Tatyana S. Zamolodchikova

Activation of recombinant proenteropeptidase by duodenase.

Duodenase, a serine proteinase from bovine Brunners (duodenal) glands that was predicted to be a natural activator of enteropeptidase zymogen, cleaves and activates recombinant single‐chain bovine proenteropeptidase (k cat/K m=2700 M−1 s−1). The measured rate of proenteropeptidase cleavage by duodenase was about 70‐fold lower compared with the rate of trypsin‐mediated cleavage of the zymogen. The role of duodenase is supposed to be the primary activator of proenteropeptidase maintaining a certain level of active enteropeptidase in the duodenum. A new scheme of proteolytic activation cascade of digestive proteases is discussed.

Crystal structure of bovine duodenase, a serine protease, with dual trypsin and chymotrypsin‐like specificities

The three‐dimensional structure of duodenase, a serine protease from bovine duodenum mucosa, has been determined at 2.4Å resolution. The enzyme, which has both trypsin‐like and chymotrypsin‐like activities, most closely resembles human cathepsin G with which it shares 57% sequence identity and similar specificity. The catalytic Ser195 in duodenase adopts the energetically favored conformation typical of serine proteinases and unlike the strained state typical of lipase/esterases. Of several waters in the active site of duodenase, the one associated with Ser214 is found in all serine proteinases and most lipase/esterases. The conservation of the Ser214 residue in serine proteinase, its presence in the active site, and participation in a hydrogen water network involving the catalytic triad (His57, Asp107, and Ser195) argues for its having an important role in the mechanism of action. It may be referred to as a fourth member of the catalytic triad. Duodenase is one of a growing family of enzymes that possesses trypsin‐like and chymotrypsin‐like activity. Not long ago, these activities were considered to be mutually exclusive. Computer modeling reveals that the S1 subsite of duodenase has structural features compatible with effective accommodation of P1 residues typical of trypsin (Arg/Lys) and chymotrypsin (Tyr/Phe) substrates. The determination of structural features associated with functional variation in the enzyme family may permit design of enzymes with a specific ratio of trypsin and chymotrypsin activities. Proteins 2000;41:8–16.

Cloning and Molecular Modeling of Duodenase with Respect to Evolution of Substrate Specificity within Mammalian Serine Proteases That Have Lost a Conserved Active-Site Disulfide Bond

Mammalian serine proteases such as the chromosome 14 (Homo sapiens, Mus musculus) located granzymes, chymases, cathepsin G, and related enzymes including duodenase collectively represent a special group within the chymotrypsin family which we refer to here as “granases”. Enzymes of this group have lost the ancient active-site disulfide bond Cys191-Cys220 (bovine chymotrypsinogen A numbering) which is strongly conserved in classic serine proteases such as pancreatic, blood coagulation, and fibrinolysis proteases and others (granzymes A, M, K and leukocyte elastases). We sequenced the cDNA encoding bovine (Bos taurus) duodenase, a granase with unusual dual trypsin-like and chymotrypsin-like specificity. The sequence revealed a 17-residue signal peptide and two-residue (GlyLys) activation peptide typical for granases. Production of the mature enzyme is apparently accompanied by further proteolytic processing of the C-terminal pentapeptide extension of duodenase. Similar C-terminal processing is known for another dual-specific granase, human cathepsin G. Using phylogenetic analysis based on 39 granases we retraced the evolution of residues 189 and 226 crucial for serine protease primary specificity. The analysis revealed that while there is no obvious link between mutability of residue 189 and the appearance of novel catalytic properties in granases, the mutability of residue 226 evidently gives rise to different specificity subgroups within this enzyme group. The architecture of the extended substrate-binding site of granases and structural basis of duodenase dual specificity based on molecular dynamic method are discussed. We conclude that the marked selectivity of granases that is crucial to their role as regulatory proteases has evolved through the fine-tuning of specificity at three levels— primary, secondary, and conformational.

Graspases—a Special Group of Serine Proteases of the Chymotrypsin Family That Has Lost a Conserved Active Site Disulfide Bond

In this report we propose a new approach to classification of serine proteases of the chymotrypsin family. Comparative structure–function analysis has revealed two main groups of proteases: a group of trypsin-like enzymes and graspases (granule-associated proteases). The most important structural peculiarity of graspases is the absence of conservative “active site” disulfide bond Cys191–Cys220. The residue at position 226 in the S1-subsite of graspases is responsible for substrate specificity, whereas the residue crucial for specificity in classical serine proteases is located at position 189. We distinguish three types of graspases on the base of their substrate specificity: 1) chymozymes prefer uncharged substrates and contain an uncharged residue at position 226; 2) duozymes possess dual trypsin-like and chymotrypsin-like specificity and contain Asp or Glu at 226; 3) aspartases hydrolyze Asp-containing substrates and contain Arg residue at 226. The correctness of the proposed classification was confirmed by phylogenic analysis.

Interaction between duodenase and α1-proteinase inhibitor

The interaction between duodenase, a newly recognized serine proteinase belonging to the small group of Janusfaced proteinases, and α1-proteinase inhibitor (α1-PI) from human serum was investigated. The stoichiometry of the inhibition was 1.2 mol/mol. The presence of a stable enzyme–inhibitor complex was shown by SDS-PAGE. The mechanism of interaction between duodenase and α1-PI was shown to be of the suicide type. The equilibrium and inhibition constants are 13 ± 3 nM and (1.9 ± 0.3)·105 M–1·sec–1, respectively. Based on the association rate constant of the enzyme–inhibitor complex and localization of duodenase and α1-PI in identical compartments, α1-PI is suggested to be a duodenase inhibitor in vivo.

