Střevní enzymy

Duodenase

Substráty

• Selective trypsin- and chymotrypsin-like substrate specificity [7]
• Natural activator of enteropeptidase zymogen [9]
• Hydrolyzes the amide bonds of substrates having Lys, Arg, Tyr, Phe and Leu residues at the P1 position
• Most active at pH 7.9-8.2.
• Hydrolyzes protein substrates yielding large and stable fragments
• V.s. belongs to the group of processing enzymes
• Primary activator of proenteropeptidase [9]

Struktura

• serine endopeptidase (duodenase) isolated from bovine duodenum mucosa
• Single chain
• Native enzyme is a monomeric protein
• Has two forms (I and II)
• Possess similar properties
• Differ in their amino acid composition
• Příbuzný/podobný:
• Chymases from mast cells
• Granzymes from cytotoxic T-lymphocytes
• Cathepsin G from leukocytes [9]

Lokalizace

• Epitheliocytes of Brunner's glands
• Mast cells within the intestinal mucosa
• Lungworm-infected lung
• Important additional role in inflammation and tissue remodelling [7]
• Duodenase in duodenal gland secretions may enter the duodenal lumen
• Contact enterocytes containing membrane-bound proenteropeptidase [9]
• Duodenase meets proenteropeptidase earlier than proenteropeptidase meets trypsin [9]

Další souvislosti

• Proteolytic action of duodenase is required to induce DNA synthesis in pulmonary artery fibroblasts [8]
• Inhibited by
• Pertussis toxin [8]
• Wortmannin [8]
• Ryba tilapia
• Duodenase-1 gene 4 of 7 SNPs
• Important role in the immuno-resistance to Streptococcus agalactiae [10]

Inhibited

• Alpha1-proteinase inhibitor (alpha1-PI) from human serum [6]
• Stable enzyme-inhibitory complex duodenase-alpha 1-PI [7]
• Human blood serpins
• Suicide mechanism of the duodenase interaction with the human blood serpins was proved [7]

Irreversibly

• Diisopropylphosphofluoridate
• Phenylmethanesulphonyl fluoride
• Alpha-N-Tosyl-L-lysine chloromethane
• Alpha-N-tosyl-L-phenylalanine chloromethane
• Inhibition of trypsin-like and chymotrypsin-like activities of duodenase
• Strongly suppressed by trypsin inhibitors from different sources
• Soybeans
• Bovine lungs
• Lima beans [8]

Bez vlivu

• Chicken egg white ovomucoid [8]

Enteropeptidase - enterokinase

Lokalizace

• Enzyme produced by cells of the duodenum

Substráty

• Involved in human and animal digestion
• Converts trypsinogen (a zymogen) into its active form trypsin
• Activation of pancreatic digestive enzymes

Stavba

• serine protease (EC 3.4.21.9)
• Chymotrypsin-clan of serine proteases
• Heavy-chain of 82-140 kDa
• Disulfide-linked
• Anchors enterokinase in the intestinal brush border membrane
• Amino-terminal membrane-spanning domain
• Mucin-like domain
• Two repeats found in complement serine proteases C1r and C1s
• MAM domain
• Macrophage scavenger receptor cysteine-rich repeat [3]
• Transmembrane part [18]
• Little influence on the recognition of small peptides [18]
• Strongly influences macromolecular substrate recognition and inhibitor specificity [18]
• Light-chain of 35–62 kDa
• Contains the catalytic subunit [3]
• Typical chymotrypsin-like serine protease
• Activation cleavage site
• Between the heavy and light chains
• Sequence Val-Ser-Pro-LysˇIle
• Might be recognized by trypsin or other trypsin-like proteases [18]
• Structurally similar to these proteins [3]
• Type II transmembrane serine protease (TTSP)
• Localized to the brush border of the duodenal and jejunal mucosa
• It is absent in crypts
• Significant in villous enterocytes
• Maximal in the upper half of the villi
• Especially on the brush border [4]
• Synthesized as a zymogen, proenteropeptidase
• Requires activation by duodenase or trypsin [3]
• TTSPs are synthesized as single chain zymogens with N-terminal propeptide sequences of different lengths
• Activated by cleavage at the carboxyl side of lysine or arginine residues present in a highly conserved activation motif.
• Once activated, TTSPs are predicted to remain membrane-bound
• Through a conserved disulfide bond linking the pro- and catalytic domains [3]

Historical significance

• Discovered by Ivan Pavlov
• Pavlov’s student demonstrated that canine duodenal secretions dramatically stimulated the digestive activity of pancreatic enzymes
• Especially trypsinogen
• Pavlov named it enterokinase
• Kunitz, who showed that the activation of trypsinogen by enterokinase was catalytic
• 1950s, cattle trypsinogen was shown to be activated autocatalytically
• By cleavage of an N-terminal hexapeptide
• Enteropeptidase since 1970

Cattle enteropeptidase

• 17 potential N-linked glycosylation sites in the heavy chain
• Three in the light chain
• Most of these are conserved in other species
• Heavy chain
• Hydrophobic section near the N-terminus that supports the transmembrane anchor
• Influences the specificity of enteropeptidase [1]

