An initial assessment of the involvement of transglutaminase2 in eosinophilic bronchitis using a disease model developed in C57BL/6 mice.

The detailed pathogenesis of eosinophilic bronchitis (EB) remains unclear. Transglutaminase 2 (TG2) has been implicated in many respiratory diseases including asthma. Herein, we aim to assess preliminarily the relationship of TG2 with EB in the context of the development of an appropriate EB model through ovalbumin (OVA) sensitization and challenge in the C57BL/6 mouse strain. Our data lead us to propose a 50 µg dose of OVA challenge as appropriate to establish an EB model in C57BL/6 mice, whereas a challenge with a 400 µg dose of OVA significantly induced asthma. Compared to controls, TG2 is up-regulated in the airway epithelium of EB mice and EB patients. When TG2 activity was inhibited by cystamine treatment, there were no effects on airway responsiveness; in contrast, the lung pathology score and eosinophil counts in bronchoalveolar lavage fluid were significantly increased whereas the cough frequency was significantly decreased. The expression levels of interleukin (IL)-4, IL-13, IL-6, mast cell protease7 and the transient receptor potential (TRP) ankyrin 1 (TRPA1), TRP vanilloid 1 (TRPV1) were significantly decreased. These data open the possibility of an involvement of TG2 in mediating the increased cough frequency in EB through the regulation of TRPA1 and TRPV1 expression. The establishment of an EB model in C57BL/6 mice opens the way for a genetic investigation of the involvement of TG2 and other molecules in this disease using KO mice, which are often generated in the C57BL/6 genetic background.

Is Endoscopic Assessment of the Esophagus and Stomach Enough to Determine the Need for Biopsy at These Sites in Pediatric Patients Undergoing Endoscopy for Elevated TTG?

OBJECTIVES: The goal of our study was to determine whether visual assessment of the esophagus and stomach could predict abnormal histology and determine the frequency of interventions based on biopsies in patients undergoing endoscopy for elevated tissue transglutaminase immunoglobulin A antibody (TTG). METHODS: Pathology records were searched for patients with biopsy performed for elevated TTG. Pathology report, endoscopy report, and follow-up were obtained and slides from the duodenum reviewed. Pathology was considered gold standard for sensitivity and specificity calculations. RESULTS: 240 patients were included. 215 patients had esophageal biopsies performed. Esophageal endoscopic visual assessment had sensitivity of 47% and specificity of 93% for abnormal histology. 16(7%) patients had therapy or referral related to results and, of these, 6(38%) had visually normal endoscopy. 237 biopsies were performed of stomach. Gastric endoscopic visual assessment had a sensitivity and specificity of 20% and 87%. 24(10%) patients had therapy based on findings and, of these, 12 (50%) had visually normal endoscopy. CONCLUSIONS: Endoscopic assessment of esophagus and stomach has low sensitivity and high specificity for pathologic abnormalities when indication for endoscopy is elevated TTG. When endoscopy is visually normal clinical interventions based on biopsy are rare, and foregoing biopsy may be considered.

Celiac Dietary Adherence Test and Standardized Dietician Evaluation in Assessment of Adherence to a Gluten-Free Diet in Patients with Celiac Disease.

Adherence to a gluten-free diet (GFD) is currently the mainstay of treatment strategy for celiac disease (CD). The aim of our study was measuring a GFD adherence in CD patients using two newly validated methods of dietary assessment-Standardized Dietician Evaluation (SDE) and the Celiac Dietary Adherence Test (CDAT). Ninety-two adults with CD were evaluated by a registered dietitian with extensive experience with the use of SDE and CDAT. Duodenal biopsy was performed and blood was drawn for serum anti-endomysial, anti-deamidated gliadin peptide and anti-tissue transglutaminase antibodies in forty four of those patients. The results of CDAT and SDE were very convergent, but SDE scores better correlated with serologic and histologic findings. As many as 24-52% of study participants did not adhere well enough to a GFD. Insufficient adherence to a GFD in CD patients is still a significant problem. The knowledge about gluten content in food ingredients and additives is very low among adults with CD. SDE is the most accurate method in assessing compliance with a GFD and is especially helpful in determining hidden sources of gluten. The CDAT may be a fast tool for screening for a GFD adherence in CD patients.

