor activation 16 . The enzymatic activity 59 of TGM2 is normally silent but during mechanical injury it becomes activated and acts as a  Whether transamidation also has a role in the formation and stabilization of intestinal 65 mucus is currently unknown. Mucus and mucins are stored highly concentrated in the 66 granules of goblet cells and expand 1,000-fold in volume upon secretion. If TGM-catalyzed 67 isopeptide cross-links contribute to mucus homoeostasis, this processing has to occur after 68 secretion and expansion. Here, we suggest that extracellular TGM activity plays a role in 69 organizing the mucus gel in the colon, especially by increasing its stability. To test this 70 hypothesis the abundance of different TGM isozymes was evaluated and their enzymatic 71 activity determined. We found that the formation of N ε -(-γ-glutamyl)-lysine isopeptide cross-72 links in colonic mucus was based on extracellular TGM3-intrinsic activity. Furthermore, 73 mice lacking this TGM isoform secrete a more protease-sensitive MUC2 molecule. In  Analyzing mRNA levels in colonic goblet cells and the remaining epithelial cell populations 89 revealed only transcripts for Tgm2 and Tgm3 genes (Fig. 1a ). Next, the TGM2 and TGM3 90 protein abundance determined by mass spectrometry (MS) in these two cell fractions was 91 extracted. This method revealed approximately 10-times lower levels of TGM3 in the goblet 92 cells compared to the non-goblet epithelial cells whereas the abundance of TGM2 was two-93 three orders of magnitude lower than TGM3 in the respective cell population (Fig. 1b). To 94 evaluate the tissue localization of TGM2 and TGM3, immunohistochemical analyses were 95 performed in WT, Tgm2 -/and Tgm3 -/animals together with the UEA1 lectin staining for the 96 highly glycosylated MUC2 mucin. None of the strains reacted with the anti-TGM2 antibody, 97 confirming the low levels of this isoform (Fig. 1c). That this antibody was functional was 98 tested on duodenal tissue sections where a signal for TGM2 was easily observed (Suppl. Fig.   99 S1a). In line with the quantitative data from mRNA expression and protein abundance, both 100 WT and Tgm2 -/animals showed a strong staining for the TGM3 isoenzyme in the epithelium 101 and as expected no signal in Tgm3 -/mice (Fig. 1d).

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As TGM3 lacks a signal sequence, we determined if TGM3 could nonetheless be 103 secreted into the mucus. To answer this, gel electrophoresis and western blot analyses for 104 TGM2 and 3 in colonic mucus were performed. Recombinantly expressed TGM2 and 105 cleaved TGM3 were also loaded as positive controls either non-activated or activated by 106 Ca 2+ -preincubation (Fig. 1e). The majority of TGM3 was represented by a band migrating 107 around 75 kDa and a weaker signal migrating at approximately 50 kDa in both WT and 108 Tgm2 -/animals. These two bands represent the zymogenic and active form of the enzyme, 109 respectively. Furthermore, several diffuse, but weak, TGM3-signals migrating between 150 110 and 250 kDa were detected in the WT and Tgm2 -/strains suggesting the self-multimerization 111 of the enzyme and/or its incorporation into substrate proteins. As similar signals were 112 detected in the activated positive control for TGM3, it is likely that self-multimerization 113 occurs in mucus. In contrast, TGM 2 was not detected in the mucus samples of any mouse 114 strain. Specificity of the used antibodies for the respective isoform was determined upon 115 western blot analyses, the anti-TGM3 antibody showed a cross-reactivity <8 % on TGM2 and  Next, we asked if TGM3 is enzymatically active in the colonic mucus and could thereby 123 contribute to its stability by the formation of additional cross-links. For that purpose, a 124 qualitative assay using the incorporation of biotinylated isoform-specific substrate peptides 125 T26 (TGM2) and E51 (TGM3) in mucus was performed. The mucus was incubated with Ca 2+ 126 and the respective peptide probe followed by gel electrophoresis and western blot using 127 streptavidin detection (Fig. 2a). Specific incorporation of the two peptides was observed in 128 WT and Tgm2 -/mucus, but not in mucus from Tgm3 -/animals. Non-specific signals were 129 observed in all samples, including control reactions where transglutaminase activity was 130 inhibited by iodoacetamide (IAA). These bands are likely due to endogenously biotinylated 131 proteins as for example pyruvate-carboxylase. Thus, the detected cross-linking activity in the 132 mucus arises from TGM3-mediated catalysis. To analyze if endogenous mucus contains 133 sufficient Ca 2+ -ions for the activation of TGM3, the experiment was repeated without 134 calcium addition. Similar results as with exogenous Ca 2+ -addition were obtained, indicating 135 the presence of intrinsic extracellular transglutaminase activity in colonic mucus (Fig. 2b). 136 These results suggest that endogenous acyl-donor protein substrates are present in colonic 137 mucus. However, the formation of a transglutaminase-catalyzed cross-linked mucus gel-138 network also requires the presence of acyl-acceptor proteins. Therefore, the Ca 2+ -free 139 experimental set up was modified by replacing the glutamine-donor with the primary amine 140 5-Biotinyl-pentylamine (5-BP) as acyl-donor. Similar to the results from the acyl-acceptor 141 experiments, specific signals were detected when the acyl-donor compound was added to 142 mucus of WT and Tgm2 -/animals, but not in the Tgm3 -/mucus or when IAA was added 143 (Fig. 2c). Together, the results show that colonic mucus contains intrinsically, active TGM3 144 as well as both acyl-acceptor and -donor molecules allowing transamidating reactions to take 145 place.

