Midgut Serine Proteases Research Articles

Midgut serine proteinases participate in dietary adaptations of the castor (Eri) silkworm Samia ricini Anderson transferred from Ricinus communis to an ancestral host, Ailanthus excelsa Roxb.

Dietary change influenced the life-history traits, nutritional utilization, and midgut serine proteinases in the larvae of the domesticated polyphagous S. ricini, transferred from R. communis (common name: castor; family Euphorbiaceae; the host plant implicated in its domestication) to A. excelsa (common name: Indian tree of heaven; family Simaroubaceae; an ancestral host of wild Samia species). Significantly higher values for fecundity and body weight were observed in larvae feeding on R. communis (Scr diet), and they took less time to reach pupation than insects feeding on A. excelsa (Scai diet). Nevertheless, the nutritional index for efficiency of conversion of digested matter (ECD) was similar for larvae feeding on the two plant species, suggesting the physiological adaptation of S. ricini (especially older instars) to an A. excelsa diet. In vitro protease assays and gelatinolytic zymograms using diagnostic substrates and protease inhibitors revealed significantly elevated levels (p ≤ 0.05) of digestive trypsins, which may be associated with the metabolic costs influencing slow growth in larvae feeding on A. excelsa. RT-PCR with semidegenerate serine proteinase gene-specific primers, and cloning and sequencing of 3' cDNA ends identified a large gene family comprising at least two groups of putative chymotrypsins (i.e., Sr I and Sr II) resembling invertebrate brachyurins/collagenases with wide substrate specificities, and five groups of putative trypsins (i.e., Sr III, Sr IV, Sr V, Sr VII, and Sr VIII). Quantitative RT-PCR indicated that transcripts belonging to the Sr I, Sr III, Sr IV, and Sr V groups, especially the Sr IV group (resembling achelase I from Lonomia achelous), were expressed differentially in the midguts of fourth instars reared on the two plant species. Sequence similarity indicated shared lineages with lepidopteran orthologs associated with expression in the gut, protein digestion, and phytophagy. The results obtained are discussed in the context of larval serine proteinases in dietary adaptations, domestication, and exploration of new host plant species for commercial rearing of S. ricini.

Phyto-inspired cyclic peptides derived from plant Pin-II type protease inhibitor reactive center loops for crop protection from insect pests

BackgroundNatural defence of plants against insect pests involves protease inhibitors (PIs) that interfere with insect digestive proteases. Pin-II type plant PIs are wound inducible upon insect damage and possess multiple inhibitory repeat domains that can inhibit trypsin and chymotrypsin-like proteases in the insect midgut. Yet, their agricultural ex-vivo application is limited due to large molecular size and environmental instability, which could be overcome by small peptides. MethodsBicyclic peptides were designed by grafting Pin-II PIs derived reactive center loop (RCL) on synthetic tris(bromomethyl)benzene scaffold. In vitro binding with trypsin-like proteases was evaluated by biochemical and biophysical assays, followed by molecular dynamics simulations. In vivo effects on two major lepidopteran insect pests, Helicoverpa armigera and Spodoptera litura were studied upon feeding with peptide treated leaves. Affinity based pull down assays were used to identify target proteins in insect gut. ResultsBicyclic RCLs showed ten-fold enhanced protease inhibition compared to their linear counterparts. They exhibited feeding deterrence and growth reduction of lepidopteran insects. Bicyclic peptides predominantly interact with midgut serine proteases. Possible binding modes involve simultaneous interaction with the active site and specificity-determining residues of insect gut trypsin. ConclusionBicyclic peptides are potent inhibitors of serine proteases in the insect midgut. They cause feeding aversion and larval growth retardation. Bi-domain cyclic peptides interact with two sites on trypsin, leading to enhanced efficacy over linear RCL peptides. General significanceBicyclic peptides mimic natural PIs by inhibiting insect proteases leading to growth reduction, thus, could be used as pest control molecules in agriculture.

Midgut serine proteases and alternative host plant utilization in Pieris brassicae L.