Assessment of expression and specific activities of transglutaminases TG1, TG2, and FXIII-A during osteoclastogenesis.

Osteoclasts are large multinucleated bone-resorbing cells derived from monocyte/macrophage lineage. Macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) drive the multi-stage osteoclastogenesis. Transglutaminases (TGs) are Ca2+- and thiol-dependent acyl transferases and protein crosslinking enzymes. TG enzyme family contains eight catalytically active enzymes TG1-7 and Factor XIII-A (FXIII-A). Recent studies have shown that TG1, TG2, and FXIII-A are present in osteoclasts and that TG2 and FXIII-A regulate osteoclastogenesis. In this study, we examined gene and protein expression and specific activities of TG1, TG2, and FXIII-A during osteoclastogenesis using "Hitomi peptides" in a day-by-day manner. We report that TG activities are highest in the differentiation and early fusion phases and then decrease dramatically. TG activities were upregulated by M-CSF and downregulated by addition of RANKL. FXIII-A was dramatically downregulated by RANKL, suggesting its involvement in M-CSF-mediated precursor commitment phase. TG1 and TG2 proteins were present throughout osteoclastogenesis, suggesting that they may have functions in both differentiation and fusion. In summary, the three TGs likely exert distinct functions at different stages of osteoclastogenesis. Our work also demonstrates that the "Hitomi peptides" are highly specific tools for detection of distinct TGs in a system where multiple TGs are present.

Adherence to a Gluten-free Diet: Assessment by Dietician Interview and Serology.

We aimed to determine whether tissue transglutaminase (tTG) autoantibody positivity was associated with dietitian-assessed adherence to a gluten-free diet in pediatric patients with celiac disease and identify areas where adherence falters. We reviewed the records of children with celiac disease who had a standardized evaluation of adherence by a registered dietitian. A negative tTG value was not associated with good adherence (P = NS). Adherent and nonadherent children differed with respect to purposeful and accidental gluten exposure (P < 0.0001), knowledge (P = 0.003), cross-contact (P = 0.003), potential exposure via medications and cosmetics (P = 0.004), and potential exposure while at restaurants (P < 0.0001), but not with respect to potential exposure at school (P = NS). Based on our findings, we suggest that negative tTG levels are not necessarily indicative of good adherence to a gluten-free diet in pediatric patients with celiac disease. A separate assessment of adherence is needed focusing on knowledge, behavior while dining out, and intent to adhere.

Strategies to improve the efficiency of celiac disease diagnosis in the laboratory.

The demand for testing to detect celiac disease (CD) autoantibodies has increased, together with the cost per case diagnosed, resulting in the adoption of measures to restrict laboratory testing. We designed this study to determine whether opportunistic screening to detect CD-associated autoantibodies had advantages compared to efforts to restrict testing, and to identify the most cost-effective diagnostic strategy. We compared a group of 1678 patients in which autoantibody testing was restricted to cases in which the test referral was considered appropriate (G1) to a group of 2140 patients in which test referrals were not reviewed or restricted (G2). Two algorithms A (quantifying IgA and Tissue transglutaminase IgA [TG-IgA] in all patients), and B (quantifying only TG-IgA in all patients) were used in each group, and the cost-effectiveness of each strategy was calculated. TG-IgA autoantibodies were positive in 62 G1 patients and 69 G2 patients. Among those positive for tissue transglutaminase IgA and endomysial IgA autoantibodies, the proportion of patients with de novo autoantibodies was lower (p=0.028) in G1 (11/62) than in G2 (24/69). Algorithm B required fewer determinations than algorithm A in both G1 (2310 vs 3493; p<0.001) and G2 (2196 vs 4435; p<0.001). With algorithm B the proportion of patients in whom IgA was tested was lower (p<0.001) in G2 (29/2140) than in G1 (617/1678). The lowest cost per case diagnosed (4.63 euros/patient) was found with algorithm B in G2. We conclude that opportunistic screening has advantages compared to efforts in the laboratory to restrict CD diagnostic testing. The most cost-effective strategy was based on the use of an appropriate algorithm.