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To quantify the intrinsic transamidating activity in colonic mucus, a colorimetric assay 147 for the incorporation of a TGM-promiscuous peptide (A25) and the two isozyme-selective 148 peptide substrates (peptides T26 21 and E51 22 ) into casein was performed (Fig. 2d). A natural 149 cross-linking activity in WT mucus of ≈ 8±2 U/mg for the promiscuous substrate was 150 determined. Substitution with the TGM3-specific substrate E51 led to a 1.5-fold increase 151 (≈12 ± 4 U/mg) of the transamidating activity, whereas a residual activity of 0.8 ± 0.3 U/mg 152 was observed for the TGM2-specific substrate. However, no measurable activity could be 153 obtained in the Tgm3 -/mucus as the detected values were below the limit of detection for our 154 assay (Fig. 2d, Suppl. Fig. S2). Blocking of the TGM-reaction with Z-DON led to an almost 155 complete (88%) inhibition for the promiscuous peptide A25. In line with our other results 156 (Fig. 1, Fig. 2 a-c), the natural cross-linking activity was related to TGM3 as the use of the 157 TGM2-specific substrate T26 led to less than 10% transglutaminase activity compared to the 158 TGM3-specific substrate in WT animals and was also below the limit of quantification of this

Loss of TGM3 alters biochemical properties of mucus/MUC2
176 The MUC2 monomer is a large glycoprotein with a mass of around 2.5 MDa (Fig. 3a). It is 177 the most abundant constituent in colonic mucus and is thus a potential target for TGM3-178 mediated cross-linking, something that could influence its biochemical properties. Colonic 179 mucus from WT, Tgm2 -/and, Tgm3 -/mice was isolated and disulfide bonds reduced  As isopeptide bonds can prevent proteolytic cleavage and secreted mucus is normally 189 exposed to numerous endogenous and bacterial proteolytic enzymes, we hypothesized that CysD2. These PTS1 and PTS2 sequences are highly glycosylated, resistant to proteolytic 210 enzymes, and not identifiable by mass spectrometry (Fig. 3a). Thus, the MUC2 mucin in the 211 Tgm3 -/mice is suggested to be already degraded in vivo due to it being more susceptible to 212 degradation in the colon lumen. The altered biochemical properties of mucus and its higher susceptibility to proteolytic 258 degradation in the absence of TGM3 activity suggested that Tgm3 -/mice could be more 259 susceptible to dextran sodium sulfate (DSS) induced colitis. To test this, age-matched 260 cohoused Tgm3 -/and WT animals were challenged with DSS. The body weight of WT mice 261 increased during the first four days whereas the Tgm3 -/animals started to lose weight from 262 day three and showed on trend decreased body weights compared to WT mice until day 6 263 (Fig. 4a). This was reflected by an earlier detection of occult blood in the feces of Tgm3 -/-264 mice one day after the start of the experiment (Fig. 4b). Consequently, the Tgm3 -/animals 265 showed a significant raised disease activity index score (DAI) from day two to day five after 266 the start of the DSS-treatment (Fig. 4c). Higher DAI was maintained in the Tgm3 -/compared 267 to WT animals until day 6, when the colitis became also established in the WT animals. 268 Finally, 50% of the Tgm3 -/animals had to be sacrificed at day 7, compared to 10 % of WT 269 mice, due to suffering and loss of weight following the ethical permit (Fig. 4d). Furthermore, 270 the colon length of Tgm3 -/mice was reduced to 88% of the WT length after 7 days of DSS early on-set of DSS effects in the Tgm3 -/supports the conclusion that the colonic mucus is 279 defect in these animals. When colonic tissue was analyzed by immunohistochemistry for 280 TGM2, this isozyme that was absent in non-treated WT and Tgm3 -/as shown in Fig. 1a, was 281 now detected in both the WT and Tgm3 -/animals after 7 days of DSS-treatment (Fig. 4h).

