Collagenolytic serine protease PC and trypsin PC from king crab Paralithodes camtschaticus: cDNA cloning and primary structure of the enzymes
© Rudenskaya et al; licensee BioMed Central Ltd. 2004
Received: 02 July 2003
Accepted: 20 January 2004
Published: 20 January 2004
In this paper, we describe cDNA cloning of a new anionic trypsin and a collagenolytic serine protease from king crab Paralithodes camtschaticus and the elucidation of their primary structures. Constructing the phylogenetic tree of these enzymes was undertaken in order to prove the evolutionary relationship between them.
The mature trypsin PC and collagenolytic protease PC contain 237 (M calc 24.8 kDa) and 226 amino acid residues (M calc 23.5 kDa), respectively. Alignments of their amino acid sequences revealed a high degree of the trypsin PC identity to the trypsin from Penaeus vannamei (approximately 70%) and of the collagenolytic protease PC identity to the collagenase from fiddler crab Uca pugilator (76%). The phylogenetic tree of these enzymes was constructed.
Primary structures of the two mature enzymes from P. camtschaticus were obtained and compared with those of other proteolytic proteins, including some enzymes from brachyurans. A phylogenetic analysis was also carried out. These comparisons revealed that brachyurins are closely related to their vertebrate and bacterial congeners, occupy an intermediate position between them, and their study significantly contributes to the understanding of the evolution and function of serine proteases.
King crab collagenolytic serine protease PC  and trypsin PC  are brachyurins (MEROPS  peptidase classification: CLAN SA, family S1; NC-IUBMB enzyme classification: EC 22.214.171.124). Brachyurin is a term recommended by NC-IUBMB in 1992 for serine endopeptidases of a distinctive type found in crabs and their relatives. The name is derived from brachyurans, the phylogenetic subgroup of "true" crabs, which are a common source of these enzymes . Early examples of the enzyme family include fiddler crab collagenase I , crayfish trypsin [6, 7] and shrimp trypsin . Other serine proteases were isolated from krill , crabs [10, 11], crayfish , shrimps [13–17], and lobster . There are at least three types of brachyurins: (Ia) the enzymes with a broad specificity, whose activities for synthetic substrates are similar to those of trypsin (Arg, Lys), chymotrypsin (Phe, Leu) and elastase (Ala, Leu) [9, 16, 19–21]; (Ib) broadly specific enzymes devoid of trypsin-like activity; and (II) the enzymes with a strict trypsin-like specificity (Arg, Lys) [10, 13]. When preparing this article, we knew the amino acid sequences of six mature brachyurins: crab collagenase I [19, 21], two crayfish trypsins [7, 12], shrimp chymotrypsins I and II , and a shrimp trypsin . The sequences encoding all the enzymes, except for crayfish trypsin, have been cloned.
In recent years, our laboratory has been engaged in the investigation of proteases from the Kamchatka king crab (P. camtschaticus) [1, 2, 23–25]. A number of proteases were isolated from the crab hepatopancreas. They are capable of collagen and fibrin lysis and are shown to exhibit a wound-healing activity in treatment of burns, trophic ulcers, and postoperative scars . A homogeneous collagenolytic protease PC  and trypsin PC  were isolated from the king crab hepatopancreas using ion-exchange and affinity chromatographies.
In this article, we describe the construction of cDNA library from the total RNA of king crab P. camtschaticus and the isolation and sequencing of genes encoding trypsin PC and collagenolytic serine protease PC. We also compare here the primary structures of the enzymes with those of other serine proteases from invertebrate and vertebrate species. The evolution of crab trypsin and collagenase are examined by constructing a phylogenetic tree.
Results and discussion
Structural features of king crab brachyurins
The coding sequences of collagenolytic protease PC and trypsin PC were cloned and their amino acid sequences were established. An analysis of their nucleotide sequences suggests that the gene products are initially synthesized as precursor proteins, which are subsequently processed to the mature enzymes. The deduced protein sequence of collagenolytic protease PC consists of 270 residues and includes initiation Met, a 15-aa signal peptide, a 29-aa precursor peptide, and the active enzyme. The deduced protein sequence of trypsin PC comprises 266 aa and includes Met, derived from the initiation methionine codon, a 15-aa signal peptide, a 14-aa precursor peptide, and the mature enzyme. A comparison of the brachyurin propeptides additionally characterizes the enzyme group, because the propeptides have variable lengths and negligible identity. Crab collagenolytic protease PC, collagenase I, shrimp chymotrypsins I and II, and shrimp trypsin are derived from propeptides that are longer (29–44 residues) than those of the vertebrate proteases (8–15 residues). The function of these large activation domains is unclear, since they are unnecessary for the heterologous expression of proteases from vertebrates. The activation site of procollagenase PC (Val-Lys-Ser-Gln-Arg-Ile-Val-Gly-Gly) is closer to those of chymotrypsinogen (Ser-Gly-Leu-Ser-Arg-Ile-Val-Val-Gly) and proelastase (Glu-Thr-Asn-Ala-Arg-Val-Val-Gly-Gly), which are activated by trypsin, than to that of trypsinogen (Asp-Asp-Asp-Asp-Lys-Ile-Val-Gly-Gly), which is activated by enteropeptidase . Interestingly, the identical activation sites of trypsin PC and shrimp trypsin (Arg-Gly-Leu-Asn-Lys-Ile-Val-Gly-Gly) are also devoid of an enteropeptidase site. This suggests that the activation cascades of the vertebrate digestive proteases have significantly diverged from those of crustaceans. Brachyurins may be self-activated, or other trypsin-like proteases in the hepatopancreas may fulfill this function.