May the assessment of baseline mucosal molecular pattern predict the development of gluten related disorders among microscopic enteritis?

AIM: To evaluate mucosal baseline mRNA expression of tissue transglutaminase 2 (tTG2), interferon gamma (IFNγ), toll-like receptor 2 (TLR2) and Myeloid Differentiation factor 88 (MyD88) in patients with microscopic enteritis (ME). METHODS: We retrospectively enrolled 89 patients with ME of different etiology, which was defined within a 2-year mean period of follow-up. Baseline histological examination was performed on Hematoxylin-Eosin stained sections and CD3 lymphocyte immunohistochemistry was used for intraepithelial lymphocyte count (IELs). ME was defined according to the criteria of Bucharest Consensus Conference. For each patient, formalin embedded biopsy samples of the duodenum referred to the period of ME diagnosis were retrieved. Real-time polymerase chain reaction (RT-PCR) was used to detect the amount of mRNA coding for tTG2, IFNγ, TLR2 and MyD88, and the quantity was expressed as fold change compared to controls. Control group was represented by duodenal normal specimens from 15 healthy subjects undergoing endoscopy for functional symptoms. Comparisons among continuous variables were performed by One way analysis of variance (ANOVA) and Bonferroni's test. The χ(2) test was used for categorical variables. Pearson's test was used to evaluate correlations. Receiver operating curves were drawn for all four markers to estimate sensitivity and specificity in discriminating the development of CD and GS. RESULTS: After a period of follow up of 21.7 ± 11.7 mo, the following diagnoses were achieved: gluten related disorders in 48 subjects (31 CD; 17 GS) and non-gluten related ones in 41 (29 Irritable Bowel Syndrome - IBS; 12 Others). CD patients had the highest tTG2 levels (8.3 ± 4.5). The ANOVA plus Bonferroni analysis showed that CD > Other ME > GS = IBS > negative controls. A cut off value of 2.258 was able to discriminate between CD and GS with a sensitivity of 52.94% and a specificity of 87.1%. Additionally, CD patients had the highest IFNγ levels (8.5 ± 4.1). ANOVA plus Bonferroni demonstrated CD > Other ME > GS = IBS > negative controls. A cut off of 1.853 was able to differentiate CD and GS with a sensitivity of 47.06% and a specificity of 96.77%. Patients with non gluten-related causes of ME exhibited the highest TLR2 levels (6.1 ± 1.9) as follows: Other ME > CD = GS = IBS > negative controls. TLR2 was unable to discriminate CD from GS. Patients with CD overexpressed MyD88 levels similarly to non gluten-related causes of DL (7.8 ± 4.9 and 6.7 ± 2.9), thus CD = Other ME > GS = IBS > negative controls. A cut off of 3.722 was able to differentiate CD from GS with a sensitivity of 52.94% and a specificity of 74.19%. IELs count (15-25 and more than 25/100 enterocytes) strongly correlated with mRNA levels of all tested molecules (P < 0.0001). CONCLUSION: Our results confirm that a single marker is unable to predict a discrimination among ME underlying conditions as well as between CD and GS. Mucosal high levels of tTG and IFNγ mRNA may predict the development of CD more than GS with high specificity, despite an expected low sensitivity. TLR2 does not discriminate the development of CD from GS. MyD88 levels indicate that intestinal permeability is more increased when a severe intestinal damage underlies ME in both gluten related and unrelated conditions. Therefore, the results of the present paper do not seem to show a clear translational value.