Pieris brassicae L. is a serious pest of cultivated crucifers in several parts of the world. Larvae of P. brassicae also feed prolifically on garden nasturtium (Tropaeolum majus L., of the family Tropaeolaceae). Proteolytic digestion was studied in larvae feeding on multiple hosts. Fourth instars were collected from cauliflower fields before transfer onto detached, aerial tissues of selected host plants in the lab. Variable levels of midgut proteases were detected in larvae fed on different hosts using protein substrates (casein and recombinant RBCL cloned from cauliflower) and diagnostic, synthetic substrates. Qualitative changes in midgut trypsin activities and quantitative changes in midgut chymotrypsin activities were implicated in physiological adaptation of larvae transferred to T. majus. Midgut proteolytic activities were inhibited to different extents by serine protease inhibitors, including putative trypsin inhibitors isolated from herbivore-attacked and herbivore-free leaves of cauliflower (CfTI) and T. majus (TpTI). Transfer of larvae to T. majus significantly influenced feeding parameters but not necessarily when transferred to different tissues of the same host. Results obtained are relevant for devising sustainable pest management strategies, including transgenic approaches using genes encoding plant protease inhibitors.

Alterations in the Helicoverpa armigera midgut digestive physiology after ingestion of pigeon pea inducible leucine aminopeptidase.

Jasmonate inducible plant leucine aminopeptidase (LAP) is proposed to serve as direct defense in the insect midgut. However, exact functions of inducible plant LAPs in the insect midgut remain to be estimated. In the present investigation, we report the direct defensive role of pigeon pea inducible LAP in the midgut of Helicoverpa armigera (Lepidoptera: Noctuidae) and responses of midgut soluble aminopeptidases and serine proteinases upon LAP ingestion. Larval growth and survival was significantly reduced on the diets supplemented with pigeon pea LAP. Aminopeptidase activities in larvae remain unaltered in presence or absence of inducible LAP in the diet. On the contrary, serine proteinase activities were significantly decreased in the larvae reared on pigeon pea LAP containing diet as compared to larvae fed on diet without LAP. Our data suggest that pigeon pea inducible LAP is responsible for the degradation of midgut serine proteinases upon ingestion. Reduction in the aminopeptidase activity with LpNA in the H. armigera larvae was compensated with an induction of aminopeptidase activity with ApNA. Our findings could be helpful to further dissect the roles of plant inducible LAPs in the direct plant defense against herbivory.

Characterization and transcriptional analyses of cDNAs encoding three trypsin- and chymotrypsin-like proteinases in Cry1Ab-susceptible and Cry1Ab-resistant strains of sugarcane borer,Diatraea saccharalis

Diatraea saccharalis is a major corn borer pest. Midgut serine proteinases are essential for insect growth and development. Alteration of midgut proteinases is responsible for Bt resistance development in some species. To clone midgut trypsin and chymotrypsin cDNAs and to test if the Cry1Ab resistance in D. saccharalis is associated with changes in midgut proteinases, total midgut tryptic and chymotryptic activities, cDNA sequences, and gene expressions of three trypsin and three chymotrypsin genes were comparatively examined between Cry1Ab-susceptible (Cry1Ab-SS) and Cry1Ab-resistant (Cry1Ab-RR) strains. Full-length cDNAs encoding three trypsin- and three chymotrypsin-like proteinases were sequenced from Cry1Ab-SS and Cry1Ab-RR larvae. These cDNAs code for active forms of midgut serine proteinases with all functional motifs, including signal peptide, conserved His-Asp-Ser for the catalytic triad, three pairs of cysteines for disulfide bridge configurations, and conserved substrate specificity determination residues. In general, cDNA and putative protein sequences are highly similar between Cry1Ab-SS and Cry1Ab-RR strains, except for a few nucleotide and predicted amino acid substitutions, whose function need to be further clarified. Total trypsin and chymotrypsin activities were also similar between Cry1Ab-SS and Cry1Ab-RR strains. Transcriptional levels of the trypsin and chymotrypsin genes had numerical difference between Cry1Ab-SS and Cry1Ab-RR strains, but the difference was not statistically significant. Data suggest that the development of Cry1Ab resistance in D. saccharalis was not significantly associated with these trypsins and chymotrypsins. Results clarified the role of six midgut proteinases and provided a foundation for continuing examination of potential involvement of other midgut proteinases in Bt resistance development and other important biochemical processes.

