Endoscopic diagnosis of a colonic localisation of a mantle cell lymphoma.

Extra-nodal localisations of mantle cell lymphomas are most frequently found in the gastrointestinal tract. It is therefore important for an endoscopist to be familiar with the endoscopic image of a mantle cell lymphoma. In this case series of three patients with colonic involvement of mantle cell lymphoma, we discuss the endoscopic diagnosis.

Olmesartan-induced enteropathy treated with budesonide.

Olmesartan, an angiotensin receptor blocker, is a widely spread antihypertensive drug. Seronegative villous atrophy of the small intestine due to olmesartan use was first described in 2012. We present a new case of olmesartan-induced enteropathy and compare it to recent literature. This case might suggest a use of budesonide for treatment.

Caramel colour and process by-products in foods and beverages: Part I - Development of a UPLC-MS/MS isotope dilution method for determination of 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (THI), 4-methylimidazole (4-MEI) and 2-methylimidazol (2-MEI).

Caramel colours are used by the food industry in a wide range of foods and beverages. During their manufacturing, low molecular weight compounds such as 4-methylimidazole (4-MEI), the structural isomer of 4-MEI, 2-methylimidazole (2-MEI) and 2-acetyl-4-tetrahydroxy-butylimidazole (THI) are generated. The presence of these inevitable by-products of caramel manufacturing can be hazardous to human health. This publication describes an isotope dilution Ultra-High-performance Liquid Chromatography tandem mass spectrometric method (UHPLC-MS/MS) that was developed and validated for the simultaneous quantification of these impurities in both beverages/liquids and foods. A limit of quantification of 5 µg/kg was obtained for 4-MEI and THI. The expanded measurement uncertainty (U; k = 2) for these compounds was below 51% in beverages/liquids and below 56% in foods. As higher measurement uncertainties were obtained for 2-MEI, the developed analytical procedure can only be used in a semi-quantitative way for this compound.

Caramel colour and process contaminants in foods and beverages: Part II - Occurrence data and exposure assessment of 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (THI) and 4-methylimidazole (4-MEI) in Belgium.

In Europe, 2-acetyl-4-(1,2,3,4-tetrahydroxybutyl)imidazole (THI) and 4-methylimidazole (4-MEI) are - to a certain level - allowed to be present in the food colours ammonia caramel (E 150c) and sulphite ammonia caramel (E 150d). Besides their presence in food colours, exposure to these contaminants may also include other dietary sources. This study describes the occurrence of THI and 4-MEI in a wide variety of food products (n = 522) purchased from the Belgian market and their dietary intake in Belgian consumers from 15 years old onwards. THI was found to be present in 22.4% of the investigated foods at a level up to 551 µg/kg. For 4-MEI (57.7% quantifiable), concentrations up to 2,835 µg/kg were observed. The average dietary intake amounted to 0.02-0.36 µg kg-1 bw-1 day for THI and 0.4-3.7 µg kg-1 bw-1 day for 4-MEI. Coffee, cola and beer were contributing most to the dietary THI and 4-MEI intake in Belgium.

The novel ADAMTS13-p.D187H mutation impairs ADAMTS13 activity and secretion and contributes to thrombotic thrombocytopenic purpura in mice.

BACKGROUND: Congenital thrombotic thrombocytopenic purpura (TTP) is characterized by mutations in the ADAMTS13 gene, which either impair protein secretion or influence ADAMTS13 (A Disintegrin-like And Metalloprotease domain with ThromboSpondin type-1 motif, member 13) activity. Phenotypic consequences of these mutations have not yet been evaluated in animal models for TTP. OBJECTIVES: To identify the in vitro effect of a novel ADAMTS13 mutation and to investigate whether this mutation induces TTP in vivo. METHODS: All 29 ADAMTS13 exons with exon-intron boundaries of a patient with pregnancy-onset TTP were sequenced. Wild-type and mutant ADAMTS13 proteins were both transiently and stably expressed in human embryonic kidney cells, and their activity was evaluated in vitro using fluorescence resonance energy transfer and flow assays. Molecular dynamics simulations were performed to study Ca(2+) stability. Adamts13(-/-) mice were hydrodynamically injected with wild-type and mutant expression plasmids and triggered with recombinant human von Willebrand factor. RESULTS: We identified a novel heterozygous c.559G>C mutation in exon 6 of the proposita's ADAMTS13 gene. This mutation resulted in a p.Asp187His substitution (p.D187H), which was located in the high affinity Ca(2+) -binding site in the metalloprotease domain of ADAMTS13. The homozygous p.D187H mutation down-regulated ADAMTS13 activity in vitro. Impaired proteolytic activity was linked to unstable Ca(2+) binding as visualized using a molecular dynamics simulation. In addition, the p.D187H mutation affects protein secretion in vitro. In Adamts13(-/-) mice, the homozygous p.D187H mutation reduced ADAMTS13 secretion and activity and contributed to TTP when these mice were triggered with recombinant human von Willebrand factor. CONCLUSIONS: Our data indicate that the p.D187H mutation impairs ADAMTS13 activity and secretion and is responsible for TTP onset in mice.