Trends and racial/ethnic disparities in gluten-sensitive problems in the United States: findings from the National Health and Nutrition Examination Surveys from 1988 to 2012.

OBJECTIVES: Racial disparities in the prevalence of celiac disease (CD) and the number of people without CD avoiding gluten (PWAG) in the United States are unknown. We aimed to describe racial differences in the prevalence of CD and PWAG, and evaluate the trends of CD in the noninstitutionalized civilian adult population of the US between 1988 and 2012. METHODS: A population-based cross-sectional study was conducted using data from the National Health and Nutrition Examination Surveys (NHANES) from 1988 to 1994, 1999 to 2004, and 2009 to 2012. Serum samples from the NHANES participants were tested for CD serology, which included IgA tissue transglutaminase (tTG IgA) and, if findings were abnormal, for IgA endomysial antibodies. Information about adherence to a gluten-free diet was obtained by means of an interviewer-administered questionnaire. RESULTS: In NHANES 2009-2012, the adjusted prevalence of CD was significantly higher (P<0.0001) among non-Hispanic whites (1.0%) than among non-Hispanic blacks (0.2%) and Hispanics (0.3%), whereas the adjusted prevalence of PWAG was significantly higher (P=0.01) in blacks (1.2%) as compared with Hispanics (0.5%) and whites (0.7%). The seroprevalence of CD in adults aged 50 years and older increased from 0.17% (95% confidence interval (CI) 0.03-0.33) in 1988-1994 to 0.44% (95% CI 0.24-0.81) in 2009-2012 (P<0.05). CONCLUSIONS: The overall prevalence of CD increased between 1988 and 2012 and is significantly more common in whites. In addition, a higher proportion of individuals maintaining a gluten-free diet in the absence of a diagnosis of CD are blacks.

Seroprevalence of celiac disease among United Arab Emirates healthy adult nationals: a gender disparity.

AIM: To determine celiac disease (CD) prevalence and associated manifestations or risk factors in healthy adult Emiratis. METHODS: It is a cross-sectional prospective study, recruiting 1197 (573 women and 624 men) healthy Emiratis between September 2007 and April 2008 among those who went to Al Ain Hospital to undertake the prenuptial examination. Test for anti-tissue transglutaminase (tTG) IgA antibodies was used for CD diagnosis. Subjects with positive results in the anti tTG antibodies assay were also tested for anti-endomysial (EMA) IgA antibodies. A structured interview was used to collect basic demographic and clinical recall data including: information on name, contact address, age, gender, education status, previous diagnosis of CD, diagnosis of CD in 1(st) degree relatives and history of "chronic diarrhea, anemia, headache, hepatitis, diabetes, tumor, and thyroid disorder". RESULTS: Fourteen blood samples (1.17%; 14/1197) were seropositive for CD. The latent CD seropositive patients were 13 women and 1 man and therefore the seroprevalence of CD was 1:86 (14/1197) for adult Emiratis: 1:44 (13/573) for women and 1:624 for men. Binary logistic regression revealed that history of chronic anemia (crude OR = 7.09; 95%CI: 2.32-21.61; P = 0.003) and being a woman (OR = 14.46; 95%CI: 1.89-110.91; P = 0.001) were associated with CD seropositivity. Whereas, the thyroid disorder showed a positive association with CD seropositivity that approach statistical significance (OR = 11.30; 95%CI: 1.32-96.95; P = 0.09) and therefore was included in the multiple logistic regression analysis, which showed that CD seropositivity is independently associated only with history of chronic anemia (OR = 4.58; 95%CI: 1.45-14.48; P = 0.01) and being a woman person (OR = 10.47; 95%CI: 1.33-82.14; P = 0.026). CONCLUSION: Compared to men the CD seroprevalence among women was remarkably higher. The CD association with women and chronic anemia is of importance from a public health perspective.

