Chemical and Enzymatic Characterization of the Collagenase from the Insect Hypodearma lineatum

Lecroisey, Anne; Boulard, Chantal; Keil, B.

doi:10.1111/j.1432-1033.1979.tb19730.x

Cited by 100 publications

(44 citation statements)

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“…This suggests that the collagenolytic enzyme is a member of the serine protease family. Many serine protease have been isolated from various sources, such as fungus (Hurion et al, 1979), insect (Lecroisey et al, 1979;Lecroisey and Keil, 1985;Lecroisey et al, 1987), bacteria (Nagano and To, 1999), crab (Eisen et al, 1973;Klimova et al, 1990;Roy et al, 1996), and fish (Yoshinaka et al, 1986;Kristjansson et al, 1995) among others.…”

Section: Discussionmentioning

confidence: 99%

“…Collagenolytic serine proteases have also been reported from Bacillus subtilis FS-2. These were isolated from traditionally fermented fish sauce (Nagano and To, 1999), greenshore crab Carcinus maenas (Roy et al, 1996), Kamchatka crab Paralithodes camtschatica (Klimova et al, 1990;, Atlantic cod Gadus morhua (Kristjansson et al, 1995), insect Hypoderma lineatum (Lecroisey et al, 1979;Lecroisey and Keil, 1985;Lecroisey et al, 1987), Antarctic krill Euphasia superba Dana (Turkiewicz et al, 1991), midgets of Penaeid shrimps Penaeus monodon (Lu et al, 1990;Chen et al, 1991;Van Wormhoudt et al, 1992), and the pancreas of catfish Parasilarus asotus (Yoshinaka et al, 1986(Yoshinaka et al, , 1987. These enzymes are involved in the production of hormones and pharmacologically active peptides and in various cellular functions such as protein digestion, blood-clotting, fibrinolysis, complement activation, and fertilization (Neurath, 1984;Bond and Butler, 1987).…”

Section: Introductionmentioning

confidence: 99%

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Purification and Characterization of a Collagenolytic Protease from the Filefish, Novoden modestrus

Kim¹,

Park²,

Kim³

et al. 2002

BMB Reports

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A serine collagenolytic protease was purified from the internal organs of filefish, Novoden modestrus, by ammonium sulfate, ion-exchange chromatography on a DEAE-Sephadex A-50, ion-exchange rechromatography on a DEAE-Sephadex A-50, and gel filtration on a Sephadex G-150 column. The molecular mass of the filefish serine collagenase was estimated to be 27.0 kDa by gel filtration and SDS-PAGE. The purified collagenase was optimally active at pH 7.0-8.0 and 55 o C. The purified enzyme was rich in Ala, Ser, Leu, and Ile, but poor in Trp, Pro, Tyr, and Met. In addition, the purified collagenolytic enzyme was strongly inhibited by N-P-toluenesulfonyl-L-lysine chloromethyl ketone (TLCK), diisopropylfluorophosphate (DFP), and soybean trypsin inhibitor.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Purification and Characterization of a Collagenolytic Protease from the Filefish, Novoden modestrus

Kim¹,

Park²,

Kim³

et al. 2002

BMB Reports

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“…Additional characterization of the proteases produced by larvae in gelatin gels indicated that there was a constant production of proteases throughout the larval development process, thus suggesting that these proteins have a crucial function in the development of the larvae and formation of the wound. This has also been observed in other Diptera species that produce myiasis (Lecroisey et al 1979, Young et al 1996, Tabouret et al 2003, Brant et al 2010.…”

Section: Discussionmentioning

confidence: 68%

“…Muharsini et al (2000Muharsini et al ( , 2001 observed that serine proteases are the main type of protein produced by Chrysomya bezziana larvae. Serine proteases have also been detected in L2 and L3 of other parasitic Diptera flies, such as Hypoderma lineatum, D. hominis and Oestrus ovis, and these proteins are believed to play an important role in larval penetration within the host tissues (Lecroisey et al 1979, Tabouret et al 2003, Brant et al 2010, as well as in modulation of the process of evasion of the host immune system (Boulard 1989, Pruett 1993, Brant et al 2010. The predominance of serine proteases observed in L2 and especially L3 C. hominivorax, as well as L2 and L3 D. hominis (Brant et al 2010), can be interpreted as an increase in the trophic activity of these growing larvae, since the viability of adult flies depends on the ability of larvae to assimilate nutrients from their host.…”