Serine proteases of small intestine mucosa — localization, functional properties, and physiological role

In this review we present data about small intestine serine proteases, which are a considerable part of the proteolytic apparatus in this major part of the gastrointestinal tract. Serine proteases of intestinal epitheliocytes, their structural-functional features, cellular localization, physiological substrates, and mechanisms of activity regulation are examined. Information about biochemical and functional properties of serine proteases is presented in a common context with morphological and physiological data, this being the basis for understanding the functional processes taking place in upper part of the intestine. Serine proteases play a key role in the physiology of the small intestine and provide the normal functioning of this organ as part of the digestive system in which hydrolysis and suction of food substances occur. They participate in renewal and remodeling of tissues, retractive activity of smooth musculature, hormonal regulation, and defense mechanisms of the intestine.

Expression of duodenase-like protein in epitheliocytes of Brunner’s glands in human duodenal mucosa

A duodenase, a protease structurally related to human cathepsin G, was found earlier in bovine duodenal mucosa. It was demonstrated that under the influence of duodenase an enteropeptidase zymogen is activated in vitro showing the possible participation of duodenase in the cascade of activation of digestive enzymes. To identify a duodenase functional analog in human duodenum, an immunofluorescence study of duodenal mucosa was conducted by confocal microscopy using antibodies to human cathepsin G and to bovine duodenase. The previously unknown place of synthesis and secretion of cathepsin G — Paneth cells located at the bottom of Lieberkuhn crypts — was revealed. Binding of cathepsin G-specific antibodies in a rough endoplasmic reticulum zone and in the cryptal duct was observed. Duodenase-specific immunofluorescence but not that of cathepsin G was found in the epitheliocytes and secretory ducts of Brunner’s glands, which are characteristic sites of duodenase biosynthesis in cattle. Binding of CD14-specific antibodies in the Brunner’s glands, where the antibodies co-localized with the antibodies to duodenase, was also demonstrated. These data indicate the presence of a protein immunologically similar to duodenase in the human duodenal mucosa. Our study demonstrated the absence of its colocalization with cathepsin G in Brunner’s glands.

Serine proteases in immune protection of the small intestine

The gastrointestinal tract is subject to a huge antigenic load, which is especially significant in the intestinal lumen. Being the connecting link between the organism and the external environment, the small intestine fulfils not only digestive and transport functions, but also protective ones and acts as a selective barrier for the flow of nutrients. This review considers proteases of the protective system of small intestine cells, their biochemical properties and activation mechanisms, and involvement in biochemical processes responsible for normal functioning and defense reactions of the intestine. Serine proteases of intestinal immunity are multifunctional enzymes making proteolytic attack aimed to immediately exterminate aggressive elements of the intestinal contents (allergens, toxins), to activate (inactivate) zymogens, receptors, and peptide hormones, and to hydrolyze protein precursors and other biologically active factors. Proteases of intestinal immunity control the inflammatory response, proliferation of B-lymphocytes, apoptosis, and secretory and contractive activity of the intestine; they release neurogenic factors, inactivate biologically active substances, and are involved in degradation of the intercellular matrix and in tissue remodeling.

Interaction between human α2-macroglobulin and duodenase, a serine proteinase with dual specificity

Interaction between a serine proteinase from bovine duodenum and human serum α2-macroglobulin (α2-MG) was studied. α2-MG is established to be one of the most effective duodenase inhibitors. The enzyme is completely inhibited in less than 30 sec at equimolar ratio of the inhibitor and enzyme (concentration 2·10−8 M). Under identical conditions, the rate of duodenase association with α2-MG is at least 2.5-fold higher than the rate of chymotrypsin association with this inhibitor. The interaction with duodenase results in proteolysis of the inhibitor subunit in the “bait region”. Similarly to other proteases, duodenase in the complex with α2-MG retains the intact catalytic apparatus and ability to hydrolyze some small substrates. But the duodenase-inhibitor complex is fully inactive to proteins (bovine serum albumin). The stoichiometry of the enzyme interaction with the inhibitor is 2: 1 (mol/mol). Based on the association rate constant and the termination time of the duodenase and α2-MG in vivo association, α2-MG is suggested to be a physiological regulator of the enzyme.

Comparative Study of the Action of Bovine Duodenal Proteinases (Duodenases) on Polypeptide Substrates

A comparative study of substrate specificity of bovine duodenal proteinases—chymotrypsin-like duodenase (ChlD) and dual-specificity duodenase (dsD)—was carried out using oligopeptide substrates (human proinsulin, glucagon, melittin, angiotensinogen fragment 1-14). ChlD displayed mainly chymotrypsin-like properties towards these substrates, hydrolyzing peptide bonds carboxy-terminally to bulky aliphatic or aromatic residues. In melittin, ChlD additionally cleaved peptide bonds after Thr and Ser residues. Dual-specificity duodenase (dsD) significantly restricted its specificity to only trypsin-like or only chymotrypsin-like or displayed full activity, combining both specificities, depending on substrate. Both ChlD and dsD efficiently hydrolyzed a single peptide bond (Phe8–His9) in angiotensinogen fragment 1-14. The kinetic parameters of angiotensinogen fragment 1-14 cleavage by ChlD and dsD were determined (kcat/Km = 80,500 M-1·sec-1 for ChlD and 103,000 M-1·sec-1 for dsD).