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rMCP-2, the Major Rat Mucosal Mast Cell Protease, an Analysis of Its Extended Cleavage Specificity and Its Potential Role in Regulating Intestinal Permeability by the Cleavage of Cell Adhesion and Junction Proteins.

Fu Z, Thorpe M, Hellman L - PLoS ONE (2015)

Bottom Line: However, no target for this effect has yet been identified.The consensus sequence obtained from the phage display analysis was used to screen the rat proteome for potential targets.Three potential targets were identified: the loop 1 of occludin, protocadherin alpha 4 and cadherin 17, which indicated that these proteins were at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.

View Article: PubMed Central - PubMed

Affiliation: Education Ministry Key Laboratory for Biomedical Engineering, Zhejiang University, 38 Zheda Road, Hangzhou, Zhejiang, China; Department of Cell and Molecular Biology, Uppsala University, Uppsala, The Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden.

ABSTRACT
Mast cells of the rat intestinal mucosa express three chymotryptic enzymes named rMCP-2, -3 and 4. rMCP-2, the most abundant of these enzymes, has been shown to increase the permeability of the intestinal epithelium, most likely by cleavage of cell adhesion and junction proteins and thereby play a role in intestinal parasite clearance. However, no target for this effect has yet been identified. To address this question we here present its extended cleavage specificity. Phage display analysis showed that it is a chymase with a specificity similar to the corresponding enzyme in mice, mMCP-1, with a preference for Phe or Tyr in the P1 position, and a general preference for aliphatic amino acids both upstream and downstream of the cleavage site. The consensus sequence obtained from the phage display analysis was used to screen the rat proteome for potential targets. A few of the most interesting candidate substrates were cell adhesion and cell junction molecules. To see if these proteins were also susceptible to cleavage in their native conformation we cleaved 5 different recombinant cell adhesion and cell junction proteins. Three potential targets were identified: the loop 1 of occludin, protocadherin alpha 4 and cadherin 17, which indicated that these proteins were at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.

No MeSH data available.


Related in: MedlinePlus

Purification and activation of recombinant rMCP-2.rMCP-2 was expressed in the human HEK 293 EBNA cell line. The proenzyme was first purified, from the conditioned media of transfected cells, on Ni-NTA beads (-EK) and then activated by removal of the His6-tag by enterokinase digestion (+EK). The purified enzyme, before and after enterokinase cleavage was analyzed on SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Panel A shows schematic drawings of the initial translated product, the product after signal sequence removal and finally the active enzyme after enterokinase cleavage. Panel B shows the Coomassie blue stained SDS-PAGE gel with a size marker, the purified enzyme before and after enterokinase cleavage (-EK and +EK). The two arrows show the proenzyme (PE) and the active enzyme (AE).
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pone.0131720.g001: Purification and activation of recombinant rMCP-2.rMCP-2 was expressed in the human HEK 293 EBNA cell line. The proenzyme was first purified, from the conditioned media of transfected cells, on Ni-NTA beads (-EK) and then activated by removal of the His6-tag by enterokinase digestion (+EK). The purified enzyme, before and after enterokinase cleavage was analyzed on SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Panel A shows schematic drawings of the initial translated product, the product after signal sequence removal and finally the active enzyme after enterokinase cleavage. Panel B shows the Coomassie blue stained SDS-PAGE gel with a size marker, the purified enzyme before and after enterokinase cleavage (-EK and +EK). The two arrows show the proenzyme (PE) and the active enzyme (AE).

Mentions: A DNA construct containing the coding region for the active rMCP-2 was designed and ordered from Genscript (Piscataway, NJ, USA). This DNA fragment also contained the coding region for a His6-tag followed by an EK site, which was positioned N-terminally of the active rMCP-2 coding sequence. This construct was cloned into the mammalian expression vector pCEP-Pu2 for expression in HEK 293 EBNA cells [17]. The His6-tag facilitates purification on Ni2+ chelating immobilized metal ion affinity chromatography columns and cleavage with EK activates the enzyme, whilst simultaneously removing the His6-tag. Samples of inactive and activated protease were separated on SDS-PAGE gels in order to ensure successful removal of the His6-tag and the EK susceptible cleavage site (Fig 1). The inactive protease migrated as 27–28 kDa bands and the EK digested enzyme was only slightly smaller (Fig 1).


rMCP-2, the Major Rat Mucosal Mast Cell Protease, an Analysis of Its Extended Cleavage Specificity and Its Potential Role in Regulating Intestinal Permeability by the Cleavage of Cell Adhesion and Junction Proteins.