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Taken together Tgm3 -/animals were significantly more susceptible towards the colitis-  That TGM3 is the dominant transglutaminase of the colon is in accordance with a previous 295 mucus proteome study 2 . mRNAseq and MS studies detected minor amounts of TGM2, but 296 based on the label-free mass spectrometric quantification TGM2 was <1% of that of TGM3 297 and could represent contaminating material from the ileum. In support of this, TGM2 was not 298 detected by immunohistochemistry or gel electrophoresis/western blot. Previous work from 299 Jeong and co-workers has claimed TGM2 as the major transglutaminase of the colon 26 . 300 However, these authors used only immunohistochemistry to demonstrate the presence of 301 TGM2 and no antibody staining against TGM3 was tested. Likely cross reactivity of the used 302 antibody can explain this observation.

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The strong TGM3 signals observed by gel electrophoresis/western blot analyses of 304 mucus represented the zymogenic form of TGM3. In addition to this, several TGM3 bands 305 with higher molecular masses were detected in the range between 150 and 250 kDa. Together 306 with control reactions performed with recombinantly activated TGM3, these signals strongly 307 suggest that the enzyme can self-multimerize and/or incorporate itself into other molecules as 308 previously observed for TGM2 27 .

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Recent reports have shown that TGM2 is extracellularly inactive and can be activated

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The pH in goblet cell granule is acidic and the Ca 2+ -ions are bound to MUC2 and the other 334 stored molecules. After secretion, the pH raises, and free Ca 2+ -ions will become available.

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For the activation of TGM3, the Ca 2+ -binding sites must be occupied and the zymogenic form  In the Tgm3 -/mice, the shortened and more degraded MUC2 still seems to be sufficient            FastQC (version 0.11.2) and filtered using Prinseq (version 0.20.3). The reads were aligned 566 against the mouse reference genome mm10 with STAR (version 2.5.2b) and the number of 567 mapped reads was calculated with HTseq (version 0.6.1p1). Data normalization, differential 568 expression and statistical analysis were made with DESeq2 (version 1.14) in R.

570
In-gel digestion and mass spectrometric analyses 571 Protein bands of interest were excised from the gel and washed with 50 % acetonitrile and 572 dried in a vacuum centrifuge followed by reduction with DTT and alkylation with IAA.

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Trypsin was added at a ratio of 1:50 and the samples incubated for 12 hours at 37˚C. 574 Afterwards, AspN was added at a ratio of 1:50 and the samples incubated for additional 5 575 hours at 37˚C. The digestion was stopped by adding TFA to a concentration of 0.5%.

576
Salt and buffer components were removed by in-house stage tips equipped with C18 577 resin 48 and the peptides dissolved in 0.1 % formic acid. The samples were analyzed on a Q-578 Exactive mass spectrometer as described earlier 49 .