A semi-probabilistic modelling approach for the estimation of dietary exposure to phthalates in the Belgian adult population.

In this study, a semi-probabilistic modelling approach was applied for the estimation of the long-term human dietary exposure to phthalates--one of world's most used families of plasticisers. Four phthalate compounds were considered: diethyl phthalate (DEP), di-n-butyl phthalate (DnBP), benzylbutyl phthalate (BBP) and di(2-ethylhexyl) phthalate (DEHP). Intake estimates were calculated for the Belgian adult population and several subgroups of this population for two considered scenarios using an extended version of the EN-forc model. The highest intake rates were found for DEHP, followed by DnBP, BBP and DEP. In the Belgian adult population, men and young adults generally had the highest dietary phthalate intake estimates. Nevertheless, predicted dietary intake rates for all four investigated phthalates were far below the corresponding tolerable daily intake (TDI) values (i.e. P99 intake values were 6.4% of the TDI at most), which is reassuring because adults are also exposed to phthalates via other contamination pathways (e.g. dust ingestion and inhalation). The food groups contributing most to the dietary exposure were grains and grain-based products for DEP, milk and dairy products for DnBP, meat and meat products or grains and grain-based products (depending on the scenario) for BBP and meat and meat products for DEHP. Comparison of the predicted intake results based on modelled phthalate concentrations in food products with intake estimates from other surveys (mostly based on measured concentrations) showed that the extended version of the EN-forc model is a suitable semi-probabilistic tool for the estimation and evaluation of the long-term dietary intake of phthalates in humans.

Autolytic degradation of ocriplasmin: a complex mechanism unraveled by mutational analysis.

Ocriplasmin, a truncated form of plasmin, is commercialized in the USA and in Europe under the trade name Jetrea(®), and indicated for the treatment of symptomatic vitreomacular adhesion and vitreomacular traction including when associated with macular hole ≤400 µm, respectively. We have shown in a previous study that ocriplasmin undergoes autolytic degradation when injected in eye vitreous, which leads to its rapid inactivation. In order to investigate this process further, we have introduced in ocriplasmin a variety of amino acid substitutions within or in the immediate vicinity of the three major autolytic cleavage sites. We demonstrate here that autolytic inactivation of ocriplasmin is a sequential process where initial cleavage occurs primarily between residues 156 and 157. Reduction or even blocking of autolysis can be achieved by mutating a limited number of key residues. In this study, we also report the identification of a series of ocriplasmin variants with improved resistance to autolysis and unimpaired catalytic activity. Such variants represent useful tools for the exploration of therapeutic approaches aiming at non-surgical resolution of vitreomacular adhesion.

Selective regulation of IP3-receptor-mediated Ca2+ signaling and apoptosis by the BH4 domain of Bcl-2 versus Bcl-Xl.

