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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 by active rMCP-2 of a number of recombinant cell adhesion and cell junctional proteins.Panel A shows schematic drawings of the basic structure of occludin and claudin. The sequence of the surface loops of occludin and claudin 7 are shown in panel B. The extracellular region is shown in black where the potential rMCP-2 cleavage sites (Tyr and Phe) have been marked in red. The yellow region shows the short region of the membrane spanning region remaining in these clones and the purple region is the kinker including a Cys that has been inserted to form a loop structure of the extracellular loops in the 2xTrx construct. In panel C the cleavage pattern of the rMCP-2 consensus 2xTrx substrate (VVLFSAVL), as positive control, occluding loop 1, occluding loop 2 and claudin 7 loop 1 is depicted. In panel D the cleavage of rat cadherin 17, rat E-cadherin and human protocadherin alpha 4 is presented where the protein is marked above each cleavage reaction and the time of cleavage in minutes is depicted above each lane. These three latter recombinant proteins were produced in mammalian cells. As can be seen from the figure cadherin 17 and protocadherin alpha 4 are very sensitive to cleavage by rMCP-2 whereas almost no cleavage is seen for E-cadherin.
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pone.0131720.g006: Analysis of the cleavage by active rMCP-2 of a number of recombinant cell adhesion and cell junctional proteins.Panel A shows schematic drawings of the basic structure of occludin and claudin. The sequence of the surface loops of occludin and claudin 7 are shown in panel B. The extracellular region is shown in black where the potential rMCP-2 cleavage sites (Tyr and Phe) have been marked in red. The yellow region shows the short region of the membrane spanning region remaining in these clones and the purple region is the kinker including a Cys that has been inserted to form a loop structure of the extracellular loops in the 2xTrx construct. In panel C the cleavage pattern of the rMCP-2 consensus 2xTrx substrate (VVLFSAVL), as positive control, occluding loop 1, occluding loop 2 and claudin 7 loop 1 is depicted. In panel D the cleavage of rat cadherin 17, rat E-cadherin and human protocadherin alpha 4 is presented where the protein is marked above each cleavage reaction and the time of cleavage in minutes is depicted above each lane. These three latter recombinant proteins were produced in mammalian cells. As can be seen from the figure cadherin 17 and protocadherin alpha 4 are very sensitive to cleavage by rMCP-2 whereas almost no cleavage is seen for E-cadherin.

Mentions: A protein that previously has been identified as potential target by rMCP-2 is occludin, as it forms the barrier in the epithelial layer of the intestine [31, 32]. Claudins are also found in close connection to occludin junctions and were also represented in the potential target list in Table 1. Claudin 7 has been shown to be expressed at high levels in mouse and/or rat intestines [33]. Both of these are complicated membrane proteins that span the membrane 4 times and are therefore difficult to produce in a soluble and properly folded form (Fig 6A). We therefore decided to produce the extracellular loops that can be targets for an enzyme that act from outside of the cell. The extracellular region including a few amino acids from the outer part of the membrane-spanning region was inserted in the two thioredoxin vector. In order to stabilize the construct and obtain a folding similar to the bended loop structure of the native protein we inserted two cysteine residues, one in each end of the loop (Fig 6B). These cysteines will with high certainty form a stable disulphide bond after opening the bacterial cell where it is produced. It may even form in the cells as the E.coli Rosetta gami has a lower reducing potential than other E.coli strains and therefore cysteine bridges may form already within the cell. Occludin has two larger extracellular loops, which we produced separately (Fig 6A and 6B). Only the larger loop of claudin 7 was produced in this form as the small loop is very small and does not contain any sequence that could be a potential target for rMCP-2. As can be seen from Fig 6, a rapid cleavage of loop 1 of occludin, protocadherin alpha 4 and cadherin 17 was observed, but no cleavage of loop 2 of occludin, loop 1 of claudin 7, and E-cadherin could be detected. Interestingly, loop 1 of occludin contains almost a perfect repeat of chymase substrate sequences, whereas loop 2 of occluding and loop 1 of claudin only contains a few relatively poor potential site and both of them also contain a putative disulphide link that may influence accessibility of these sites (Fig 6A).