Serological assessment for celiac disease in IgA deficient adults.

PURPOSE: Selective immunoglobulin A deficiency is the most common primary immunodeficiency disorder that is strongly overrepresented among patients with celiac disease (CD). IgG antibodies against tissue transglutaminase (tTG) and deamidated gliadin peptides (DGP) serve as serological markers for CD in IgA deficient individuals, although the diagnostic value remains uncertain. The aim of this study was to investigate the prevalence of these markers in a large cohort of IgA deficient adults with confirmed or suspected CD and relate the findings to gluten free diet. METHODS: Sera from 488,156 individuals were screened for CD in seven Swedish clinical immunology laboratories between 1998 and 2012. In total, 356 out of 1,414 identified IgA deficient adults agreed to participate in this study and were resampled. Forty-seven IgA deficient blood donors served as controls. Analyses of IgG antibodies against tTG and DGP as well as HLA typing were performed and a questionnaire was used to investigate adherence to gluten free diet. Available biopsy results were collected. RESULTS: Out of the 356 IgA deficient resampled adults, 67 (18.8%) were positive for IgG anti-tTG and 79 (22.2%) for IgG anti-DGP, 54 had biopsy confirmed CD. Among the 47 IgA deficient blood donors, 4 (9%) were positive for IgG anti-tTG and 8 (17%) for anti-DGP. Four were diagnosed with biopsy verified CD, however, 2 of the patients were negative for all markers. Sixty-eight of 69 individuals with positive IgG anti-tTG were HLA-DQ2/DQ8 positive whereas 7 (18.9%) of the 37 individuals positive for IgG anti-DGP alone were not. CONCLUSIONS: IgG anti-tTG seems to be a more reliable marker for CD in IgA deficient adults whereas the diagnostic specificity of anti-DGP appears to be lower. High levels of IgG antibodies against tTG and DGP were frequently found in IgA deficient adults despite adhering to gluten free diet.

Barriers impeding serologic screening for celiac disease in clinically high-prevalence populations.

BACKGROUND: Celiac disease is present in ~1% of the general population in the United States and Europe. Despite the availability of inexpensive serologic screening tests, ~85% of individuals with celiac disease remain undiagnosed and there is an average delay in diagnosis of symptomatic individuals with celiac disease that ranges from ~5.8-11 years. This delay is often attributed to the use of a case-based approach for detection rather than general population screening for celiac disease, and deficiencies at the level of health care professionals. This study aimed to assess if patient-centered barriers have a role in impeding serologic screening for celiac disease in individuals from populations that are clinically at an increased risk for celiac disease. METHODS: 119 adults meeting study inclusion criteria for being at a higher risk for celiac disease were recruited from the general population. Participants completed a survey/questionnaire at the William K. Warren Medical Research Center for Celiac Disease that addressed demographic information, celiac disease related symptoms (gastrointestinal and extraintestinal), family history, co-morbid diseases and conditions associated with celiac disease, and patient-centered barriers to screening for celiac disease. All participants underwent serologic screening for celiac disease using the IgA tissue transglutaminase antibody (IgA tTG) and, if positive, testing for IgA anti-endomysial antibody (IgA EMA) as a confirmatory test. RESULTS: Two barriers to serologic testing were significant across the participant pool. These were participants not knowing they were at risk for celiac disease before learning of the study, and participants not knowing where to get tested for celiac disease. Among participants with incomes less than $25,000/year and those less than the median age, not having a doctor to order the test was a significant barrier, and this strongly correlated with not having health insurance. Symptoms and co-morbid conditions were similar among those whose IgA tTG were negative and those who tested positive. CONCLUSION: There are significant patient-centered barriers that impede serologic screening and contribute to the delayed detection and diagnosis of celiac disease. These barriers may be lessened by greater education of the public and health care professionals about celiac disease symptoms, risk factors, and serologic testing.

