Primary structure of two distinct rat pancreatic preproelastases determined by sequence analysis of the complete cloned messenger ribonucleic acid sequences

The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.     

Molecular cloning and expression in Escherichia coli of a cDNA encoding human pancreatic elastase 2

We have cloned a DNA that is complementary to the messenger RNA that encodes human pancreatic elastase 2 from a human pancreatic cDNA library using a cloned cDNA for rat pancreatic elastase 2 messenger RNA. This complementary DNA contains the entire protein coding region of 807 nucleotides which encodes preproelastase of 269 amino acids, and 4 and 82 nucleotides of the 5'- and 3'-untranslated sequences, respectively. When this deduced amino acid sequence was compared with known amino acid sequences it showed 82% homology with rat pancreatic elastase 2. This deduced sequence also contains a 16-amino-acid peptide identical with the N-terminal sequence determined for native human pancreatic proelastase 2. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 241 amino acids including 16 and 12 amino acids for a signal peptide and an activation peptide, respectively. Moreover, the predicted key amino acid residues involved in determining the substrate specificity of mammalian pancreatic elastase 2 are retained in the human enzyme. Cloned human pancreatic elastase 2 cDNA was expressed in E. coli as a mature and pro-form protein. Both resulting proteins showed immunoreactivity toward anti-elastase serum and enzymatic activity. We have also cloned and sequenced a porcine pancreatic elastase 2 cDNA.     

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.     

Investigation of the active center of rat pancreatic elastase

We have isolated rat pancreatic elastase I (EC 3.4.21.36) using a fast two-step procedure and we have investigated its active center with p-nitroanilide substrates and trifluoroacetylated inhibitors. These ligands were also used to probe porcine pancreatic elastase I whose amino acid sequence is 84% homologous to rat pancreatic elastase I as reported by MacDonald, et al. (Biochemistry 21, (1982) 1453-1463). Both proteinases exhibited non-Michaelian kinetics for substrates composed of three or four residues: substrate inhibition was observed for most enzyme substrate pairs, but with Ala3-p-nitroanilide, rat elastase showed substrate inhibition, whereas porcine elastase exhibited substrate activation. With most of the longer substrates, Michaelian kinetics were observed. The kcat/Km ratio was used to compare the catalytic efficiency of the two elastases on the different substrates. For both elastases, occupancy of subsite S4 was a prerequisite for efficient catalysis, occupancy of subsite S5 further increased the catalytic efficiency, P2 proline favored catalysis and P1 valine had an unfavorable effect. Rat elastase has probably one more subsite (S6) than its porcine counterpart. The rate-limiting step for the hydrolysis of N-succinyl-Ala3-p-nitroanilide by rat elastase was essentially acylation, whereas both acylation and deacylation rate constants participated in the turnover of this substrate by porcine elastase. For both enzymes, trifluoroacetylated peptides were much better inhibitors than acetylated peptides and trifluoroacetyldipeptide anilides were more potent than trifluoroacetyltripeptide anilides. A number of quantitative differences were found, however, and with one exception, trifluoroacetylated inhibitors were less efficient with rat elastase than with the porcine enzyme.     

Novel gene exon homologous to pancreatic phospholipase A2: sequence and chromosomal mapping of both human genes

We described previously the cloning and DNA sequence of the human gene encoding pancreatic phospholipase A2 [DNA 5, 519]. When pancreatic phospholipase A2 (PLA2) cDNA was used to screen a human genomic library, two classes of clones were obtained. One class encoded the pancreatic enzyme, and a second class encoded one exon of an apparently related PLA2. No additional PLA2 gene exons displayed sufficient homology to be detected by the probe. A homologous sequence in both rat and porcine genomic DNA was detected by DNA blot hybridization, and the corresponding gene fragments were cloned and sequenced. Within the deduced amino acid sequences, the presence of known functional residues along with the high degree of interspecies conservation suggests the genes encode a functional PLA2 enzyme form. The encoded sequence lacks Cys11, as do the "type II" viperid venom and other nonpancreatic mammalian PLA2 enzymes. The sequence is distinct from porcine intestinal PLA2 and appears not to be a direct homolog of the recently published rabbit ascites and rat platelet enzymes. Hybridization of DNA probes containing sequences from these genes to genomic DNA blots of mouse/human somatic cell hybrids permitted chromosomal assignment for both. The pancreatic gene mapped to human chromosome 12, and the homologous gene mapped to chromosome 1.     

