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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

Analysis of the cleavage of purified rat intestinal epithelium by rMCP-2.Panel A shows a one dimensional gel of purified rat epithelial layer incubated for 2 hours with inactive (C1) and active enzyme (T1). In panels C2 and T2 have 10 ug of the recombinant substrate, the rMCP-2 consensus substrate (VVLFSAVL), been added to the cleavage reaction. The samples have then been incubated with inactive and active enzyme for 2 hours as for C1 and T1. In the left four lanes in panel A the recombinant substrate have been incubated with inactive enzyme (control) for 0 or 2 hours (C(0h) and C(2h)) and with active enzyme for 0 or 2 hours (T(0h) and T(2h)). As can be seen from the figure cleavage of the recombinant substrate can only be seen in the T(2h) lane. In panels B and C the results from the 2D gel analysis is shown. In panel B and C the inactive and active enzymes have been used, respectively.
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pone.0131720.g005: Analysis of the cleavage of purified rat intestinal epithelium by rMCP-2.Panel A shows a one dimensional gel of purified rat epithelial layer incubated for 2 hours with inactive (C1) and active enzyme (T1). In panels C2 and T2 have 10 ug of the recombinant substrate, the rMCP-2 consensus substrate (VVLFSAVL), been added to the cleavage reaction. The samples have then been incubated with inactive and active enzyme for 2 hours as for C1 and T1. In the left four lanes in panel A the recombinant substrate have been incubated with inactive enzyme (control) for 0 or 2 hours (C(0h) and C(2h)) and with active enzyme for 0 or 2 hours (T(0h) and T(2h)). As can be seen from the figure cleavage of the recombinant substrate can only be seen in the T(2h) lane. In panels B and C the results from the 2D gel analysis is shown. In panel B and C the inactive and active enzymes have been used, respectively.

Mentions: As a next step in trying to identify potential intestinal targets we decided to test purified rat intestinal epithelium for its sensitivity to cleavage by rMCP-2. We used a recently published method to recover intact epithelial layers from rat intestines and thereby reduce the number of potential targets and the complexity of a 2D analysis [23]. Using a solution that dissolves the basement membrane combined with the air pressure makes it possible to isolate an intact epithelial layer as a sheet. This cell material was then exposed to active and inactive enzyme in separate reaction vials. The two samples were dissolved in sample buffer and separated on both one and two dimensional gels (Fig 5). By the 2D analysis an average of approximately 300 protein spots were reproducibly detected on gels by using silver staining (Fig 5). The proteins spanned a broad range of pI from 3 to 10 and apparent molecular mass from 10 to 100 kDa. However, after quantitative and qualitative analysis, no differential protein spots between rat intestinal epithelium incubated with inactive and active rMCP2 were detected, which have at least 2.0 fold increase or 0.5-fold decrease (Fig 5). As no difference could be detected the experiment was repeated and the consensus substrate was added to the tubes to ensure that the enzyme could cleave under these conditions (Fig 5A). The recombinant (thioredoxin) consensus substrate was efficiently cleaved under these conditions. However, we also observed some cleavage, although to a lesser degree, in the tube where inactive enzyme had been added indicating that active rMCP-2 or another enzyme with similar specificity was present in the tissue sample. One possibility would have been to add protease inhibitors to the sample during preparation. However, then the sample would have been swamped with inhibitors, which would most likely affected the active rMCP-2 when added to the sample. We therefore decided to try to look at pure recombinant cell adhesion and junction proteins for their susceptibility to cleavage by rMCP-2.


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)

Analysis of the cleavage of purified rat intestinal epithelium by rMCP-2.Panel A shows a one dimensional gel of purified rat epithelial layer incubated for 2 hours with inactive (C1) and active enzyme (T1). In panels C2 and T2 have 10 ug of the recombinant substrate, the rMCP-2 consensus substrate (VVLFSAVL), been added to the cleavage reaction. The samples have then been incubated with inactive and active enzyme for 2 hours as for C1 and T1. In the left four lanes in panel A the recombinant substrate have been incubated with inactive enzyme (control) for 0 or 2 hours (C(0h) and C(2h)) and with active enzyme for 0 or 2 hours (T(0h) and T(2h)). As can be seen from the figure cleavage of the recombinant substrate can only be seen in the T(2h) lane. In panels B and C the results from the 2D gel analysis is shown. In panel B and C the inactive and active enzymes have been used, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131720.g005: Analysis of the cleavage of purified rat intestinal epithelium by rMCP-2.Panel A shows a one dimensional gel of purified rat epithelial layer incubated for 2 hours with inactive (C1) and active enzyme (T1). In panels C2 and T2 have 10 ug of the recombinant substrate, the rMCP-2 consensus substrate (VVLFSAVL), been added to the cleavage reaction. The samples have then been incubated with inactive and active enzyme for 2 hours as for C1 and T1. In the left four lanes in panel A the recombinant substrate have been incubated with inactive enzyme (control) for 0 or 2 hours (C(0h) and C(2h)) and with active enzyme for 0 or 2 hours (T(0h) and T(2h)). As can be seen from the figure cleavage of the recombinant substrate can only be seen in the T(2h) lane. In panels B and C the results from the 2D gel analysis is shown. In panel B and C the inactive and active enzymes have been used, respectively.
Mentions: As a next step in trying to identify potential intestinal targets we decided to test purified rat intestinal epithelium for its sensitivity to cleavage by rMCP-2. We used a recently published method to recover intact epithelial layers from rat intestines and thereby reduce the number of potential targets and the complexity of a 2D analysis [23]. Using a solution that dissolves the basement membrane combined with the air pressure makes it possible to isolate an intact epithelial layer as a sheet. This cell material was then exposed to active and inactive enzyme in separate reaction vials. The two samples were dissolved in sample buffer and separated on both one and two dimensional gels (Fig 5). By the 2D analysis an average of approximately 300 protein spots were reproducibly detected on gels by using silver staining (Fig 5). The proteins spanned a broad range of pI from 3 to 10 and apparent molecular mass from 10 to 100 kDa. However, after quantitative and qualitative analysis, no differential protein spots between rat intestinal epithelium incubated with inactive and active rMCP2 were detected, which have at least 2.0 fold increase or 0.5-fold decrease (Fig 5). As no difference could be detected the experiment was repeated and the consensus substrate was added to the tubes to ensure that the enzyme could cleave under these conditions (Fig 5A). The recombinant (thioredoxin) consensus substrate was efficiently cleaved under these conditions. However, we also observed some cleavage, although to a lesser degree, in the tube where inactive enzyme had been added indicating that active rMCP-2 or another enzyme with similar specificity was present in the tissue sample. One possibility would have been to add protease inhibitors to the sample during preparation. However, then the sample would have been swamped with inhibitors, which would most likely affected the active rMCP-2 when added to the sample. We therefore decided to try to look at pure recombinant cell adhesion and junction proteins for their susceptibility to cleavage by rMCP-2.

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