Yield and cost of performing screening tests for constipation in children.

BACKGROUND: Chronic constipation is one of the most common reasons for pediatric outpatient visits. Clinical guidelines recommend that the work-up for chronic refractory constipation include thyroid function tests, celiac serology, and measurement of calcium and lead levels. Data to justify routine screening of constipated children using these laboratory tests are lacking. OBJECTIVES: To study the prevalence of celiac disease, hypothyroidism, hypercalcemia and lead poisoning in children with chronic constipation; and to estimate the health care costs of applying the guideline recommendations. METHOD: Charts of constipated children from 2007 to 2011 were reviewed for the present retrospective cohort study. Results and costs of thyroid function tests, celiac panel, total immunoglobulin (Ig) A, and determination of lead and calcium levels were analyzed. RESULTS: A total of 7472 children (mean age 7.9 years; 3908 female) were evaluated: 1731 patients were screened for celiac antibodies; 55 had elevated tissue transglutaminase IgA levels and 29 had biopsy-positive celiac disease. Only three celiac patients had constipation as the sole presenting symptom; 1703 patients were screened for total IgA levels; 55 had IgA deficiency and two had biopsy-positive celiac disease; 2332 had free T4 and/or thyroid-stimulating hormone levels; and 14 had hypothyroidism. Only two patients had constipation as the sole presenting symptom; 4651 patients had calcium levels measured, 10 of whom had high levels but normal repeat values. Three patients had normal lead levels. The mean cost per patient was USD$1,014. Total screening cost for all patients was USD$4.7 million. CONCLUSION: Constipation alone did not increase the likelihood of celiac disease or hypothyroidism above the population prevalence. No benefit of screening for hypercalcemia was found. High health care costs were associated with the use of screening tests for organic constipation.

Proteomic analyses lead to a better understanding of celiac disease: focus on epitope recognition and autoantibodies.

Proteomic technologies are being used with increasing frequency in the scientific community. In this review, we have highlighted their use in celiac disease (CD). The available techniques, which include two-dimensional (2D) gel electrophoresis, mass spectrometry, and antibody and tissue arrays, have been used to identify proteins or changes in protein expression specific to gut tissue from patients with CD. A number of studies have employed proteomic methodologies to determine the diagnostic biomarkers in body fluids or to examine changes in protein expression and posttranslational modifications during signaling. A fast technological development of these methods, along with the combination of classic techniques with proteomics, will lead to new discoveries, which will consent a better understanding of the pathogenesis of CD.

Coeliac disease in primary care: evaluation of a case-finding strategy.

BACKGROUND: Coeliac disease is still under-diagnosed as a consequence of poor physician awareness of the clinical spectrum of the disease. We evaluated the feasibility and the cost-effectiveness of a case-finding approach for early identification of cases, carried out by primary care practitioners. METHODS: We developed a case-finding strategy based on testing for anti-tissue transglutaminase IgA antibodies in subjects showing predefined signs and symptoms or belonging to at-risk groups. RESULTS: Sixty-nine primary care doctors and 60 primary care paediatricians agreed to participate. One thousand forty-one adults and 447 children were selected for anti-tissue transglutaminase testing during the year of the study (2001). Thirty-one (2.08%, 19 adults and 12 children) were ultimately diagnosed as coeliac patients. While no cases of coeliac disease had been diagnosed by the participating doctors in the previous year, 29 subjects were diagnosed as coeliacs in the year after the completion of the study (2002). The prevalence of confirmed coeliac disease in the population under study increased from 1:1,506 to 1:1,073 in adults and from 1:827 to 1:687 in children from year 2000 to 2001. When cases diagnosed in 2002 are included, the prevalence is 1:832 and 1:602, respectively. We calculated a cost of 923.25 euros for each new case diagnosed. CONCLUSIONS: Case-finding is a feasible and successful strategy for detecting undiagnosed coeliac patients and has the important added value of increasing the awareness of the disease among primary care physicians; it represents a cost-effective alternative to population screening for reducing the burden of undiagnosed coeliac disease.