In vitro activation and enzyme kinetic analysis of recombinant midgut serine proteases from the Dengue vector mosquito Aedes aegypti

BackgroundThe major Dengue virus vector Aedes aegypti requires nutrients obtained from blood meal proteins to complete the gonotrophic cycle. Although bioinformatic analyses of Ae. aegypti midgut serine proteases have provided evolutionary insights, very little is known about the biochemical activity of these digestive enzymes.ResultsWe used peptide specific antibodies to show that midgut serine proteases are expressed as zymogen precursors, which are cleaved to the mature form after blood feeding. Since midgut protein levels are insufficient to purify active proteases directly from blood fed mosquitoes, we engineered recombinant proteins encoding a heterologous enterokinase cleavage site to permit generation of the bona fide mature form of four midgut serine proteases (AaET, AaLT, AaSPVI, AaSPVII) for enzyme kinetic analysis. Cleavage of the chromogenic trypsin substrate BApNA showed that AaET has a catalytic efficiency (kcat/KM) that is ~30 times higher than bovine trypsin, and ~2-3 times higher than AaSPVI and AaSPVII, however, AaLT does not cleave BApNA. To measure the enzyme activities of the mosquito midgut proteases using natural substrates, we developed a quantitative cleavage assay based on cleavage of albumin and hemoglobin proteins. These studies revealed that the recombinant AaLT enzyme was indeed catalytically active, and cleaved albumin and hemoglobin with equivalent efficiency to that of AaET, AaSPVI, and AaSPVII. Structural modeling of the AaLT and AaSPVI mature forms indicated that AaLT is most similar to serine collagenases, whereas AaSPVI appears to be a classic trypsin.ConclusionsThese data show that in vitro activation of recombinant serine proteases containing a heterologous enterokinase cleavage site can be used to investigate enzyme kinetics and substrate cleavage properties of biologically important mosquito proteases.

Biochemical characterization of digestive proteases and carbohydrases of the carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae)

Digestive proteinases and carbohydrases of Ectomyelois ceratoniae (Zeller) larvae were investigated using appropriate substrates and inhibitors. Midgut pH in larvae was determined to be slightly alkaline. Midgut extracts showed optimum activity for proteolysis of hemoglobin at pH 9–12. Midgut proteinases also hydrolyzed the synthetic substrates of trypsin, chymotrypsin, and elastase at pH 8–11. Maximum digestive α-amylase activity was also observed at pH 8–11. However, optimum activity for α- and β-glucosidase occurred at pH 5–8. Alpha- and β-galactosidases optimum activities occurred at pH 5 and pH 6, respectively. Inhibitors of serine proteases were effective on midgut serine proteases (trypsin and chymotrypsin proteases). Zymogram analyses revealed at least five bands of total proteolytic activity in the larval midgut. Protease-specific zymogram analyses revealed at least four, two, and one isozymes for trypsin-, chymotrypsin-, and elastase-like activities respectively. Two α-amylase isozymes were found in the midgut of fifth instar larvae and in the whole bodies of 1st through 5th instar larvae. Zymogram studies also revealed the presence of one and two bands of activity for β- and α-glucosidase, respectively. Recycling of α-amylase and proteases in the larval midgut was not complete. At least one isozyme of trypsin, chymotrypsin, elastase, and α-amylase were not recycled and were observed in the larval hindgut.

Expression of diverse midgut serine proteinases in the sericigenous Lepidoptera Antheraea assamensis (Helfer) is influenced by choice of host plant species

Antheraea assamensis is reared on various species of the Lauraceae family from north-east India for its distinctive cocoon silk. We demonstrate differential expression of digestive trypsin and chymotrypsins in larvae feeding on a primary host, Persea bombycina Kosterm., in comparison to larvae feeding on Litsea monopetala Roxb. using in vitro proteolytic assays, zymogram analyses with proteinase inhibitors, restriction digestion of RNA-PCR amplicons and quantitative real-time PCR (RT-PCR). Eight novel members of the serine proteinase gene family were identified, including an intron-spliced trypsin (AaPb4) and seven putative chymotrypsins (AaPb2, AaPb4, AaPb12, AaLm4, AaLm6, AaLm19 and AaLm29). Midgut transcript levels of the putative trypsin were higher in larvae fed P. bombycina whereas levels of transcripts encoding putative chymotrypsins were higher in larvae reared on L. monopetala. Complex, differential expression of sequence divergent midgut serine proteinases may reflect the ability of lepidopteran larvae to feed on different species of host plants. Possible implications of host plant choice on the digestive physiology of A. assamensis are discussed.

