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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

Phage displayed nonamers susceptible to cleavage by rMCP-2 after five biopannings.After the last selection step (round 5), phages released by proteolytic cleavage of the protease were isolated and the sequences encoding the nonamers were determined. The general sequence of the T7 phage capsid proteins are PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers. The protein sequences were aligned into a P4-P4´ consensus, where cleavage occurs between positions P1 and P1´. The aa are color coded according to the side chain properties as indicated in the figure, bottom right corner. For comparison the previously determined phage display results from rMCP-4, mMCP-1 and mMCP-4 are included in the figure [18, 20, 34].
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pone.0131720.g002: Phage displayed nonamers susceptible to cleavage by rMCP-2 after five biopannings.After the last selection step (round 5), phages released by proteolytic cleavage of the protease were isolated and the sequences encoding the nonamers were determined. The general sequence of the T7 phage capsid proteins are PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers. The protein sequences were aligned into a P4-P4´ consensus, where cleavage occurs between positions P1 and P1´. The aa are color coded according to the side chain properties as indicated in the figure, bottom right corner. For comparison the previously determined phage display results from rMCP-4, mMCP-1 and mMCP-4 are included in the figure [18, 20, 34].

Mentions: After the last biopanning, 120 individual phage clones were picked as individual plaques and the region encoding the randomly synthesized nona-peptides were PCR amplified. The 96 clearest PCR bands were selected for sequencing, and the nucleotide sequences were then translated into nona-peptides and aligned (Fig 2). Based on the alignments, the distribution of aa in positions P4 to P4´ were calculated (Fig 3). Of the 96 clones sequenced 83 provided good readable sequences. Alignment of these sequences resulted in a relatively well-defined extended specificity. All of these clones contained a Phe or a Tyr in the P1 position and a high number of aliphatic aa were seen both N and C terminal of this aromatic residue (Fig 2). Overall, very few acidic aa were found among the nonapeptide region of these 83 clones. A relatively broad selection of different aa was observed for the P1´position with high numbers of both hydrophilic aa as represented by Ser, Thr and Met, and also basic aa as primarily represented by Arg (Fig 2). In addition, aliphatic aa as Leu, Val, Ala and Gly were also seen at a relatively high number (Fig 2). This relatively broad spectrum of aa is also reflected in the frequency diagram of Fig 3. The relatively high number of His residues in positions P3´and P4´is somewhat artificial as after the random nonamer region comes a His-tag of six residues, which results in an overrepresentation of His residues in these two positions. The general pattern is a strict preference for Phe and Tyr in the P1 position, a high frequency of aliphatic aa both N and C terminal of the cleavage site from position P2 to P4 and P2´to P4´. In the P1’ position there was a relatively broad selection of aa. However, no aromatic and no negatively charged residues were observed in this position. In general we also observed a very low number of negatively charged aa in the entire region from P4 to P4´. The phage display results from the analysis of two additional rodent mucosal mast cell proteases rMCP-4 and mMCP-1 and the major connective tissue mast cell chymase in mouse mMCP-4 are also added to the figure for a comparison of similarities and differences in sequence preferences (Fig 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)

Phage displayed nonamers susceptible to cleavage by rMCP-2 after five biopannings.After the last selection step (round 5), phages released by proteolytic cleavage of the protease were isolated and the sequences encoding the nonamers were determined. The general sequence of the T7 phage capsid proteins are PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers. The protein sequences were aligned into a P4-P4´ consensus, where cleavage occurs between positions P1 and P1´. The aa are color coded according to the side chain properties as indicated in the figure, bottom right corner. For comparison the previously determined phage display results from rMCP-4, mMCP-1 and mMCP-4 are included in the figure [18, 20, 34].
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131720.g002: Phage displayed nonamers susceptible to cleavage by rMCP-2 after five biopannings.After the last selection step (round 5), phages released by proteolytic cleavage of the protease were isolated and the sequences encoding the nonamers were determined. The general sequence of the T7 phage capsid proteins are PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers. The protein sequences were aligned into a P4-P4´ consensus, where cleavage occurs between positions P1 and P1´. The aa are color coded according to the side chain properties as indicated in the figure, bottom right corner. For comparison the previously determined phage display results from rMCP-4, mMCP-1 and mMCP-4 are included in the figure [18, 20, 34].
Mentions: After the last biopanning, 120 individual phage clones were picked as individual plaques and the region encoding the randomly synthesized nona-peptides were PCR amplified. The 96 clearest PCR bands were selected for sequencing, and the nucleotide sequences were then translated into nona-peptides and aligned (Fig 2). Based on the alignments, the distribution of aa in positions P4 to P4´ were calculated (Fig 3). Of the 96 clones sequenced 83 provided good readable sequences. Alignment of these sequences resulted in a relatively well-defined extended specificity. All of these clones contained a Phe or a Tyr in the P1 position and a high number of aliphatic aa were seen both N and C terminal of this aromatic residue (Fig 2). Overall, very few acidic aa were found among the nonapeptide region of these 83 clones. A relatively broad selection of different aa was observed for the P1´position with high numbers of both hydrophilic aa as represented by Ser, Thr and Met, and also basic aa as primarily represented by Arg (Fig 2). In addition, aliphatic aa as Leu, Val, Ala and Gly were also seen at a relatively high number (Fig 2). This relatively broad spectrum of aa is also reflected in the frequency diagram of Fig 3. The relatively high number of His residues in positions P3´and P4´is somewhat artificial as after the random nonamer region comes a His-tag of six residues, which results in an overrepresentation of His residues in these two positions. The general pattern is a strict preference for Phe and Tyr in the P1 position, a high frequency of aliphatic aa both N and C terminal of the cleavage site from position P2 to P4 and P2´to P4´. In the P1’ position there was a relatively broad selection of aa. However, no aromatic and no negatively charged residues were observed in this position. In general we also observed a very low number of negatively charged aa in the entire region from P4 to P4´. The phage display results from the analysis of two additional rodent mucosal mast cell proteases rMCP-4 and mMCP-1 and the major connective tissue mast cell chymase in mouse mMCP-4 are also added to the figure for a comparison of similarities and differences in sequence preferences (Fig 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