• Native enteropeptidase
• Resistant to soybean trypsin inhibitor
• Isolated light chain is subtle
• Native enteropeptidase and the isolated light chain
• Have similar activity toward Gly-(Asp)4-Lys-NHNap
• Secluded light chain has distinctly decreased activity toward trypsinogen
• Analogous selective defect in the recognition of trypsinogen can be produced in two-chain enteropeptidase by
• Heating
• Acetylation [1]

Human enteropeptidase

• Intrinsic membrane protein of enterocytes of the proximal small intestine
• Key role in the digestive cascade, cleaving the N-terminal activation peptide from trypsinogen to produce trypsin
• Subsequent activation of other pancreatic zymogens by trypsin [9]
• Single-chain precursor proenteropeptidase
• Activated by duodenase
• A serine protease expressed in the duodenum
• Catalyses the hydrolysis of peptide bonds in proteins
• Does not catalyze transfer of phosphate groups
• Exhibits trypsin-like activity
• Cleaving proteins following a lysine at a specific cleavage site (Asp-Asp-Asp-Asp-Lys)
• Trypsindependent activation of other pancreatic zymogens
• Chymotrypsinogen
• Proelastase
• Procarboxypeptidase
• Prolipase [1]

Enterokinase deficiency

• Seriously impairs protein absorption [4]

Proteinase-activated receptor 2

• Present at the apical and basolateral membrane of enterocytes
• Activation of this receptor by trypsin
• Stimulates enterocytes to secrete eicosanoids
• Act locally in the intestinal wall
• Regulate epithelial growth [4]
• Enterokinase localization on the luminal surface of the duodenal villi
• Contributes to enterocyte growth
• By generating active trypsin on the cell surface [4]
• Congenital enteropeptidase deficiency, as a novel target for obesity treatment [16]

Genetics and disease relevance

• In humans, enteropeptidase is encoded by the PRSS7 gene (also known as ENTK) on chromosome 21q21
• Rare recessive disorder with enteropeptidase deficiency
• MRNA expression is limited to the proximal small intestine
• protein is found in enterocytes of duodenum and proximal jejunum
• Trypsinogen encounters enteropeptidase and is activated
• Trypsin then cleaves and activates
• Other pancreatic serine protease zymogens
• Metalloprotease zymogens (procarboxypeptidases)
• Prolipases [3]
• Duodenase mutations
• Defective activation of proenteropeptidase
• May possibly lead to disease, similar to enterokinase deficiency [4]
• Severe intestinal malabsorption caused by congenital deficiency of enteropeptidase
• Can be life-threatening
• Responds to oral supplementation with pancreatic extract [3]

Klinika

• Only 13 cases of primary enterokinase deficiency have been reported
• Three additional patients were reported with a similar clinical picture
• But with unmeasured intestinal enterokinase activity [4]
• Present at birth with diarrhea and failure to thrive [4]
• Unknown reasons, protein digestion improves with time
• Can be adequate in the adult [4]
• Adulthood, patients have normal body weight and no GI symptoms
• Even in the absence of pancreatic enzyme supplements. [4]
• Almost all patients present at birth with diarrhea and failure to thrive.
• All patients exhibit hypoproteinemia.
• Vomiting has been reported in approximately 50% of patients [4]
• Children present with
• Malabsorption syndrome
• Muscle wasting
• Failure to thrive
• Hypoproteinemia (eg, kwashiorkor)
• Generalized during the disease's early phase and includes:
• Steatorrhea - probably secondary to protein malnutrition
• Generalized edema -in one half of affected patients [4]
• Severe anemia - presumably secondary to protein malabsorption and vitamin E deficiency [4]

Secondary enterokinase/enteropeptidase deficiency

• In patients with partial or total villous atrophy
• Usually not significantly affected in these conditions
• Patient developed celiac disease at age 25 years
• Enterokinase levels remained low after normalization of intestinal mucosa with gluten-free diet [4]
• Deficiency causes severe protein malabsorption with poor growth and development [4]

Duodenopancreatic reflux

• Duodenal contents in ductus pancreaticus
• Could result in trypsinogen activation by enteropeptidase within the pancreas
• Followed by acute pancreatitis
• Beta-site APP-cleaving enzyme1 (BACE1)
• A protease closely linked to the pathogenesis of Alzheimer disease [4]
• Highly expressed in pancreatic acinar cells
• Could possibly protect the pancreas from premature trypsinogen activation [4]

Aktivace enteropeptidázy

• Activation cleavage site between the heavy and light chains
• Sequence Val-Ser-Pro-LysˇIle
• Might be recognized by
• Trypsin
• Other trypsin-like proteinases
• Duodenase [9]

• Enteropeptidase was released into the small intestine lumen
• When brush-border membranes were treated with
• Bile-acids
• Cholescystokinin–pancreozymin [19]
• Enzymes responsible for enteropeptidase shedding have not been characterized [19]