Expression profiling and comparative analyses of seven midgut serine proteases from the yellow fever mosquito, Aedes aegypti

Aedes aegypti utilizes blood for energy production, egg maturation and replenishment of maternal reserves. The principle midgut enzymes responsible for bloodmeal digestion are endoproteolytic serine-type proteases within the S1.A subfamily. While there are hundreds of serine protease-like genes in the A. aegypti genome, only five are known to be expressed in the midgut. We describe the cloning, sequencing and expression profiling of seven additional serine proteases and provide a genomic and phylogenetic assessment of these findings. Of the seven genes, four are constitutively expressed and three are transcriptionally induced upon blood feeding. The amount of transcriptional induction is strongly correlated among these genes. Alignments reveal that, in general, the conserved catalytic triad, active site and accessory catalytic residues are maintained in these genes and phylogenetic analysis shows that these genes fall within three distinct clades; trypsins, chymotrypsins and serine collagenases. Interestingly, a previously described trypsin consistently arose with other serine collagenases in phylogenetic analyses. These results suggest that multiple gene duplications have arisen within the S1.A subfamily of midgut serine proteases and/or that A. aegypti has evolved an array of proteases with a broad range of substrate specificities for rapid, efficient digestion of bloodmeals.

A Kunitz-type proteinase inhibitor from the midgut of the ixodid tick, Haemaphysalis longicornis, and its endogenous target serine proteinase

Although previous studies strongly suggested the involvement of serine proteases in blood digestion in the midgut of ticks, the regulating molecules of these proteinases are still unidentified. A novel Haemaphysalis longicornis Kunitz-type serine proteinase inhibitor with a single Kunitz-domain (HlMKI) has been identified and its co-localization with a midgut-derived serine proteinase (HlSP) within the epithelial cells has been demonstrated. Recombinant HlMKI inhibited the hydrolytic activity of HlSP, suggesting that HlMKI is a possible inhibitor of HlSP and may be part of a regulatory system of midgut serine proteinases.

Molecular genetic analysis of midgut serine proteases in Aedes aegypti mosquitoes

Digestion of blood meal proteins by midgut proteases provides anautogenous mosquitoes with the nutrients required to complete the gonotrophic cycle. Inhibition of protein digestion in the midgut of blood feeding mosquitoes could therefore provide a strategy for population control. Based on recent reports indicating that the mechanism and regulation of protein digestion in blood fed female Aedes aegypti mosquitoes is more complex than previously thought, we used a robust RNAi knockdown method to investigate the role of four highly expressed midgut serine proteases in blood meal metabolism. We show by Western blotting that the early phase trypsin protein (AaET) is maximally expressed at 3 h post-blood meal (PBM), and that AaET is not required for the protein expression of three late phase serine proteases, AaLT (late trypsin), AaSPVI (5G1), and AaSPVII. Using the trypsin substrate analog BApNA to analyze in vitro enzyme activity in midgut extracts from single mosquitoes, we found that knockdown of AaSPVI expression caused a 77.6% decrease in late phase trypsin-like activity, whereas, knockdown of AaLT and AaSPVII expression had no significant effect on BApNA activity. In contrast, injection of AaLT, AaSPVI, and AaSPVII dsRNA inhibited degradation of endogenous serum albumin protein using an in vivo protease assay, as well as, significantly decreased egg production in both the first and second gonotrophic cycles ( P < 0.001). These results demonstrate that AaLT, AaSPVI, and AaSPVII all contribute to blood protein digestion and oocyte maturation, even though AaSPVI is the only abundant midgut late phase serine protease that appears to function as a classic trypsin enzyme.

The Effects of Midgut Serine Proteases on Dengue Virus Type 2 Infectivity of Aedes aegypti

Dengue viruses (DENV) cause significant morbidity and mortality worldwide and are transmitted by the mosquito Aedes aegypti. Mosquitoes become infected after ingesting a viremic bloodmeal, and molecular mechanisms involved in bloodmeal digestion may affect the ability of DENV to infect the midgut. We used RNA interference (RNAi) to silence expression of four midgut serine proteases and assessed the effect of each RNAi phenotype on DENV-2 infectivity of Aedes aegypti. Silencing resulted in significant reductions in protease mRNA levels and correlated with a reduction in activity except in the case of late trypsin. RNA silencing of chymotrypsin, early and late trypsin had no effect on DENV-2 infectivity. However, silencing of 5G1 or the addition of soybean trypsin inhibitor to the infectious bloodmeals significantly increased midgut infection rates. These results suggest that some midgut serine proteases may actually limit DENV-2 infectivity of Ae. aegypti.

