The body wall collagen of an edible sea cucumber, Stichopus japonicus, was studied with respect to its chemical composition and subunit structure. About 70% of the total body wall protein was accounted for by highly insoluble collagen fibers. The disaggregation with b-mercaptoethanol, 0.1 M NaOH treatment, and limited pepsin digestion of these collagen fibers resulted in complete solubilization. The solubilized collagen was isolated and characterized; it had 2 distinct subunits, a1 and a2, which formed (a1) 2 a2 heterotrimers and was rich in glutamic acid when compared with other fibrillar collagens. The unique textural properties of cooked sea cucumber seem to be due to thermal denaturation of the insoluble collagen fibers.
The subunit compositions of skin and muscle type I collagens from rainbow trout were found to be α1(I)α2(I)α3(I) and [α1(I)]2α2(I), respectively. The occurrence of α3(I) has been observed only for bonyfish. The skin collagen exhibited more susceptibility to both heat denaturation and MMP‐13 digestion than the muscle counterpart; the former had a lower denaturation temperature by about 0.5 °C than the latter. The lower stability of skin collagen, however, is not due to the low levels of imino acids because the contents of Pro and Hyp were almost constant in both collagens. On the other hand, some cDNAs coding for the N‐terminal and/or a part of triple‐helical domains of proα(I) chains were cloned from the cDNA library of rainbow trout fibroblasts. These cDNAs together with the previously cloned collagen cDNAs gave information about the complete primary structure of type I procollagen. The main triple‐helical domain of each proα(I) chain had 338 uninterrupted Gly‐X‐Y triplets consisting of 1014 amino acids and was unique in its high content of Gly‐Gly doublets. In particular, the bonyfish‐specific α(I) chain, proα3(I) was characterized by the small number of Gly‐Pro‐Pro triplets, 19, and the large number of Gly‐Gly doublets, 38, in the triple‐helical domain, compared to 23 and 22, respectively, for proα1(I). The small number of Gly‐Pro‐Pro and the large number of Gly‐Gly in proα3(I) was assumed to partially loosen the triple‐helical structure of skin collagen, leading to the lower stability of skin collagen mentioned above. Finally, phylogenetic analyses revealed that proα3(I) had diverged from proα1(I). This study is the first report of the complete primary structure of fish type I procollagen.
Matrix metalloproteinases (MMPs) are widely distributed in vertebrate tissues and form a large family consisting of at least four distinct subfamilies. Higher vertebrate MMP-13 is well-known as collagenase-3, which represents the third member of a collagenase subfamily. In this study, we cloned cDNA coding for a unique fish homologue of human MMP-13 from a rainbow trout fibroblast cDNA library. The cDNA was 2.1 kb long and contained an open reading frame encoding a protein of 475 amino acids. The catalytic domain of the protein was 66% identical to the human counterpart with the greatest degree of identity occurring in the zinc binding site. In addition, it possessed three amino-acid residues (Tyr122, Asp233 and Gly235) characteristic of the collagenase subfamily, together with a six residue insertion which did not occur in the collagenase subfamily. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and skin type I collagen. It is worth noting that rainbow trout type I collagen was more susceptible to proteolysis with the recombinant protein when compared with the calf one. It appeared that the recombinant protein also cleaved the nonhelical regions of rainbow trout muscle type V collagen. These results have revealed that the cDNA encodes a unique MMP-13 of rainbow trout. This is the first report of cDNA coding for fish MMP capable of degrading type I collagen.Keywords: rainbow trout; fish; MMP; MMP-13; collagenase.Matrix metalloproteinases (MMPs) are assumed to play a central role in degrading collagens. The structure and function of MMPs has been extensively studied; they comprise a grand family and 16 distinct molecular species have been identified in human tissues [1±3]. The MMP family is classified into at least four subfamilies, collagenase, gelatinase, stromelysin, and membrane type-MMP. Degradation of fibrillar collagens such as types I, II, and III at neutral pH is achieved by members of the collagenase subfamily such as MMP-1 (interstitial collagenase, EC 3.4.24.7), EC 3.4.24.34),. An amphibian MMP also belongs to the collagenase subfamily [7]. MMPs of the collagenase subfamily have distinct substrate preferences toward the fibrillar collagens; MMP-1, 2 8, and 2 13 are especially active against types III, I and II collagens, respectively [4±6]. In addition, these MMPs share the unique ability to initially cleave the triple helical domain of the collagens, generating about three-fourths and one-fourth length collagen fragments as a result of the hydrolysis of a single Gly-Ile/Leu bond in each a chain of the collagen molecule. On the other hand, serine and cysteine proteinases cleave only a part of these fibrillar collagens such as the nonhelical regions [8,9]. The generated fragments are degraded further by these MMPs themselves as well as by gelatinolytic enzymes such as MMP-2 (gelatinase A, EC 3.4.24.24), MMP-9 (gelatinase B, EC 3.4.24.35), neutrophil elastase (EC 3.4.21.37) and plasmin (EC 3.4.21.7) [10±12]. Degradation of both fragme...
SUMMARY: Matrix metalloproteinases (MMP) are widely distributed in vertebrate tissues. One of the well‐characterized gelatinases from higher vertebrates is MMP‐2, named gelatinase A or 72 kDa type IV collagenase, which is active for the cleavage of denatured collagens, type IV collagen, type V collagen, and other matrix proteins. To investigate the primary structure and properties of MMP‐2 from teleost as lower vertebrates, a cDNA library was prepared from mRNA of rainbow trout fibroblast and screened. Using polymerase chain reaction and degenerate oligonucleotide primers, which are specific for two highly conserved sequences found in MMP of higher vertebrates, a resultant cDNA fragment was used as a probe. A cDNA clone 3.0 kb long was isolated and found to contain an open‐reading frame coding for a polypeptide of 655 amino acids. The rainbow trout polypeptide was 73% identical, at the level of amino acid sequence, to human proMMP‐2 with the greatest degree of similarity occurring in the propeptide and catalytic domains and was denoted as rainbow trout proMMP‐2. Then the isolated cDNA was expressed in Escherichia coli and the recombinant protein was found to degrade gelatin and human type V collagen, providing support to the hypothesis that the cDNA codes for the authentic rainbow trout proMMP‐2. In contrast to human proMMP‐2, rainbow trout proMMP‐2 was not activated by 4‐amino‐phenylmercuric acetate. This is the first report of cDNA for fish proMMP to our knowledge.
We have investigated the inhibitory effects of polyphenols from natural products, such as green tea, bilberry, grape, ginkgo, and apple, on rainbow trout gelatinase activities. Gelatinases from the skin, muscle, and blood of rainbow trout contained serine proteinase, metalloproteinase, and other proteinase activities as measured by gelatin zymography. The polyphenols of green tea caused the strong inhibition of some gelatinase activities when compared with those of the other products. This inhibition was quite similar to that of metalloproteinase by ethylenediaminetetraacetic acid, suggesting that the effects of green tea polyphenols on proteinase activities are specific for metalloproteinases. The major catechins of green tea polyphenols were then separated and identified by reverse-phase chromatography to be (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin, (-)-epicatechin gallate, and (-)-epicatechin. The effects of these catechins on gelatinase activities were examined; the most potent inhibitor of metalloproteinase activities was found to be EGCG. These results have indicated that green tea polyphenols including EGCG are useful for regulating metalloproteinase activities of fish meat.
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