Subcellular Localization, Substrate Specificity and Crystallization of Duodenase, A Potential Activator of Enteropeptidase

Abstract

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold iinmunocytochemical method. Duodenase exhibits trypsin-like and chyinotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be a-N-tosylglycylprolyllysine 4-nitroanilide (kJK,,, of 35 000 M-' s-'), a-N-succinylthreonylprolyllysine 4-nitroanilide (kc4,/K1,! of 18 000 M-' s-') and a-N-serylprolyllysine 4-nitroanilide (k,JK,, of 2600 M-' s-I), all of which contain the P I -P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9 -PS'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (K,,, of 87 pM; k,;,, of 1.4 s-I; k,,,/K,,, of 16000 M-' s-I). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron Xrays to 0.2-nm resolution.

Proteolytic action of duodenase is required to induce DNA synthesis in pulmonary artery fibroblasts

“…It was possible to isolate duodenase from bovine jejunum using methodology identical to that employed for the purification of sMCP-1 from gastrointestinal tissues. However, duodenase has previously been localized only to the epithelial cells of Brunner's glands located in the duodenal wall [4]. This suggests either that duodenase is present in both cell types, or that each site produces distinct enzymes that are nonetheless highly similar structurally, functionally and immunologically.…”

“…The first of two peptide substrates analysed, bee venom melittin, was cleaved preferentially at Lys7, with secondary cleavage at Lys23, as previously described for duodenase [17]. Porcine angiotensinogen (1-14) was rapidly and specifically cleaved at Phe8, as has been shown for duodenase [4]. Therefore, the jejunal enzyme we purified was identified as duodenase, or a highly similar variant of the enzyme.…”

“…Anti-duodenase serum and affinity-purified anti-(sMCP-1) IgG were prepared as described previously [4,8]. Anti-(p85 PtdIns 3-kinase) Ig was obtained from TCS Biologicals (Botolph Claydon, UK) and [c-32 P]ATP from Amersham (Amersham).…”

“…The closely related enzyme, sheep mast cell proteinase-1 (sMCP-1) is 85% identical at the amino-acid level [2] and, due to close similarity of the primary substrate binding region, has a strikingly similar cleavage specificity [3]. Duodenase was originally immunolocalized to epithelial cells of Brunner's glands within the duodenum, and was an activator of enteropeptidase [4]. Other studies employing esterase staining have provided evidence for the expression of an enzyme with trypsin-like properties, distinct from tryptase, in intestinal mucosal mast cells, and in the lung around bronchioles and within the alveolar septa [5].…”

Characterization of the duodenase-1 gene and its associations with resistance to Streptococuus agalactiae in hybrid tilapia (Oreochromis spp.)

“…It is synthesized in Brunner's glands and mast cells [2]. In mammals, there is only one duodenase gene, which has been extensively studied at the protein level, including purification and some catalytic properties [3], primary structure [1], and subcellular location [4]. The proinflammatory effect of duodenase has been experimentally confirmed in mammals [5].…”