Characterization of digestive proteolytic activity in Lygus hesperus Knight (Hemiptera: Miridae)

The tarnished plant bug, Lygus hesperus Knight, is a pest that causes considerable economic losses to vegetables, cotton, canola, and alfalfa. Detailed knowledge of its digestive physiology will provide new opportunities for a sustainable pest management approach to control this insect. Little is known about the different protease class contributions to the overall digestion of a specific protein. To this end, the proteolytic activities in female adult L. hesperus salivary gland and midgut homogenates were quantified over a range of pH's and time points, and the contribution of different classes of proteases to the degradation of FITC-casein was determined. In the salivary gland, serine proteases were the predominant class responsible for caseinolytic activity, with the rate of activity increasing with increasing pH. In contrast, both aspartic and serine proteases contributed to caseinolytic activity in the midgut. Aspartic protease activity predominated at pH 5.0 and occurred immediately after incubation, whereas serine protease activity predominated at pH 7.5 after a 9 h delay and was resistant to aprotinin. The salivary serine proteases were distinctly different from midgut serine proteases, based on the tissue-specific differential susceptibility to aprotinin and differing pH optima. Collectively, the caseinolytic activities complement one another, expanding the location and pH range over which digestion can occur.

Cloning and characterization of trypsin- and chymotrypsin-like proteases from the midgut of the sand fly vector Phlebotomus papatasi

Trypsin and chymotrypsin serine proteases are the main digestive proteases in Diptera midguts and are also involved in many aspects of the vector–parasite relationship. In sand flies, these proteases have been shown to be a potential barrier to Leishmania growth and development within the midgut. Here we describe the sequence and partial characterization of six Phlebotomus papatasi midgut serine proteases: two chymotrypsin-like (Ppchym1 and Ppchym2) and four trypsin-like (Pptryp1–Pptryp4). All six enzymes show structural features typical to each type, including the histidine, aspartic acid, and serine (H/D/S) catalytic triad, six conserved cysteine residues, and other amino acid residues involved in substrate specificity. They also show a high degree of homology (40–60% identical residues) with their counterparts from other insect vectors, such as Anopheles gambiae and Aedes aegypti. The mRNA expression profiles of these six proteases vary considerably: two trypsin-like proteases (Pptryp1 and Pptryp2) are downregulated and one (Pptryp4) upregulated upon blood feeding. The two chymotrypsin-like enzymes display expression behavior similar to that of the early and late trypsins from Ae. aegypti.

Three serine proteinases from midguts of the hard tick Rhipicephalus appendiculatus; cDNA cloning and preliminary characterization.

Rhipicephalus appendiculatus is one of the most economically important ticks distributed in south central and eastern Africa where little or no progress has been made on attempts to develop a vaccine. We have used a combination of RT-PCR, the 3' and 5'rapid amplification of cDNA ends (RACE) to clone and sequence three cDNAs encoding full-length R. appendiculatus midgut serine proteinases (RAMSP). RT-PCR degenerate primers were designed from amino acid sequences surrounding active sites, His57 and Ser195 conserved among most known serine proteinase-like genes (Mulenga et al. 2001). Northern blotting analysis of total RNA extracted from unfed and partially fed adult ticks revealed that mRNAs for RAMSP-1 and -2 were expressed only in partially fed ticks, while RAMSP-3 mRNA was not only expressed in both unfed and partially fed ticks, it was also up-regulated as tick feeding progressed. Expression analysis by RT-PCR revealed that RAMSP-3 was predominantly expressed in midguts when compared to salivary glands. For RAMSP-1 and -2, they were expressed at equivalent levels in both midguts and salivary glands. Based on key amino acid sequence features as well as similarity comparisons from the database, we speculated that polypeptides encoded by RAMPSP-1 to -3 are structurally more closely related to chymotrypsin- than trypsin-like serine proteinases. We have based our comments on the potential of serine proteinases as candidates for tick vaccines.

Differences in midgut serine proteinases from larvae of the bruchid beetles Callosobruchus maculatus and Zabrotes subfasciatus.