Antiapoptotic B-cell lymphoma 2 (Bcl-2) targets the inositol 1,4,5-trisphosphate receptor (IP(3)R) via its BH4 domain, thereby suppressing IP(3)R Ca(2+)-flux properties and protecting against Ca(2+)-dependent apoptosis. Here, we directly compared IP(3)R inhibition by BH4-Bcl-2 and BH4-Bcl-Xl. In contrast to BH4-Bcl-2, BH4-Bcl-Xl neither bound the modulatory domain of IP(3)R nor inhibited IP(3)-induced Ca(2+) release (IICR) in permeabilized and intact cells. We identified a critical residue in BH4-Bcl-2 (Lys17) not conserved in BH4-Bcl-Xl (Asp11). Changing Lys17 into Asp in BH4-Bcl-2 completely abolished its IP(3)R-binding and -inhibitory properties, whereas changing Asp11 into Lys in BH4-Bcl-Xl induced IP(3)R binding and inhibition. This difference in IP(3)R regulation between BH4-Bcl-2 and BH4-Bcl-Xl controls their antiapoptotic action. Although both BH4-Bcl-2 and BH4-Bcl-Xl had antiapoptotic activity, BH4-Bcl-2 was more potent than BH4-Bcl-Xl. The effect of BH4-Bcl-2, but not of BH4-Bcl-Xl, depended on its binding to IP(3)Rs. In agreement with the IP(3)R-binding properties, the antiapoptotic activity of BH4-Bcl-2 and BH4-Bcl-Xl was modulated by the Lys/Asp substitutions. Changing Lys17 into Asp in full-length Bcl-2 significantly decreased its binding to the IP(3)R, its ability to inhibit IICR and its protection against apoptotic stimuli. A single amino-acid difference between BH4-Bcl-2 and BH4-Bcl-Xl therefore underlies differential regulation of IP(3)Rs and Ca(2+)-driven apoptosis by these functional domains. Mutating this residue affects the function of Bcl-2 in Ca(2+) signaling and apoptosis.

Inventory of experiences from national/regional dietary monitoring surveys using EPIC-Soft.

BACKGROUND/OBJECTIVES: The EPIC-Soft 24-h recall (the software developed to conduct 24-h dietary recalls (24-HDRs) in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study) has been used in several regional/national dietary monitoring surveys. The main objective of the study was to present and discuss design, settings, logistics, data management and quality controls of dietary monitoring surveys that used EPIC-Soft for the collection of food consumption data. SUBJECTS/METHODS: Within European Food Consumption Validation (EFCOVAL), a questionnaire including questions on current/past EPIC-Soft experiences and requirements for the future was developed and sent to all institutes that used EPIC-Soft in their food consumption survey(s) (five surveys in four different countries). RESULTS: EPIC-Soft was used in the national food consumption survey in Belgium (≥ 15-97 years), Germany (14-80 years), the Netherlands (19-30 years and 2-6 years) and Spain (regional only; 4-18 years). Participation rates in these surveys were 46% (Belgium), 42% (Germany), 42% (Dutch survey in adults), 79% (Dutch survey in children) and 77% (Basque survey). Two 24-HDRs were collected by conducting face-to-face interviews in Belgium and Spain, and through telephone interviews in Germany and the Netherlands. Except the Netherlands (19-30 years), where the study was conducted only in autumn, in all other countries the study was conducted throughout the four seasons, including all days of the week. Interviews were conducted by dietitians, except in Germany and Spain. Mean EPIC-Soft interview time was 20-34 min. The dropout rate between the first and second interviews was low (<7.5%) in all surveys. CONCLUSION: EPIC-Soft has been used in different study settings and populations for nutritional exposure assessments. To guarantee the comparability of data across countries, recommendations for the design of future pan-European dietary monitoring surveys using EPIC-Soft should be drawn.

The standardized computerized 24-h dietary recall method EPIC-Soft adapted for pan-European dietary monitoring.

BACKGROUND/OBJECTIVES: The EPIC-Soft program (the software initially developed to conduct 24-h dietary recalls (24-HDRs) in the European Prospective Investigation into Cancer and Nutrition (EPIC) Study) was recommended as the best way to standardize 24-HDRs for future pan-European dietary monitoring. Within European Food Consumption Validation (EFCOVAL), EPIC-Soft was adapted and further developed on various aspects that were required to optimize its use. In this paper, we present the structure and main interview steps of the EPIC-Soft program, after implementation of a series of new specifications deemed to satisfy specific requirements of pan-European monitoring surveys and other international studies. SUBJECTS/METHODS: Updates to optimize the EPIC-Soft program were ascertained according to the following stepwise approach: (1) identification of requested specifications to be potentially implemented through an ad hoc 'EPIC-Soft specifications questionnaire' sent to past, current and possible future users of the software; (2) evaluation of the specifications in collaboration with two ad hoc task force groups and through a workshop; (3) development of a technical solution for each retained specification; (4) implementation of the specifications by software developers; (5) testing and amendment of bugs. RESULTS: A number of new specifications and facilities were implemented to EPIC-Soft program. In addition, the software underwent a full reprogramming and migration to a modern Windows environment, including changes in its internal architecture and user interface. Although the overall concept and structure of the initial software were not changed substantially, these improvements ease the current and future use of EPIC-Soft and increase further its adaptation to other countries and study contexts. CONCLUSIONS: EPIC-Soft is enriched with further functions and facilities expected to fulfil specific needs of pan-European dietary monitoring and risk assessment purposes. The validity, feasibility and relevance of this software for different national and international study designs, and the logistical aspects related to its implementation are reported elsewhere.

