Enterokinase (enteropeptidase): comparative aspects

Light, Albert; Janska, Hanna

doi:10.1016/0968-0004(89)90133-3

Cited by 88 publications

(70 citation statements)

References 17 publications

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“…EK is a highly specific enzyme that cleaves the Lys-Ile bond in its Asp-Asp-Asp-Lys-Ile recognition motif (24). Because Ile is the essential N-terminal amino acid of mature mMCP-7 and because EK is a relatively stable enzyme at pH 5.0, it was anticipated that the secreted recombinant pseudozymogen could be activated under conditions where the generated tryptase would have very little enzymatic activity until the pH is raised to 7.0.…”

Section: Expression Of Pro-enterokinase (Ek)-mmcp-7 and Pro-ek-mmcp-7mentioning

confidence: 99%

The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

Huang

Wong

Ghildyal

et al. 1997

Journal of Biological Chemistry

100

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Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used this in vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34 -55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the ␣-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptasespecific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the ␣-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions.

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Section: Expression Of Pro-enterokinase (Ek)-mmcp-7 and Pro-ek-mmcp-7mentioning

confidence: 99%

The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

Huang

Wong

Ghildyal

et al. 1997

Journal of Biological Chemistry

100

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“…Enterokinase (EK, also known as enteropeptidase) is a membrane-bound serine protease found in the duodenum and initiates activation of pancreatic hydrolases by cleaving and activating trypsinogen. Although the canonical target site for enterokinase is DDDDK, it is known that EK does not exhibit high stringency in its specificity for this sequence [3][4][5][6]. For instance, Liew et al showed that EK preferentially cleaved at an unexpected LKGDR site that was near the carboxyl terminus of N-terminal proCNP and more accessible than the internal canonical DDDDK sequence [5].…”

Section: Introductionmentioning

confidence: 99%

Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag

Shahravan

Chan

et al. 2008

Protein Expression and Purification

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In our work with designed minimalist proteins based on the bZIP motif, we have found our Histagged proteins to be prone to inclusion body formation and aggregation; we suspect this problem is largely due to the His tag, known to promote aggregation. Using AhR6-C/EBP, a hybrid of the AhR basic region and C/EBP leucine zipper, as representative of our bZIP-like protein family, we attempted removal of the His tag with enterokinase (EK) but obtained the desired cleavage product in very small yield. EK is known for proteolysis at noncanonical sites, and most cleavage occurred at unintended sites. We manipulated experimental conditions to improve specificity of proteolysis and analyzed the cleavage products; no effect was observed after changing pH, temperature, or the amount of EK. We then suspected the accessibility of the EK site was impeded due to protein aggregation. We found that the easily implemented strategy of addition of urea (1-4 M) greatly improved EK cleavage specificity at the canonical site and reduced adventitious cleavage. We believe that this enhancement in specificity is due to a more "open" protein structure, in which the now accessible canonical target can compete effectively with adventitious cleavage sites of related sequence.

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“…A specific binding function is supported by the high conservation of the SEA module in agrins from different species (Tsim et al, 1992). The digestion initiator enterokinase is physiologically the only enzyme that converts trypsinogen into trypsin in pancreatic fluid (Light & Janska, 1989). It is thought to interact with the intestinal brush border membrane through its modular noncatalytic heavy chain.…”

mentioning

confidence: 99%

The SEA module: A new extracellular domain associated with O‐glycosylation

1995

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Using a variety of homology search methods and multiple alignments, a new extracellular module was identified in (1) agrin, (2) enterokinase, (3) a 63‐kDa sea urchin sperm protein, (4) perlecan, (5) the breast cancer marker MUC1 (episialin), (6) the cell surface antigen 114/A10, and (7/8) two functionally uncharacterized, probably extracellular, Caenorhabditis elegans proteins. Despite the functional diversity of these adhesive proteins, a common denominator seems to be their existence in heavily glycosylated environments. In addition, the better characterized proteins mentioned above contain all O‐glycosidiclinked carbohydrates such as heparan sulfate that contribute considerably to their molecular masses. The common module might regulate or assist binding to neighboring carbohydrate moieties.

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Enterokinase (enteropeptidase): comparative aspects

Cited by 88 publications

References 17 publications

The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

Enhancing the specificity of the enterokinase cleavage reaction to promote efficient cleavage of a fusion tag

The SEA module: A new extracellular domain associated with O‐glycosylation

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