Fu Z, Thorpe M, Hellman L - PLoS ONE (2015)

Purification and activation of recombinant rMCP-2.rMCP-2 was expressed in the human HEK 293 EBNA cell line. The proenzyme was first purified, from the conditioned media of transfected cells, on Ni-NTA beads (-EK) and then activated by removal of the His6-tag by enterokinase digestion (+EK). The purified enzyme, before and after enterokinase cleavage was analyzed on SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Panel A shows schematic drawings of the initial translated product, the product after signal sequence removal and finally the active enzyme after enterokinase cleavage. Panel B shows the Coomassie blue stained SDS-PAGE gel with a size marker, the purified enzyme before and after enterokinase cleavage (-EK and +EK). The two arrows show the proenzyme (PE) and the active enzyme (AE).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482586&req=5

pone.0131720.g001: Purification and activation of recombinant rMCP-2.rMCP-2 was expressed in the human HEK 293 EBNA cell line. The proenzyme was first purified, from the conditioned media of transfected cells, on Ni-NTA beads (-EK) and then activated by removal of the His6-tag by enterokinase digestion (+EK). The purified enzyme, before and after enterokinase cleavage was analyzed on SDS-PAGE and visualized with Coomassie Brilliant Blue staining. Panel A shows schematic drawings of the initial translated product, the product after signal sequence removal and finally the active enzyme after enterokinase cleavage. Panel B shows the Coomassie blue stained SDS-PAGE gel with a size marker, the purified enzyme before and after enterokinase cleavage (-EK and +EK). The two arrows show the proenzyme (PE) and the active enzyme (AE).
Mentions: A DNA construct containing the coding region for the active rMCP-2 was designed and ordered from Genscript (Piscataway, NJ, USA). This DNA fragment also contained the coding region for a His6-tag followed by an EK site, which was positioned N-terminally of the active rMCP-2 coding sequence. This construct was cloned into the mammalian expression vector pCEP-Pu2 for expression in HEK 293 EBNA cells [17]. The His6-tag facilitates purification on Ni2+ chelating immobilized metal ion affinity chromatography columns and cleavage with EK activates the enzyme, whilst simultaneously removing the His6-tag. Samples of inactive and activated protease were separated on SDS-PAGE gels in order to ensure successful removal of the His6-tag and the EK susceptible cleavage site (Fig 1). The inactive protease migrated as 27–28 kDa bands and the EK digested enzyme was only slightly smaller (Fig 1).

Bottom Line: However, no target for this effect has yet been identified.The consensus sequence obtained from the phage display analysis was used to screen the rat proteome for potential targets.Three potential targets were identified: the loop 1 of occludin, protocadherin alpha 4 and cadherin 17, which indicated that these proteins were at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.

View Article: PubMed Central - PubMed

Affiliation: Education Ministry Key Laboratory for Biomedical Engineering, Zhejiang University, 38 Zheda Road, Hangzhou, Zhejiang, China; Department of Cell and Molecular Biology, Uppsala University, Uppsala, The Biomedical Center, Box 596, SE-751 24 Uppsala, Sweden.

ABSTRACT
Mast cells of the rat intestinal mucosa express three chymotryptic enzymes named rMCP-2, -3 and 4. rMCP-2, the most abundant of these enzymes, has been shown to increase the permeability of the intestinal epithelium, most likely by cleavage of cell adhesion and junction proteins and thereby play a role in intestinal parasite clearance. However, no target for this effect has yet been identified. To address this question we here present its extended cleavage specificity. Phage display analysis showed that it is a chymase with a specificity similar to the corresponding enzyme in mice, mMCP-1, with a preference for Phe or Tyr in the P1 position, and a general preference for aliphatic amino acids both upstream and downstream of the cleavage site. The consensus sequence obtained from the phage display analysis was used to screen the rat proteome for potential targets. A few of the most interesting candidate substrates were cell adhesion and cell junction molecules. To see if these proteins were also susceptible to cleavage in their native conformation we cleaved 5 different recombinant cell adhesion and cell junction proteins. Three potential targets were identified: the loop 1 of occludin, protocadherin alpha 4 and cadherin 17, which indicated that these proteins were at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.

No MeSH data available.


Related in: MedlinePlus