A comparison of mature collagenases and chymotrypsins. Figures indicate the identity percentage in alignment of the sequences presented in Fig. 1. The percentages are calculated using the TreeTop http://www.genebee.msu.su/services/phtree_reduced.html program
A comparison of mature trypsins. Figures indicate the identity percentage in alignment of the compared sequences presented in Fig. 2. The percentages are calculated using the TreeTop http://www.genebee.msu.su/services/phtree_reduced.html program
A comparative structural and functional analysis indicates that these invertebrate enzymes are closely related to their analogues from vertebrates and bacteria but differ from them [11, 19]. Thus, brachyurins demonstrate a high degree of structural similarity (25–36 kDa, 35–40% of amino acid sequence identity) to other members of the chymotrypsin family of serine proteases. However, these invertebrate enzymes have a lesser number of disulfide bonds than their analogues from vertebrates [12, 19, 22] and are particularly unstable at low pH values probably due to this reason [5, 14]. Exceptionally acidic pI values are another special feature of them [5, 13].
For example, the Jones–Taylor–Thornton matrix shows that the P. camtschaticus trypsin is separated by 1.32 from Drosophila melanogaster trypsin, by 1.16 from Bos taurus trypsin, and by 0.41 from P. vannamei trypsin. King crab collagenase is 1.45 away from the king crab trypsin and 1.48 away from the bovine chymotrypsin, while the distance between the prawn chymotrypsin II and the king crab collagenase is 0.32. King crab trypsin and king crab collagenase are separated from the S. griseus trypsin by 1.57 and 1.78, respectively. Fungal trypsins are also more separated from the P. camtschaticus collagenase than from the P. camtschaticus trypsin.
A comparative sequence analysis of brachyurins, bacterial and vertebrate chymotrypsins, and trypsins allows us to understand the evolution of this serine protease family . A closer inspection suggests that brachyurins share more sequence identity with vertebrate trypsins and chymotrypsins than with their bacterial analogues, while some important structural features, such as disulfide bonds, are conserved between the brachyurins and bacterial enzymes. Therefore, brachyurins occupy an intermediate evolutionary position between the bacterial and vertebrate trypsins and chymotrypsins. The major types of brachyurins can be distinguished on the basis of sequence similarity. The study of brachyurins significantly contributes to our understanding of the evolution of serine protease structure and function. Comparative structural and functional analyses indicate that these invertebrate enzymes are closely related to their vertebrate and bacterial analogues, but differ from them.
RNA isolation and cDNA library construction
A live king crab (P. camtschaticus) was obtained from the local market. Total RNA was isolated from 0.5 g of hepatopancreas of king crab by guanidine–phenol–chloroform extraction . A cDNA library was obtained from 0.1 μg of total RNA and amplified by a SMART PCR cDNA Synthesis Kit (CLONTECH) using manufacturer's protocol. The amplified cDNA sample was 20-fold diluted with water and used in the following RACE procedures.
Isolation of the crab collagenase cDNA and trypsin cDNA
The GenBank accession numbers for two sequences determined in this study are AF461035 and AF461036 for collagenolytic serine protease PC and trypsin PC, respectively.
Alignment of amino acid sequences and a phylogenetic analysis of the crab collagenase and trypsin
The primary structures of enzymes used for the alignment were taken from Swiss-Prot database. Multiple sequence alignments were performed using ClustalW program with manual verification . To construct the phylogenetic tree on the basis of the multiple sequence alignment, a pairwise distance matrix was set up by the Protdist program within the PHYLIP package . The construction of the unrooted phylogenetic tree was performed by the Bionj program  according to the aforementioned matrix. The unrooted phylogenetic tree was drawn by TreeView program .
List of abbreviations
- Collagenolytic serine protease PC:
colladenolytic serine protease from Paralithodes camtschaticus
- Trypsin PC:
trypsin from Paralithodes camschaticus
- and aa:
number of amino acid residues.
This work was supported by the Russian Foundation for Basic Research, project no. 02-04-48699.
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