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Isolation and partial characterization of collagenolytic enzyme from the mosquito-parasitizing fungus, Lagenidium giganteum

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Abstract

Lagenidium giganteum Couch produced an extracellular collagenolytic enzyme when grown on peptone-yeast extract-glucose (PYG) broth. The enzyme was purified to apparent homogeneity by chromatography on soybean trypsin inhibitor Sepharose 4B and DEAE cellulose. The purified enzyme hydrolyzed Azocoll, insoluble bovine Achilles tendon collagen, and acid soluble calfskin collagen at neutral pH. No other substrates appeared to be hydrolyzed.

The purified enzyme had an apparent molecular weight of 100,000 and required calcium for optimal activity. The inhibitors PMSF, TPCK, TLCK, D-penicillamine, 8-hydroxyquinoline, cysteine, and 1,10-phenanthroline had little or no effect on enzyme activity.

SDS polyacrylamide gel electrophoresis of collagenase digested collagen indicated that the enzyme cleaved in the helical region of the collagen molecule, although the exact site of cleavage was not determined.

The properties of the Oomycete collagenase, the first ever reported, were found to differ consider considerably from those collagenases reported in higher fungi, with regard to metal-dependence, molecular weight and mode of action.

A possible role for this enzyme was discussed in relation to pathogenesis produced in mosquito larvae by this fungus.

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Abbreviations

BAPA:

N-α-benzoyl-Dl-arginine p-nitroanilide HCl

Bis:

N,N1-methylene-bis-acrylamide

CAPS:

3-(cyclohexylamino)-propane sulfonic acid

EDTA:

disodium ethylenediamine tetraacetate

EGTA:

ethylene glycol-bis-(B-aminoethyl ether)-N,N1 tetraacetic acid

I-RT-63:

α,α′,α″-tris (3-amidinophenoxy) mesitylene-3 HCl

MOPS:

3-(N-morpholino)-propane sulfonic acid

PMSF:

phenylmethylsulfonyl fluoride

PZ-peptide:

4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg

SDS:

sodium dodecylsulfate

TAME:

p-toluenesulfonyl-L-arginine methylester

TEMED:

N,N,N′,N′-tetramethylethylenediamine

TES:

2-(tris-hydroxymethyl)-aminoethane sulfonic acid

TLCK:

N-tosyl-L-lysine chloromethyl ketone

TPCK:

N-tosyl-L-phenylalanine chloromethyl ketone

Tris:

tris-(hydroxymethyl)-aminomethane

References

  • Ashhurst DE (1968) The connective tissues of insects. Ann Rev Entomol 13:45–74

    Article  Google Scholar 

  • Ashhurst DE, Bailey AJ (1980) Locust collagen: Morphological and biochemical characterization. Eur J Biochem 103:75–83

    PubMed  Google Scholar 

  • Ashhurst DE, Costin NM (1976) The secretion of collagen by insects: Uptake of [H3]-proline by collagen synthesizing cells in Locusta migratoria and Galleria mellonella. J Cell Sci 20:377–403

    PubMed  Google Scholar 

  • Aston WP, Chadwick JS, Henderson MJ (1976) Effect of cobra venom factor on the in vivo immune response in Galleria mellonella to Pseudomonas aeruginosa. J Invert Pathol 27:171–176

    Google Scholar 

  • Boulard C, Garrone R (1978) Characterization of a collagenolytic enzyme from larvae of Hypoderma lineatum. Comp Biochem Physiol 59B:251–255

    Google Scholar 

  • Carrick L, Berk RS (1975) Purification and partial characterization of a collagenolytic enzyme from Pseudomnas aeruginosa. Biochim Biophys Acta 391:422–434

    PubMed  Google Scholar 

  • Cuatrecasas P, Wilchek M, Anfinsen CB (1968) Selective enzyme purification by affinity chromatography. Proc Natl Acad Sci USA 61:636–643

    PubMed  Google Scholar 

  • Dean DD, Domnas AJ (1983) The extracellular proteolytic enzymes of the mosquito-parasitizing fungus, Lagenidium giganteum. Exptl Mycol 7

  • Domnas AJ, Giebel PE, McInnis TM (1974) Biochemistry of mosquito infection: Preliminary studies of biochemical change in Culex pipiens quinquefasciatus following infection with Lagenidium giganteum. J Invert Pathol 24:293–304

    Google Scholar 

  • Eisen AZ, Jeffrey JJ (1969) An extractable collagenase from crustacean hepatopancreas. Biochim Biophys Acta 191:517–526

    PubMed  Google Scholar 

  • Eisen AZ, Henderson KO, Jeffrey JJ, Bradshaw RA (1973) A collagenolytic protease from the hepatopancreas of the fiddler crab, Uca pugilator. Purification and properties. Biochem 12:1814–1822

