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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: 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.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.

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 specificity by the use of recombinant protein substrates.Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the MC. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His6-tag positioned in their C termini. The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one Bam HI and one SalI site, which are indicated in the bottom of panel A. Panels C-G show the cleavage of a number of substrates by rMCP-2. The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage in minutes is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of approximately 25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa (Panel B).
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pone.0131720.g004: Analysis of the cleavage specificity by the use of recombinant protein substrates.Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the MC. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His6-tag positioned in their C termini. The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one Bam HI and one SalI site, which are indicated in the bottom of panel A. Panels C-G show the cleavage of a number of substrates by rMCP-2. The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage in minutes is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of approximately 25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa (Panel B).

Mentions: In order to verify the results from the phage display analysis and to get quantitative estimates of the importance of particular amino acids in positions surrounding the cleavage site we used a new type of recombinant substrate. The consensus sequence obtained from the phage display analysis was first inserted in a linker region between two E.coli thioredoxin molecules by ligating a double stranded oligonucleotide encoding the actual sequence into a BamHI and a SalI site of the vector construct (Fig 4A). For purification purposes a His6-tag was added to the C-terminal of this protein (Fig 4A). A number of related and unrelated substrate sequences were also later produced with this system, by ligating the corresponding oligonuclotides into the BamHI/SalI sites of the vector. All of these substrates were expressed as soluble proteins in a bacterial host, E.coli Rosetta gami, and purified on IMAC columns to obtain a protein with a purity of 90–95%. These recombinant proteins were then used to study the preference of rMCP-2 for these different sequences (Fig 4B–4G). The results showed that rMCP-2 very efficiently cleaved the consensus sequence obtained from the phage display analysis (VVLFSAVL). By changing the serine residue in the P1´position into a leucine (VVLFLGVL) the efficiency in cleavage by rMCP-2 drops significantly by a factor of 10 (Fig 4C). In contrast, change in the same position, P1´, to an arginine (VVLFRGVL) only marginally affected the efficiency of cleavage, by a factor 2–3 (Fig 4C). In contrast, changing the same position into a negatively charged aa, aspartic acid (VVLFDGVL), had a major effect on cleavage (Fig 4C). The activity dropped by approximately 20 times. This showed that negatively charged aa are not liked in the near vicinity of the cleavage site. Testing a few additional substrates that previously have been identified as optimal substrates for other MC enzymes, the human chymase (VVLFSEVL), the human chymase double mutant (human chymase variant VVLFSGVL) and the dog chymase (VVRFLSLL), showed that all three were cleaved relatively efficiently [26–28]. They were cleaved at a rate of three to ten times less efficiently compared to the rMCP-2 consensus (Fig 4D). The dog chymase sequence was the least effectively cleaved with approximately 10 times lower cleavage rate (Fig 4D). Changing the aromatic aa at the P1 position, the cleavage site, to a valine, dramatically lowered the cleavage activity. Almost no cleavage was seen with three substrates having a valine in the cleavable position (VVLVLEVL, VLLVSEVL and VVSVSEVL) (Fig 4E). However, when increasing the enzyme concentration by 40 times we observed cleavage of one of them (VLLVSEVL) possibly by low efficiency cleavage at double leucines. Testing 4 additional substrates, the activation site in prothrombin (MTPRSEGS), the human thrombin consensus cleavage site (LTPRGVRL), a site with three arginines (VRARARAAG) and the opossum MC chymase consensus cleavage site (VGLWLDRV) that has a tryptophan in the P1 position, did not result in any detectable cleavage even when using the higher enzyme concentration (40x) (Fig 4G) [29, 30]. This showed the very high specificity of the enzyme. Two aromatic aa, phenylalanine and tyrosine were strongly favored at the P1 position and another aromatic aa acid tryptophan, or valine or any other aa with the possible exception of multiple leucines were not tolerated. Negatively charges in or around the cleavage sites also had a very negative effect on the cleavage activity.