Discovery of novel mechanisms and molecular targets for the inhibition of activated thrombin activatable fibrinolysis inhibitor.

BACKGROUND: Thrombin activatable fibrinolysis inhibitor (TAFI) is an important regulator of fibrinolysis and an attractive target to develop profibrinolytic drugs. OBJECTIVE: To analyze the (inhibitory) properties of five monoclonal antibodies (mAbs) directed towards rat TAFI (i.e. MA-RT13B2, MA-RT30D8, MA-RT36A3F5, MA-RT36B2 and MA-RT82F12). METHODS AND RESULTS: Direct interference of the mAb with rat activated TAFI (TAFIa) activity was assayed using a chromogenic activity assay. This revealed reductions of 79% +/- 1%, 54% +/- 4%, and 19% +/- 2% in activity in the presence of a 16-fold molar excess of MA-RT13B2, MA-RT36A3F5, and MA-RT82F12, respectively whereas MA-RT30D8 and MA-RT36B2 had no direct inhibitory effect. Additionally, MA-RT13B2 and MA-RT36A3F5 reduced rat TAFIa half-life by 56% +/- 2% and 61% +/- 3%. Tissue-type plasminogen activator mediated in vitro clot lysis was determined using rat plasma. Compared to potato tuber carboxypeptidase inhibitor, MA-RT13B2, MA-RT30D8, MA-RT36A3F5, and MA-RT82F12 reduced clot lysis times by 86% +/- 14%, 100% +/- 5%, 100% +/- 10%, and 100% +/- 11%, respectively. During epitope mapping, Arg(227) and Ser(251) were identified as major residues interacting with MA-RT13B2. Arg(188) and His(192) contribute to the interaction with MA-RT36A3F5. Arg(227), Ser(249), Ser(251), and Tyr(260) are involved in the binding of MA-RT30D8 and MA-RT82F12 with rat TAFI(a). The following mechanisms of inhibition have been deduced: MA-RT13B2 and MA-RT36A3F5 have a destabilizing effect on rat TAFIa whereas MA-RT30D8 and MA-RT82F12 partially block the access to the active site of TAFIa or interact with the binding of TAFIa to the blood clot. CONCLUSIONS: The described inhibitory mAb towards rat TAFIa will facilitate TAFI research in murine models. Additionally, we reveal novel molecular targets for the direct inhibition of TAFIa through different mechanisms.

Comparative evaluation of stable TAFIa variants: importance of alpha-helix 9 and beta-sheet 11 for TAFIa (in)stability.

BACKGROUND: Activated thrombin activatable fibrinolysis inhibitor (TAFIa) plays a pivotal role in fibrinolysis. TAFIa activity is regulated by a temperature-dependent instability. This instability has not only complicated the study of structure-function relationships of TAFIa but has also prevented the crystallization of TAFIa. Furthermore, the TAFIa instability has severely compromised the development of activity inhibiting monoclonal antibodies. Recently, we combined all known stabilizing mutations (i.e. S305C, T325I, T329I, H333Y and H335Q) resulting in a synergistic (one hundred and eightyfold) stabilization of TAFIa at 37 degrees C. All these residues are located in an amino acid region (AA297-335) consisting of alpha-helix 9 and beta-sheet 11. OBJECTIVES: To provide a comparative evaluation of the characteristics of a panel of stable TAFIa mutants and an energy-minimized model of the most stable TAFI variant. RESULTS: The catalytic efficiency for activation of TAFI by thrombin/thrombomodulin was higher for all TAFI mutants compared with TAFI-wild type (wt). Except for TAFI variants carrying T325I-T329I, S305C-T325I or S305C-T325I-T329I mutations, the catalytic efficiency for Hip-Arg hydrolysis by TAFIa was similar for the TAFI mutants compared with the wild type. All TAFIa variants were equally well inhibited by potato tuber carboxypeptidase inhibitor (PTCI) and showed a significantly increased antifibrinolytic potential in accordance with their increased stability. Based on the intrinsic fluorescence decay of TAFIa, two independent structural transitions were found to be associated with the loss of functional activity. CONCLUSIONS: Using molecular dynamic calculations on both TAFI-wt and TAFI-S305C-T325I-T329I-H333Y-H335Q models, we were able to identify the molecular interactions that contribute to the increased stability of the mutants.