Section: Discussionmentioning

confidence: 99%

Proteolytic activity of excretory/secretory products of Cochliomyia hominivorax larvae (Diptera: Calliphoridae)

et al. 2016

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The protein profiles and proteolytic activity of the excretory secretory products (E/SP) of the first (L1), second (L2) and third (L3) larval stages of Cochliomyia hominivorax were studied in the laboratory. Analysis on the E/SP protein profile was carried out using polyacrylamide gel containing sodium dodecyl sulfate (SDS-PAGE). The E/SP of each larval stage (L1, L2 and L3) treated with protease inhibitors, containing 30μg, 40μg and 50μg of protein, was applied to the 10% polyacrylamide gel. The proteolytic activity of the crude E/SP was analyzed in gels copolymerized with gelatin and by colorimetric assays using azocasein as a substrate, with the characterization of the proteases using synthetic inhibitors. Different protein profiles were observed for the larval instars, with L1 presenting the most complex profile. Nevertheless, various protein bands were observed that were common to all the larval instars. The E/SP of all the instars showed proteolytic activity on gelatin, evidenced by proteolysis zones, predominantly with apparently higher molecular masses in L1, while for L2 and L3 the proteolysis zones could also be observed in regions with lower masses. Tests with protease inhibitors using gelatin as substrate showed that the E/SP of larvae were mainly composed of serine proteases. Additionally, inhibition was observed in L2 E/SP treated previously with EDTA, an inhibitor of metalloproteases. The assays with azocasein revealed a gradual increase of proteolytic activity on this substrate with larval development progress, with the strongest inhibitions being observed after treatments with 3,4-dichloroisocoumarin (DCI) for E/SP of L1, L2 and L3. These results suggest that C. hominivorax larvae produce different proteases, a fact that can be related to the parasite's vital processes for survival, such as penetration into the host's tissues and nutrition during the larval stage.

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“…Crude and homogeneous collagenase from Hypoderma lineatum [3] and hypodermin A 151 were prepared as previously described.…”

Section: Methodsmentioning

confidence: 99%

Hypodermin B, a Trypsin‐Related Enzyme from the Insect Hypoderma lineatum

Lecroisey

Tong

Keil

1983

European Journal of Biochemistry

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Hypodermin B, a serine proteinase with a molecular weight of 23000, was purified to homogeneity from the larvae Hypoderma lineatum. It is stoichiometrically inhibited by diisopropylfluorophosphate and fully inactivated by N-tosyllysine chloromethyl ketone and soya bean and bovine pancreatic trypsin inhibitors. N-Tosylphenylalanine chloromethyl ketone and ovomucoid are without effect on its activity. Hypodermin B hydrolyses both amide and ester substrates of trypsin but does not display any chymotryptic activity on synthetic substrates. Its specificity on the B chain of insulin is slightly broader than that of bovine trypsin. Its amino acid composition and N-terminal sequence suggest structural homology with serine proteinases of the trypsin family and with two other serine proteinases, hypodermin A and Hypoderma collagenase, previously isolated from the same larvae. Hypodermins A and B are very similar however strongly from Hypoderma collagenase.Although proteolytic enzymes have been reported in many insects, they are usually poorly characterized due to the scarceness of the material available. With respect to the amount and the diversity of their enzymatic equipment, larvae from Hypoderma lineaturn represent an advantageous source. These endoparasites of cattle enter through the skin of their host and pursue a migration through its connective tissue; this action is helped by excretion of proteolytic enzymes. The larvae partially reabsorb these enzymes together with the degradation products of the connective tissue and stock them in their midgut which is closed in its hind end and can thus act as a reservoir. After eight months the gut opens and its contents are excreted during the first moult [I].Two enzymes were recently isolated and characterized from the midgut of the first instar larvae : first, a collagenase which cleaves native collagen in a manner similar to that of vertebrate collagenases but which belongs to serine enzymes of the trypsin family [2 -41 ; secondly, a trypsin-like proteinase, hypodermin A 151. In the present paper, we report the purification and the characterization of a third enzyme from the same larval organ, hypodermin B. We demonstrate that it is a trypsin-like proteinase and we compare it with the two other larval proteinases and with other serine enzymes of the trypsin family. MATERIALS AND METHODS MaterialsChemical reagents were purchased from the following sources : Bz-Arg-OEt, Bz-Tyr-OEt and Dip-F from Fluka ;Abbreviations. Bz-Arg-OEt, N-ct-benzoyl-L-arginine ethyl ester; BzTyr-OEt, N-benzoyl-L-tyrosine ethyl ester; Bz-Arg-Nan, N-a-benzoyl-Larginine p-nitroanilide; Ac-Tyr-Nan, N-acetyl-L-tyrosine p-nitroanilide; Tos-Lys-CH,Cl, N-tosyllysine chloromethyl ketone; Tos-Phe-CH,Cl, Ntosyl-phenylalanine chloromethyl ketone ; BPTI, bovine pancreatic trypsin inhibitor; STI, soya bean trypsin inhibitor; Dip-F, diisopropylfluorophosphate; Pth, phenylthiohydantoin.Enzyme. Hypoderma collagenase (EC 3.4.21.49).with respect to their inhibition and specificity, they differ Bz-Arg-Nan, Ac-Tyr-Nan...

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Chemical and Enzymatic Characterization of the Collagenase from the Insect Hypodearma lineatum

Cited by 100 publications

References 43 publications

Purification and Characterization of a Collagenolytic Protease from the Filefish, Novoden modestrus

Purification and Characterization of a Collagenolytic Protease from the Filefish, Novoden modestrus

Proteolytic activity of excretory/secretory products of Cochliomyia hominivorax larvae (Diptera: Calliphoridae)

Hypodermin B, a Trypsin‐Related Enzyme from the Insect Hypoderma lineatum

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