Tissue transglutaminase antibodies in celiac disease: assessment of a commercial kit.

OBJECTIVE: Tissue transglutaminase was identified as the autoantigen eliciting endomysial antibody. A homemade enzyme-linked immunosorbent assay (ELISA)-based test was recently developed to determine quantitative titers of IgA antitissue transglutaminase antibody. Our objective in this study was to assess the suitability of a newly developed commercial kit for quantitative determination of antibody in patients with untreated celiac disease. MATERIALS: We tested serum samples from 79 untreated celiac patients, 42 healthy blood donors, and 18 patients with nonceliac intestinal disorders evaluated in two different centers. Samples were tested for antitissue transglutaminase, and antiendomysial and antigliadin antibodies in the center where diagnosis was performed. To assess interlaboratory variability of methods, 24 samples randomly selected were blindly tested in both centers. Antitissue transglutaminase antibodies were determined using a commercial kit (INOVA Diagnostics, Inc., San Diego, CA). RESULTS: Untreated celiac patients had significantly higher titers of antitissue transglutaminase than healthy and disease controls (p < 0.00001). According to the cut-off provided by the manufacturers (20 AU/mL), overall sensitivity was 92% (85% for one center and 100% for the other) and specificity was 98% (100% and 95%, respectively). Antiendomysial antibody was 86% sensitive and 100% specific. Discordance between antitissue transglutaminase and antiendomysial antibodies was detected in 13% of patients. Although two antitissue transglutaminase-negative cases had a positive antiendomysial antibody, the inverse situation was found in eight cases. A blind determination of antitissue transglutaminase on the same samples evidenced a good agreement (kappa statistic: 0.66) between both centers when assessment was qualitative (based on the decision of positive or negative). Although correlation of titers for both determinations was highly significant (r: 0.902, p < 0.00001), a very wide interlaboratory variability (median: 50%) was detected when absolute values were considered. CONCLUSIONS: The quantitative determination of antitissue transglutaminase using a commercial kit was highly sensitive and specific for detection of celiac disease. We observed an incomplete overlapping with antiendomysial antibody. The very high variability of values between laboratories still remains to be solved so as to propose the commercial ELISA assay for the screening of celiac disease.

Ligand-induced conformational changes in tissue transglutaminase: Monte Carlo analysis of small-angle scattering data.

Small-angle neutron and x-ray scattering experiments have been performed on type 2 tissular transglutaminase to characterize the conformational changes that bring about Ca(2+) activation and guanosine triphosphate (GTP) inhibition. The native and a proteolyzed form of the enzyme, in the presence and in the absence of the two effectors, were considered. To describe the shape of transglutaminase in the different conformations, a Monte Carlo method for calculating small-angle neutron scattering profiles was developed by taking into account the computer-designed structure of the native transglutaminase, the results of the Guinier analysis, and the essential role played by the solvent-exposed peptide loop for the conformational changes of the protein after activation. Although the range of the neutron scattering data is rather limited, by using the Monte Carlo analysis, and because the structure of the native protein is available, the distribution of the protein conformations after ligand interaction was obtained. Calcium activation promotes a rotation of the C-terminal with respect to the N-terminal domain around the solvent-exposed peptide loop that connects the two regions. The psi angle between the longest axes of the two pairs of domains is found to be above 50 degrees, larger than the psi value of 35 degrees calculated for the native transglutaminase. On the other hand, the addition of GTP makes possible conformations characterized by psi angles lower than 34 degrees. These results are in good agreement with the proposed enzyme activity regulation: in the presence of GTP, the catalytic site is shielded by the more compact protein structure, while the conformational changes induced by Ca(2+) make the active site accessible to the substrate.