Proteinase activities in the larval midguts of the bruchids Callosobruchus maculatus and Zabrotes subfasciatus were investigated. Both midgut homogenates showed a slightly acidic to neutral pH optima for the hydrolysis of fluorogenic substrates. Proteolysis of epsilon-aminocaproil-Leu-Cys(SBzl)-MCA was totally inhibited by the cysteine proteinase inhibitors E-64 and leupeptin, and was activated by 1.5 mM DTT in both insects, while hydrolysis of the substrate Z-ArgArg-MCA was inhibited by aprotinin and E-64, which suggests that it is being hydrolysed by serine and cysteine proteinases. Gel assays showed that the proteolytic activity in larval midgut of C. maculatus was due to five major cysteine proteinases. However, based on the pattern of E-64 and aprotinin inhibition, proteolytic activity in larval midgut of Z. subfasciatus was not due only to cysteine proteinases. Fractionation of the larval midgut homogenates of both bruchids through ion-exchange chromatography (DEAE-Sepharose) revealed two peaks of activity against Z-ArgArg-MCA for both bruchid species. The fractions from C. maculatus have characteristics of cysteine proteinases, while Z. subfasciatus has one non-retained peak of activity containing cysteine proteinases and another eluted in a gradient of 250-350 mM NaCl. The proteolytic activity of the retained peak is higher at pH 8.8 than at pH 6.0 and corresponds with a single peak that is active against N-p-tosyl-GlyGlyArg-MCA, and sensitive to 250 microM aprotinin (90% inhibition). The peak contains a serine proteinase which hydrolyzes alpha-amylase inhibitor 1 from the common bean (Phaseolus vulgaris). Arch.

Identification of six chymotrypsin cDNAs from larval midguts of Helicoverpa zea and Agrotis ipsilon feeding on the soybean (Kunitz) trypsin inhibitor

Lepidopteran insects like Helicoverpa zea and Agrotis ipsilon produce STI-insensitive trypsins in the midgut following ingestion of dietary plant proteinase inhibitors like STI [ Broadway, R. M., J. Insect Physiol. 43(9) (1997) 855–874]. In this paper, the effects of dietary STI on a related family of midgut serine proteinases, the chymotrypsins, were investigated. STI-insensitive midgut chymotrypsins were detected in larvae of H. zea and A. ipsilon feeding on diets containing 1% STI while STI-sensitive chymotrypsins were present in larvae feeding on diets containing 0% STI. These chymotrypsins were unaffected by TPCK, a diagnostic inhibitor of mammalian chymotrypsins but were fully inhibited by chymostatin. Four midgut cDNA libraries were constructed from larvae of each species fed either 0% STI or 1% STI diets. Six full-length cDNAs 1 GenBank Accessions # AF233728-AF233737 1 encoding diverse preprochymotrypsins were isolated (three from H. zea and three from A. ipsilon) with certain sequence motifs that set them apart from their mammalian counterparts. Northern blots showed that some chymotrypsin mRNA were detected at higher levels while others were down-regulated when comparing insects reared on 0% STI and 1% STI diets. Southern hybridizations suggested that (like mammals) both species contained several chymotrypsin genes. A full-length chymotrypsin gene 1 from H. zea was sequenced for the first time and the presence of four introns was deduced. A first time comparison of 5′ upstream regions 1 from three chymotrypsin genes and two trypsin genes of A. ipsilon indicated the presence of putative TATA boxes and regulatory elements. However a lack of consensus motifs in these upstream regions suggested the likelihood of multiple trans factors for regulation of genes encoding digestive proteinases and a complex response mechanism linked to ingestion of proteinase inhibitors.

Collagenolytic activity of crustacean midgut serine proteases: Comparison with the bacterial and mammalian enzymes

1. 1. We have investigated the collagenolytic activity of the following serine proteases: proteinase K, subtilisin Novo, Staphylococcal endoproteinase Glu-C, Streptomyces pronases, the trypsins and chymotrypsins from shrimp midgut and bovine pancreas. 2. 2. By assays on both the insoluble 3H-collagen fibrils and the soluble type I collagen, it was demonstrated that the shrimp midgut serine proteases, and less efficiently, the pronases from Streptomyces griseus, could hydrolyze collagen while the other serine proteases tested could not. 3. 3. Our data indicate that the trypsins and chymotrypsins of shrimp ( Penaeus monodon) directly and indirectly digest native collagen, and that the indirect pathway probably involves activation of procollagenase in the native collagen by these serine proteases.