Substrate specificity of human pancreatic elastase 2

The substrate specificity of human pancreatic elastase 2 was investigated by using a series of peptide p-nitroanilides. The kinetic constants, kcat and Km, for the hydrolysis of these peptides revealed that this serine protease preferentially hydrolyzes peptides containing P1 amino acids which have medium to large hydrophobic side chains, except for those which are disubstituted on the first carbon of the side chain. Thus, human pancreatic elastase 2 appears to be similar in peptide bond specificity to the recently described porcine pancreatic elastase 2 [Gertler, A., Weiss, Y., & Burstein, Y. (1977) Biochemistry 16, 2709] but differs significantly in specificity from porcine elastase 1. The best substrates for human pancreatic elastase 2 were glutaryl-Ala-Ala-Pro-Leu-p nitroanilide and succinyl-Ala-Ala-Pro-Met-p-nitroanilide. However, there was little difference among substrates with leucine, methionine, phenylalanine, tyrosine, norvaline, or norleucine in the P1 position. Changes in the hydrolysis rate of peptides with differing P5 residues indicate that this enzyme has an extended binding site which interacts with at least five residues of peptide substrates. The overall catalytic efficiency of human pancreatic elastase 2 is significantly lower than that of porcine elastase 1 or bovine chymotrypsin with the compounds studied.     

Molecular cloning of complementary DNA encoding one of the human pancreatic protease E isozymes

We have cloned a DNA from a human pancreatic cDNA library using a cloned rat pancreatic elastase 1 cDNA as a probe, and determined its nucleotide sequence. This cDNA contains a coding region of 810 nucleotides which encodes a 270-amino-acid protein. The deduced amino acid sequence shows less than 60% homologies with rat and porcine pancreatic elastase 1, although its substrate binding region is homologous with those of the above elastases 1. When this deduced amino acid sequence was compared with known amino acid sequences of pancreatic proteases other than elastases, it was found to contain an amino acid sequence which was highly homologous with the N-terminal amino acid sequence of porcine pancreatic protease E. We also purified human pancreatic protease E isozymes from human pancreatic juice, and determined their N-terminal amino acid sequences. One of the isozymes does not hydrolyze elastin but does hydrolyze a synthetic substrate. Endoglycosidase F digests glycoside bonds of the isozyme. These results suggest that the cDNA cloned by us corresponded to one of the human protease E isozymes.     

Studies on the specificity of the cholesterol-binding pancreatic proteinase and identification as human pancreatic elastase 1

The proteolytic attack of the cholesterol-binding pancreatic proteinase (CBPP) on the oxidized insulin A and B chains as well as on glucagon was investigated by kinetic studies. The reaction products were isolated by high-pressure liquid chromatography and identified by amino acid analysis. The combined results reveal a pronounced selectivity of CBPP for the peptide bonds at the carboxy ends of Ala, Val, Leu, Ser, His and Thr residues with Ala, Val and Leu most favoured, indicating a close catalytic relationship to porcine pancreatic elastase [Narayanan, A. S. & Anwar, R. A. (1969) Biochem. J. 114, 11-17] and the anionic porcine pancreatic protease E [Kobayashi R., Kobayashi, Y. & Hirs, C. H. W. (1981) J. Biol. Chem. 256, 2460-2465] which resembles human pancreatic elastase 1. The immunological comparison indeed disclosed the identity of CBPP with human pancreatic elastase 1.     

Isolation and characterization of rat pancreatic elastase

Proelastase has been purified to homogeneity from rat pancreatic tissue by a combination of CM-Sephadex and immobilized protease inhibitor affinity resins. Trypsin activation yields an elastolytic enzyme that possesses a specificity toward small hydrophobic residues in synthetic amide substrates, similar to those of porcine elastase 1 and canine elastase. However, the rat enzyme also rapidly hydrolyzes a substrate containing tyrosine in the P1 position. N-Terminal sequence analysis reveals that rat proelastase has an identical activation peptide with that of porcine proelastase 1 and has two conservative amino acid sequence differences from the activation peptide of canine proelastase. The sequence data established that rat proelastase corresponds to the elastase 1 mRNA clone isolated by MacDonald et al. [MacDonald, R. J., Swift, G. H., Quinto, C., Swain, W., Pictet, R. L., Nikovits, W., & Rutter, W. J. (1982) Biochemistry 21, 1453]. The sequence and substrate data obtained for rat and canine elastases suggest that there is a family of pancreatic elastases with properties similar to those of the classically described porcine elastase 1.     