    Google Scholar 

  • Esslinger JH, Chandler AC (1959) Studies on the properties of the metabolic products of the screw-worm, Callitroga hominivorax. Exptl Parasitol 8:527–538

    Google Scholar 

  • Fürthmayr H, Timpl R (1971) Characterization of collagen peptides by sodium dodecylsulfate-polyacrylamide electrophoresis. Anal Biochem 41:510–516

    PubMed  Google Scholar 

  • Gabriel BP (1968) Histochemical study of the insect cuticle infected by the fungus Entomophthora coronata. J Invert Pathol 11:82–89

    Google Scholar 

  • Gabriel JD (1974) A study of proteolytic activities in the mosquito parasite lagenidium giganteum. Honor's thesis. University of North Carolina at Chapel Hill

  • Giebel PE, Domnas AJ (1976) Soluble trehalases from larvae of the mosquito, Culex pipiens, and the fungal parasite, Lagenidium giganteum. Insect Biochem 6:303–311

    Article  Google Scholar 

  • Grant GA, Eeisen AZ (1980) Substrate specificity of the collagenolytic serine protease from Uca pugilator: Studies with noncollagenous substrates. Biochem 19:6089–6095

    Google Scholar 

  • Gross J, Lapiere CM (1962) Collagenolytic activity in amphibian tissues: A tissue culture assay. Proc Natl Acad Sci USA 48:1014–1022

    PubMed  Google Scholar 

  • Haff LA, Easterday RL (1978) Sephacryl gels: Physical properties and evaluation of performance in gel filtration. J Liq Chrom 1:811–832

    Google Scholar 

  • Harper E (1980) Collagenases. Ann Rev Biochem 49:1063–1078

    Article  PubMed  Google Scholar 

  • Hobson RP (1931) On an enzyme from blowfly larvae (Lucilia sericata) which digests collagen in alkaline solution. Biochem J 25:1458–1463

    Google Scholar 

  • Hurion N, Fromentin H, Keil B (1977) Proteolytic enzymes of Entomophthora coronata. Characterization of a collagenase. Comp Biochem Physiol 56B: 259–264

    Google Scholar 

  • Hurion N, Fromentin H, Keil B (1979) Specificity of the collagenolytic enzyme from the fungus Entomophthora coronata: Comparison with the bacterial collagenase from Achromobacter iophagus. Arch Biochem Biophys 192:438–445

    PubMed  Google Scholar 

  • Klemm N (1976) Histochemistry of putative transmitter substances in the insect brain. Prog Neurobiol 7:99–169

    Article  PubMed  Google Scholar 

  • Labadie J, Montel MC (1982) Purification et étude de quelques propríetés d'une collagénase produite par Empedobacter collagenolyticum. Biochimie 64:49–53

    PubMed  Google Scholar 

  • Landsperger W, Peters E, Dresden M (1981) Properties of a collagenolytic enzyme from Bipalium kewense. Biochim Biophys Acta 661:213–220

    PubMed  Google Scholar 

  • Lecroisey A, Keil B (1979) Differences in the degradation of native collagen by two microbial collagenases. Biochem J 179:53–58

    PubMed  Google Scholar 

  • Lecroisey A, Boulard C, Keil B (1979) Chemical and enzymatic characterization of the collagenase from the insecet Hypoderma lineatum. Eur J Biochem 101:385–393

    PubMed  Google Scholar 

  • Leibovich SJ, Weiss JB (1973) Elucidation of the exact sites of cleavage of tropocollagen by rheumatoid synovial collagenase: Correlation of cleavage sites with fibril structure. Conn Tiss Res 2:11–19

    Google Scholar 

  • Lienert E, Throsell W (1955) Untersuchungen über die Aktivität von Autolysaten aus Wandeslarven auf Elemente des Bindegewebes. Exptl Parasitol 4:117–122

    Article  Google Scholar 

  • Lewert RM, Lee Cl (1956) Quantitative studies of the collagenase-like enzymes of cercariae of Schistosoma mansoni and the larvae of Strongyloides ratti. J Infect Dis 99:1–14

    PubMed  Google Scholar 

  • Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275

    PubMed  Google Scholar 

  • Lwebuga-Mukasa JS, Harper E, Taylor P (1976) Collagenase enzymes from Clostridium: Characterization of individual enzymes. Biochem 15:4736–4741

    Google Scholar 

  • McInnis TM, Domnas AJ (1973) The properties of trehalase from the mosquito-parasitizing water mold, Lagenidium giganteum. J Invert Pathol 22:313–320

    Google Scholar 

  • McInnis TM, Domnas AJ (1974) An aryl-B-D-glucosidase of the aquatic fungus, Lagenidium giganteum, a parasite of mosquito larvae. Arch Microbiol 101:343–350