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 specificity by the use of recombinant protein substrates.Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the MC. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His6-tag positioned in their C termini. The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one Bam HI and one SalI site, which are indicated in the bottom of panel A. Panels C-G show the cleavage of a number of substrates by rMCP-2. The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage in minutes is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of approximately 25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa (Panel B).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131720.g004: Analysis of the cleavage specificity by the use of recombinant protein substrates.Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the MC. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His6-tag positioned in their C termini. The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one Bam HI and one SalI site, which are indicated in the bottom of panel A. Panels C-G show the cleavage of a number of substrates by rMCP-2. The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage in minutes is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of approximately 25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa (Panel B).
Mentions: In order to verify the results from the phage display analysis and to get quantitative estimates of the importance of particular amino acids in positions surrounding the cleavage site we used a new type of recombinant substrate. The consensus sequence obtained from the phage display analysis was first inserted in a linker region between two E.coli thioredoxin molecules by ligating a double stranded oligonucleotide encoding the actual sequence into a BamHI and a SalI site of the vector construct (Fig 4A). For purification purposes a His6-tag was added to the C-terminal of this protein (Fig 4A). A number of related and unrelated substrate sequences were also later produced with this system, by ligating the corresponding oligonuclotides into the BamHI/SalI sites of the vector. All of these substrates were expressed as soluble proteins in a bacterial host, E.coli Rosetta gami, and purified on IMAC columns to obtain a protein with a purity of 90–95%. These recombinant proteins were then used to study the preference of rMCP-2 for these different sequences (Fig 4B–4G). The results showed that rMCP-2 very efficiently cleaved the consensus sequence obtained from the phage display analysis (VVLFSAVL). By changing the serine residue in the P1´position into a leucine (VVLFLGVL) the efficiency in cleavage by rMCP-2 drops significantly by a factor of 10 (Fig 4C). In contrast, change in the same position, P1´, to an arginine (VVLFRGVL) only marginally affected the efficiency of cleavage, by a factor 2–3 (Fig 4C). In contrast, changing the same position into a negatively charged aa, aspartic acid (VVLFDGVL), had a major effect on cleavage (Fig 4C). The activity dropped by approximately 20 times. This showed that negatively charged aa are not liked in the near vicinity of the cleavage site. Testing a few additional substrates that previously have been identified as optimal substrates for other MC enzymes, the human chymase (VVLFSEVL), the human chymase double mutant (human chymase variant VVLFSGVL) and the dog chymase (VVRFLSLL), showed that all three were cleaved relatively efficiently [26–28]. They were cleaved at a rate of three to ten times less efficiently compared to the rMCP-2 consensus (Fig 4D). The dog chymase sequence was the least effectively cleaved with approximately 10 times lower cleavage rate (Fig 4D). Changing the aromatic aa at the P1 position, the cleavage site, to a valine, dramatically lowered the cleavage activity. Almost no cleavage was seen with three substrates having a valine in the cleavable position (VVLVLEVL, VLLVSEVL and VVSVSEVL) (Fig 4E). However, when increasing the enzyme concentration by 40 times we observed cleavage of one of them (VLLVSEVL) possibly by low efficiency cleavage at double leucines. Testing 4 additional substrates, the activation site in prothrombin (MTPRSEGS), the human thrombin consensus cleavage site (LTPRGVRL), a site with three arginines (VRARARAAG) and the opossum MC chymase consensus cleavage site (VGLWLDRV) that has a tryptophan in the P1 position, did not result in any detectable cleavage even when using the higher enzyme concentration (40x) (Fig 4G) [29, 30]. This showed the very high specificity of the enzyme. Two aromatic aa, phenylalanine and tyrosine were strongly favored at the P1 position and another aromatic aa acid tryptophan, or valine or any other aa with the possible exception of multiple leucines were not tolerated. Negatively charges in or around the cleavage sites also had a very negative effect on the cleavage activity.

Bottom Line: 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.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.

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