Isolation and amino-terminal sequence analysis of two dissimilar pancreatic proelastases from the African lungfish, Protopterus aethiopicus

C, de Haën; Gertler, Arieh

doi:10.1021/bi00710a002

Cited by 30 publications

(15 citation statements)

References 23 publications

(20 reference statements)

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“…The foregut-midgut (Try-G2) trypsins may have developed insensitivity to PIs because they are the first to access and digest food sources containing PIs, thereby protecting the midgut-hindgut (Try-G3) trypsins, which are PI sensitive. It may also be possible that foregut-midgut trypsins play roles in activation of other zymogens, like trypsinogen, chymotrypsinogen and proelastase [67], or in initiating a cascade of events that subsequently regulate late trypsin expression [68]. Indeed, in Aedes aegypti adult female mosquitoes, multiple trypsin transcripts are sequentially induced with succeeding feeding periods, and an early trypsin may play a role in initiating a cascade of events with the subsequent expression of late trypsins [69], [70].…”

Section: Discussionmentioning

confidence: 99%

Characterization of cDNAs Encoding Serine Proteases and Their Transcriptional Responses to Cry1Ab Protoxin in the Gut of Ostrinia nubilalis Larvae

et al. 2012

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Serine proteases, such as trypsin and chymotrypsin, are the primary digestive enzymes in lepidopteran larvae, and are also involved in Bacillus thuringiensis (Bt) protoxin activation and protoxin/toxin degradation. We isolated and sequenced 34 cDNAs putatively encoding trypsins, chymotrypsins and their homologs from the European corn borer (Ostrinia nubilalis) larval gut. Our analyses of the cDNA-deduced amino acid sequences indicated that 12 were putative trypsins, 12 were putative chymotrypsins, and the remaining 10 were trypsin and chymotrypsin homologs that lack one or more conserved residues of typical trypsins and chymotrypsins. Reverse transcription PCR analysis indicated that all genes were highly expressed in gut tissues, but one group of phylogenetically-related trypsin genes, OnTry-G2, was highly expressed in larval foregut and midgut, whereas another group, OnTry-G3, was highly expressed in the midgut and hindgut. Real-time quantitative PCR analysis indicated that several trypsin genes (OnTry5 and OnTry6) were significantly up-regulated in the gut of third-instar larvae after feeding on Cry1Ab protoxin from 2 to 24 h, whereas one trypsin (OnTry2) was down-regulated at all time points. Four chymotrypsin and chymotrypsin homolog genes (OnCTP2, OnCTP5, OnCTP12 and OnCTP13) were up-regulated at least 2-fold in the gut of the larvae after feeding on Cry1Ab protoxin for 24 h. Our data represent the first in-depth study of gut transcripts encoding expanded families of protease genes in O. nubilalis larvae and demonstrate differential expression of protease genes that may be related to Cry1Ab intoxication and/or resistance.

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Section: Discussionmentioning

confidence: 99%

Characterization of cDNAs Encoding Serine Proteases and Their Transcriptional Responses to Cry1Ab Protoxin in the Gut of Ostrinia nubilalis Larvae

et al. 2012

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“…This enzyme did not exhibit any elastolytic activity towards elastinϪCongo-red, but had specificity on synthetic substrates with bulky residues, indicating an enlarged binding pocket compared with elastase I. Elastases from several different fish species have been purified and characterized [12Ϫ20]. In some studies, two enzyme species have been separated [12,21], but chymotrypsinlike specificity has not been reported for a fish elastase so far.We have previously purified and characterized a cationic type-I elastase [17] and two isozymes of chymotrypsin from Atlantic cod (variants A and B) [22]. Recently, two elastase-like polypeptide sequences, designated elastases A and B, were described resulting from a search for serine proteinase genes in a cod cDNA library [23].…”

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confidence: 99%

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confidence: 99%

The third serine proteinase with chymotrypsin specificity isolated from Atlantic cod (Gadus morhua) is a type‐II elastase