    Google Scholar 

  • Menzel EJ, Smolen J, Reid K (1981) Interaction of collagen with Clq via its collagen-like portion. Biochim Biophys Acta 670:265–273

    PubMed  Google Scholar 

  • Merkel JR, Dreisbach JH (1978) Purification and characterization of a marine bacterial collagenase. Biochem 17:2857–2863

    Google Scholar 

  • Monboissé JC, Labadie J, Bouet P (1979) Purification et proprietés physico-chimiques d'une collagènase synthetiseé par une bacterie du genre Acinètobacter sp. Biochimie 61:1169–1175

    PubMed  Google Scholar 

  • Morales TI, Woessner JF (1977) PZ-Peptidase from chick embryos. J Biol Chem 252:4855–4860

    PubMed  Google Scholar 

  • Mũnoz Ma De L, Calderon J, Rojkind M (1982) The collagenase of Enamoeba histolytica. J Exp Med 155:42–51

    Article  PubMed  Google Scholar 

  • Nordwig A, Bretschneider G (1971) Nonspecific collagenolytic activity: Hydrolysis of native collagen by a mold protease. Biochim Biophys Acta 250:408–415

    PubMed  Google Scholar 

  • Nordwig A, Jahn WF (1968) A collagenolytic enzyme from Aspergillus oryzae. Eur J Biochem 3:519–529

    PubMed  Google Scholar 

  • Rasmuson T, Boman H (1979) Insect immunity-V purification and some properties of immune protein P4 from haemolymph of Hyalophora cecropia pupae. Insect Biochem 9:259–264

    Article  Google Scholar 

  • Rippon JW (1968) Extracellular collagenase from Trichophyton schoenleinii. J Bact 95:43–46

    PubMed  Google Scholar 

  • Seltzer JL, Jeffrey JJ, Eisen AZ (1977) Evidence for mammalian collagenase as zinc ion metalloenzymes. Biochim Biophys Acta 485:179–187

    PubMed  Google Scholar 

  • Söderhäll K, Svensson E, Unestam T (1978) Chitinase and protease activities in germination zoospore cysts of a parasitic fungus, Aphanomyces astaci, oomycetes. Mycopathol 64:9–11

    Google Scholar 

  • Tidwell RR, Fox LL, Geratz JD (1976) Aromatic tris-amidines: A new class of hightly active inhibitors of trypsin-like proteases. Biochim Biophys Acta 445:729–738

    PubMed  Google Scholar 

  • Walters DK (1974) Dissociation of lepidoterous tissues with proteolytic enzymes: Comparison of collagenase, trypsin and pronase. J Insect Physiol 20:49–54

    Article  Google Scholar 

  • Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412

    PubMed  Google Scholar 

  • Weber K, Pringle JR, Osborn M (1972) Measurement of molecular weights by electrophoresis on SDS-acrylamide gels. In: Hirs CHW, Timasheff SN (eds) Methods in enzymology, vol. 26. Academic Press, New York, pp 3–27

    Google Scholar 

  • Welgus HG, Grant GA, Jeffrey JJ, Eisen AZ (1982) Substrate specificity of then collagenolytic serine protease from Uca pugilator: Studies with collagenous substrates. Biochem 21:5183–5189

    PubMed  Google Scholar 

  • Whitaker JR (1963) Determination of molecular weights of proteins by gel filtration on sephadex. Anal Chem 35:1950–1953

    Google Scholar 

  • Wilder WH (1970) Histochemical localization of cholinesterase in larvae of Culiseta inornata (Diptera: Culicidae). Ann Entom Soc Am 63:1620–1624

    Google Scholar 

  • Woessner JF (1980) Literature on vertebrate collagenases. In: Wooley DE, Evanson JM (eds) Collagenase in normal and pathological connective tissues. John Wiley and Sons, New York, pp 251–285

    Google Scholar 

Download references

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Author notes

  1. David D. Dean

    Present address: Department of Biochemistry (R-127), University of Miami School of Medicine, P.O. Box 016960, 33101, Miami, FL, USA

Authors and Affiliations

  1. Biochemistry Laboratory, Department of Botany, University of North Carolina at Chapel Hill, 27514, Chapel Hill, NC, USA

    David D. Dean & Aristotle J. Domnas

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  2. Aristotle J. Domnas
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Dean, D.D., Domnas, A.J. Isolation and partial characterization of collagenolytic enzyme from the mosquito-parasitizing fungus, Lagenidium giganteum . Arch. Microbiol. 136, 212–218 (1983). https://doi.org/10.1007/BF00409847

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  • DOI: https://doi.org/10.1007/BF00409847

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