Breakfast habits affect overall nutrient profiles in adolescents.

OBJECTIVE: To describe breakfast consumption patterns, on a nutrient and food item level, in Belgian adolescents. DESIGN: A 7-day estimated food record was administered in a cross-sectional survey. SETTING: Secondary schools in Ghent, Belgium. SUBJECTS: A total of 341 adolescents (13-18 years old), multistage clustered sampling. RESULTS: The energy contribution of breakfast to daily energy intake was on average 15.7% in boys and 14.9% in girls. Significantly more overweight girls and significantly more girls following vocational training were categorised as eating a low-quality breakfast. In boys, the energy contribution of polysaccharides was significantly higher in consumers of good-quality breakfasts. The intake of all selected micronutrients was significantly higher in consumers of good-quality breakfasts. In girls, the total energy intake and the proportional intake of proteins and polysaccharides were significantly higher in consumers of good-quality breakfasts, while the proportional contribution of total fat, monounsaturated and polyunsaturated fatty acids was significantly lower in these girls. The intake of all micronutrients was significantly higher in girls consuming a good-quality breakfast. In all adolescents, consumers of a good-quality breakfast had significantly higher intakes of bread, fruit, vegetables, milk and milk products, and fruit juice, while intake of soft drinks was significantly lower than in consumers of low-quality breakfasts. CONCLUSIONS: Consumers of a good-quality breakfast had a better overall dietary pattern - on a nutrient and food group level - than consumers of a low-quality breakfast. A daily breakfast, including whole-grain products, fruit and (semi-) skimmed milk products or an alternative source of calcium, is recommended.

Sources of saturated fatty acids in Belgian adolescents' diet: implications for the development of food-based dietary guidelines.

The objectives of the present study are to describe the dietary sources of total fat and of saturated fatty acids (SFA) and to formulate food-based dietary guidelines for SFA in Belgian adolescents. A random sample of 13-18-year-old adolescents was drawn from secondary schools in the region of Ghent. A 7 d estimated food record method was used to quantify nutrient and food intake. The average daily SFA intake is 4 % above the recommended 10 % of the total energy contribution. The most important contributors of SFA on food group level were 'fats, oils and savoury sauces', 'meat and meat products', 'sugar, confectionery, sweet fillings and sauces', 'cheese', 'milk and milk products' and 'bread, rusk and breakfast rolls'. On food subgroup level 'fresh meat', 'high-fat margarine' and 'high-fat cheese' had the highest contribution to SFA intake in all adolescents. Adolescents with a low SFA intake (lowest tertile) were compared with adolescents with a high intake (highest tertile). In the lowest tertile the intake of total fat and MUFA was significantly lower than in the highest tertile, while the intake of total carbohydrates, mono- and disaccharides and complex carbohydrates was significantly higher. Overall, the high-fat cheese intake is significantly lower in the lowest tertile, while the fruit intake is higher. The present analysis shows that the nutritional profile of Belgian adolescents could be potentially improved by decreasing the portion sizes of fresh meat (in boys), high-fat margarine, high-fat cheese and reducing intake of commercially prepared baked goods and processed foods, including fast foods.

Iron intake and dietary sources of iron in Flemish adolescents.

OBJECTIVE: To investigate the dietary iron intake and food sources of iron in Flemish adolescents. DESIGN: Cross-sectional survey; dietary assessment method: a 7-day estimated food record. SETTING: Private and public secondary schools in Ghent, a city in the Dutch-speaking part of Belgium. SUBJECTS: A total of 341 adolescents (129 boys and 212 girls), 13-18 y, randomly selected by a multistage clustered sampling technique. RESULTS: The mean total iron intake (s.d.) for boys was 13.4 (+/- 2.91) mg/day and for girls 10.1 (+/- 2.79) mg/day. A proportion of 38.8% of the boys and 99.5% of the girls had a mean total iron intake below the Belgian Recommended Dietary Allowance and 3.1% of the boys and 71.2% of the girls below the British Estimated Average Requirement. When bioavailable iron intake is considered, 84.5% of the boys and only 16.5% of the girls met the age-specific requirement. The food groups with the highest mean proportional contribution to total iron intake in both males and females were bread, meat and meat products, cereals and potatoes. A comparison of adolescents from the highest tertile of iron intake (mg/day) with adolescents from the lowest tertile showed a significantly higher energy-adjusted intake of brown bread and a significantly lower intake of soft drinks in the former group in both boys and girls. A significantly higher energy-adjusted intake of breakfast cereals in adolescents of the highest tertile than those of the lowest tertile was seen in girls only. Analyses in consumers only did not change this overall picture. CONCLUSIONS: One can conclude that the mean iron intake of Flemish girls is considerably lower than the current recommendations. An increased iron intake in this subgroup of the population is therefore advisable.