Ásgeirsson

Leth-Larsen²,

Thórólfsson

et al. 1998

European Journal of Biochemistry

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Preparations of chymotrypsin from Atlantic cod are heterogeneous and previously gave rise to two active peaks when subjected to pH-gradient chromatography. Extension of the pH-gradient resolved a third protein peak with benzoyltyrosine ethylester hydrolytic activity. The first two peaks have been characterized as chymotrypsin variants and designated A and B, whereas the identity of the third peak was not clear. Analysis of this protein by Edman sequencing and mass spectrometry has now confirmed a high degree of identity with the predicted protein sequence from a recently described cDNA clone. That sequence was named elastase B by sequence comparison. As the present elastase deviates in 16 positions from that of elastase B, we have named it elastase C. The elastase C was active in hydrolysing typical substrates used by chymotrypsin, namely benzoyl-L-tyrosine ethylester and succinyl-Ala-Ala-ProPhe-p-nitroanilide, but inactive against the typical elastase substrates succinyl-Ala-Ala-Ala-p-nitroanilide and orcein-elastin. Comparison of the kinetic properties of the cod elastase C with bovine chymotrypsin and cod chymotrypsin variants A and B, using succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, showed a lower catalytic efficiency of elastase C. The effects of several inhibitors on cod elastase C were identical to effects on chymotrypsins variants A and B, but dissimilar when compared with porcine pancreatic elastase. On the basis of the specificity and amino acid sequence, we conclude that the enzyme under study is most correctly classified as a type-II elastase.Keywords : elastase; serine protease ; Atlantic cod ; amino acid sequence; cold adaptation.Variants of elastases have been identified in different animals and in different animal tissues such as the pancreas, leucocytes, platelets, and aorta [1]. Elastase was initially characterized as a mammalian pancreatic serine protease with the ability to dissolve elastin [2], but elastase can digest a wide variety of other protein substrates in addition to native elastin [3]. Some elastases in the serine proteinases family turn out to be specific for non-bulky amino acid residues (e.g. porcine pancreatic elastase 1, human leucocyte elastase) while others have a chymotrypsinlike specificity (e.g. porcine pancreatic elastase 2, human pancreatic elastase 2). Pancreatic elastase type I was originally isolated from hog pancreas and is strongly cationic with an isoelectric point near pH 9.5 [2]. Another anionic enzyme, called elastase type II, was also obtained from the same tissue and found to have a characteristic chymotrypsin activity [4,5]. Human pancreatic tissue and juice also contain two enzymes, one cationic and the other anionic, which can hydrolyse a specific substrate for porcine pancreatic elastase and undyed elastin [6,7]. The cationic enzyme is the major elastase in human pancreas and has similar properties to porcine pancreatic elastase 1, whereas elastase 2 preferentially hydrolysed peptides at sites of medium to large hydrophobic side chains [8]. Human elastase 1 ...

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“…A similar situation was encountered in the case of lungfish proelastase A (de Haen & Gertler, 1974). Some artiodactyl trypsinogens (goat, sheep, deer) contain both an eight-residue and a six-residue activation peptide (Bricteux-GrCgoire et al, 1966;Schyns et al, 1969;Bricteux-GrCgoire, 1970;Bricteux-GrCgoire et al, 197 1 a,b).…”

Section: Discussionmentioning

confidence: 68%

Isolation and amino-terminal sequence analysis of a new pancreatic trypsinogen of the African lungfish Protopterus aethiopicus

C¹,

Ka²,

Neurath³

1977

Biochemistry

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The purification and characterization of three pancreatic trypsinogens A1, A2, and A3, from the African lungfish, Protopterus aethiopicus, is reported. These zymogens are activated by trypsin, by enterokinase, by an acid protease from Aspergillus oryzae, and by autoactivation. The three trypsinogens contain the same amino-terminal amino acid sequence, beginning with the activation peptide Leu-Pro-Leu-Glu-Asp-Asp-Lys-. Like the activation peptide of the previously characterized trypsinogen B [Reeck, G. R., & Neurath, H. (1972) Biochemistry 11, 503] of the same organism, it lacks the tetraaspartyl sequence characteristic of other vertebrate trypsinogens. Two of the corresponding lungfish trypsins were found to have identical amino-terminal sequences for at least 27 residues. These data suggest that the three enzymes are allelic variants. In contrast, the amino acid sequences differ sufficiently from that of trypsinogen B of the same organism to indicate that trypsinogens A and B are the products of different gene loci.

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Isolation and amino-terminal sequence analysis of two dissimilar pancreatic proelastases from the African lungfish, Protopterus aethiopicus

Cited by 30 publications

References 23 publications

Characterization of cDNAs Encoding Serine Proteases and Their Transcriptional Responses to Cry1Ab Protoxin in the Gut of Ostrinia nubilalis Larvae

Characterization of cDNAs Encoding Serine Proteases and Their Transcriptional Responses to Cry1Ab Protoxin in the Gut of Ostrinia nubilalis Larvae

The third serine proteinase with chymotrypsin specificity isolated from Atlantic cod (Gadus morhua) is a type‐II elastase

Isolation and amino-terminal sequence analysis of a new pancreatic trypsinogen of the African lungfish Protopterus aethiopicus

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