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 by active rMCP-2 of a number of recombinant cell adhesion and cell junctional proteins.Panel A shows schematic drawings of the basic structure of occludin and claudin. The sequence of the surface loops of occludin and claudin 7 are shown in panel B. The extracellular region is shown in black where the potential rMCP-2 cleavage sites (Tyr and Phe) have been marked in red. The yellow region shows the short region of the membrane spanning region remaining in these clones and the purple region is the kinker including a Cys that has been inserted to form a loop structure of the extracellular loops in the 2xTrx construct. In panel C the cleavage pattern of the rMCP-2 consensus 2xTrx substrate (VVLFSAVL), as positive control, occluding loop 1, occluding loop 2 and claudin 7 loop 1 is depicted. In panel D the cleavage of rat cadherin 17, rat E-cadherin and human protocadherin alpha 4 is presented where the protein is marked above each cleavage reaction and the time of cleavage in minutes is depicted above each lane. These three latter recombinant proteins were produced in mammalian cells. As can be seen from the figure cadherin 17 and protocadherin alpha 4 are very sensitive to cleavage by rMCP-2 whereas almost no cleavage is seen for E-cadherin.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131720.g006: Analysis of the cleavage by active rMCP-2 of a number of recombinant cell adhesion and cell junctional proteins.Panel A shows schematic drawings of the basic structure of occludin and claudin. The sequence of the surface loops of occludin and claudin 7 are shown in panel B. The extracellular region is shown in black where the potential rMCP-2 cleavage sites (Tyr and Phe) have been marked in red. The yellow region shows the short region of the membrane spanning region remaining in these clones and the purple region is the kinker including a Cys that has been inserted to form a loop structure of the extracellular loops in the 2xTrx construct. In panel C the cleavage pattern of the rMCP-2 consensus 2xTrx substrate (VVLFSAVL), as positive control, occluding loop 1, occluding loop 2 and claudin 7 loop 1 is depicted. In panel D the cleavage of rat cadherin 17, rat E-cadherin and human protocadherin alpha 4 is presented where the protein is marked above each cleavage reaction and the time of cleavage in minutes is depicted above each lane. These three latter recombinant proteins were produced in mammalian cells. As can be seen from the figure cadherin 17 and protocadherin alpha 4 are very sensitive to cleavage by rMCP-2 whereas almost no cleavage is seen for E-cadherin.
Mentions: A protein that previously has been identified as potential target by rMCP-2 is occludin, as it forms the barrier in the epithelial layer of the intestine [31, 32]. Claudins are also found in close connection to occludin junctions and were also represented in the potential target list in Table 1. Claudin 7 has been shown to be expressed at high levels in mouse and/or rat intestines [33]. Both of these are complicated membrane proteins that span the membrane 4 times and are therefore difficult to produce in a soluble and properly folded form (Fig 6A). We therefore decided to produce the extracellular loops that can be targets for an enzyme that act from outside of the cell. The extracellular region including a few amino acids from the outer part of the membrane-spanning region was inserted in the two thioredoxin vector. In order to stabilize the construct and obtain a folding similar to the bended loop structure of the native protein we inserted two cysteine residues, one in each end of the loop (Fig 6B). These cysteines will with high certainty form a stable disulphide bond after opening the bacterial cell where it is produced. It may even form in the cells as the E.coli Rosetta gami has a lower reducing potential than other E.coli strains and therefore cysteine bridges may form already within the cell. Occludin has two larger extracellular loops, which we produced separately (Fig 6A and 6B). Only the larger loop of claudin 7 was produced in this form as the small loop is very small and does not contain any sequence that could be a potential target for rMCP-2. As can be seen from Fig 6, a rapid cleavage of loop 1 of occludin, protocadherin alpha 4 and cadherin 17 was observed, but no cleavage of loop 2 of occludin, loop 1 of claudin 7, and E-cadherin could be detected. Interestingly, loop 1 of occludin contains almost a perfect repeat of chymase substrate sequences, whereas loop 2 of occluding and loop 1 of claudin only contains a few relatively poor potential site and both of them also contain a putative disulphide link that may influence accessibility of these sites (Fig 6A).

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