Molecular mechanisms of mild and moderate hemophilia A.

Mutations responsible for mild/moderate hemophilia A were extensively characterized over the last 15 years and more than 200 mutations have been identified. However, most of the molecular mechanisms responsible for the reduced factor (F)VIII levels in patients' plasma were determined only recently. Recent progresses in the study of the FVIII molecule three-dimensional structure provided a major insight for understanding molecular events leading to mild/moderate hemophilia A. This allowed prediction of mutations impairing FVIII folding and intracellular processing, which result in reduced FVIII secretion. Mutations potentially slowing down FVIII activation by thrombin were also identified. A number of mutations were also predicted to result in altered stability of activated FVIII. Biochemical analyses allowed identification of mutations reducing FVIII production. Mutations impairing FVIII stability in plasma, by reducing FVIII binding to von Willebrand factor (VWF) were also characterized. Defects in FVIII activity, notably slow activation by thrombin, or abnormal interaction with FIXa, were also recently demonstrated. Biochemical analysis of FVIII variants provided information regarding the structure/function relationship of the FVIII molecule and validated predictions of the three-dimensional structure of the molecule. These observations also contributed to explain the discrepant activities recorded for some FVIII variants using different types of FVIII assays. Altogether, the study of the biochemical properties of FVIII variants and the evaluation of the effects of mutations in three-dimensional models of FVIII identified molecular mechanisms potentially explaining reduced FVIII levels for a majority of patients with mild/moderate hemophilia A. It is expected that these studies will improve diagnosis and treatment of this disease.

Critical role of glutamic acid 202 in the enzymatic activity of stromelysin-1 (MMP-3).

To test the hypothesis that Glu202, adjacent to the His201 residue that participates in the coordination of Zn(2+) in matrix metalloproteinase-3 (MMP-3 or stromelysin-1), plays a role in its enzymatic activity it was substituted with Ala, Lys or Asp by site-specific mutagenesis. Wild-type proMMP-3, proMMP-3(E202A), proMMP-3(E202K) and proMMP-3(E202D) were expressed in Escherichia coli and purified to apparent homogeneity. Whereas 33-kDa wild-type proMMP-3 (consisting of the propeptide and catalytic domains) was quantitatively converted to 24-kDa active MMP-3 by treatment with p-aminophenyl-mercuric acetate (APMA), proMMP-3(E202A) and proMMP-3 (E202K) were fully resistant to APMA and proMMP-3 (E202D) was quantitatively converted into a 14-kDa species. In contrast, treatment with plasmin quantitatively converted the wild-type and the three mutant proMMP-3 moieties into the corresponding 24-kDa MMP-3 moieties. Biospecific interaction analysis revealed comparable affinity for binding to plasminogen of wild-type and mutant proMMP-3 (K(a) of 2.6-6.3 x 10(6) M(-1)) or MMP-3 (K(a) of 33-58 x 10(6) M(-1)) moieties. The affinity for binding to single-chain urokinase-type plasminogen activator (scu-PA) was also similar for wild-type and mutant proMMP-3 (K(a) of 5.0-6.9 x 10(6) M(-1)) or MMP-3 (K(a) of 37-72 x 10(6) M(-1)) moieties. However, MMP-3(E202A) and MMP-3(E202K) did not hydrolyze plasminogen whereas MMP-3(E202D) showed an activity of 20--30% of wild-type MMP-3. All three mutants were inactive towards scu-PA under conditions where this was quantitatively cleaved by wild-type MMP-3. Furthermore, MMP-3(E202A) and MMP-3(E202K) were inactive toward a fluorogenic substrate and MMP-3 (E202D) displayed about 15% of the activity of wild-type MMP-3. Taken together, these data suggest that Glu202 plays a crucial role in the enzymatic activity of MMP-3.