Tissue-specific expression of the rat pancreatic elastase I gene in transgenic mice

The gene for rat pancreatic elastase I is selectively expressed to high levels in the rat exocrine pancreas. When the cloned rat elastase I gene with 7 kb upstream and 5 kb downstream flanking sequences was introduced into mice by microinjection into fertilized eggs, the gene was expressed in a pancreas-specific manner. In four of five transgenic mice, the level of rat elastase I mRNA in the pancreas was equal to or greater than the normal rat level (10,000 mRNAs per cell) and correlated with the number of integrated gene copies. In nonpancreatic tissues the levels were at least 10³-fold lower, except for expression in the liver of one mouse. Thus transfer of a 23 kb genomic DNA segment containing the rat elastase I gene to a foreign chromosomal location in the mouse can give rise to qualitatively and quantitatively normal expression.     

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.     

Human serum alpha 1-antichymotrypsin is an inhibitor of pancreatic elastases

Incubation of human serum alpha 1-antichymotrypsin with human pancreatic elastase 2 or porcine pancreatic elastase results in the complete inhibition of each enzyme as determined by spectrophotometric assays. alpha 1-Antichymotrypsin reacts much more rapidly with the human than with the porcine enzyme. The inhibitor: enzyme molar ratio, required to obtain full inhibition of enzymatic activity, is equal to 1.25/1 when alpha 1-antichymotrypsin reacts with human pancreatic elastase 2 while it is markedly higher with porcine pancreatic elastase (5.5/1). Patterns obtained by SDS/polyacrylamide gel electrophoresis of the reaction products show the formation with both enzymes of an equimolar complex (Mr near 77 000) and the release of a fragment migrating as a peptide of Mr near 5000. Moreover a free proteolytically modified form of alpha 1-antichymotrypsin, electrophoretically identical with that obtained in the reaction with cathepsin G or bovine chymotrypsin, is produced in the reaction with each elastase but in a much greater amount when alpha 1-antichymotrypsin reacts with porcine elastase than with human elastase. As a consequence of our findings, the specificity of alpha 1-antichymotrypsin, so far limited to the inhibition of chymotrypsin-like enzymes from pancreas and leukocyte origin, has to be extended to the two pancreatic elastases investigated in this work. A contribution of alpha 1-antichymotrypsin to the regulatory balance between plasma inhibitors and human pancreatic elastase 2 in pancreatic diseases is suggested.     

Further studies on human cholesterol-binding pancreatic protease/elastase 1. Immunological detection of analogous enzymes in several animal species and identification of the porcine-derived enzyme as protease E

Antibodies against the human cholesterol-binding pancreatic protease/elastase 1 (Sziegoleit, A., Linder, D., Schlüter, M., Ogawa, M., Nishibe, S. & Fujimoto, K. 1985) Eur. J. Biochem. 151, 595-599) recognize a distinct protein in the pancreas homogenate of various animal species. The CBPP/elastase 1-related porcine protease was purified and characterized. Its properties, including specificity, proved to be the same as those of the well classified porcine pancreatic protease E (Kobayashi, R., Kobayashi, Y. & Hirs, C.H.W. (1981) J. Biol. Chem. 256, 2460-2465). While the common features of all these proteins seem to be the proteolysis with elastase-like specificities (investigated for the human-, porcine-, dog- and rat-derived protein) and an isoelectric point at about pH 5 (determined for the pancreatic proteins from man, swine, rat dog and cattle), the bile salt and cholesterol-binding capacity varies significantly among the animal species. Charge shift crossed immunoelectrophoresis of the pancreatic proteins from rat, dog, cat, swine, horse, zebra, cattle and rabbit reveals that only the protein from rat pancreas binds the negatively charged bile salt sodium deoxycholate to an extent comparable to that of human CBPP/elastase 1. Thus, within the diverse elastase family, there seems to be a distinct enzyme which merits distinct classification.     

Identification of a novel class of elastase isozyme, human pancreatic elastase III, by cDNA and genomic gene cloning

By using porcine elastase I cDNA as a probe, we have isolated two different but closely related cDNAs encoding elastase-like proteases from a human pancreatic cDNA library. The amino acid sequences deduced from the cloned cDNA sequences showed 56-61% identity with those of both pancreatic elastases I and II, similar to the homology between elastases I and II. The active form of the elastase-like proteases appeared to be composed of 242 amino acids and preceded by a signal peptide and propeptide of 28 amino acids. Dot blot analysis of various tissue mRNAs demonstrated that the genes for the cloned cDNAs are expressed at a high level only in the pancreas. In addition, sequence analysis of the cloned genomic genes corresponding to one of the cDNAs showed that they are members of the elastase gene family. These results indicate that the two enzymes encoded by the cDNAs should be classified into a third class of elastase isozyme. Therefore, we designated them as human pancreatic elastases IIIA and IIIB. They strongly resembled cholesterol-binding pancreatic protease, suggesting that they may possess not only a digestive function but also function(s) related to cholesterol metabolism or transport in the intestine.