Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA

Abstract

Although protease E was isolated from human pancreas over 10 years ago [Mallory, P. A., & Travis, J. (1975) Biochemistry 14, 722-729], its amino acid sequence and relationship to the elastases have not been established. We report the isolation of a cDNA clone for human pancreatic protease E and determination of the nucleic acid sequence coding for the protein. The deduced amino acid sequence contains all of the features common to serine proteases. The substrate binding region is highly homologous to those of porcine and rat elastases 1, explaining the similar specificity for alanine reported for protease E and these elastases. However, the amino acid sequence outside the substrate binding region is less than 50% conserved, and there is a striking difference in the overall net charge for protease E (6-) and elastases 1 (8+). These findings confirm that protease E is a new member of the serine protease family. We have attempted to identify amino acid residues important for the interaction between elastases and elastin by examining the amino acid sequence differences between elastases and protease E. In addition to the large number of surface charge changes which are outside the substrate binding region, there are several changes which might be crucial for elastolysis: Leu-73/Arg-73; Arg-217A/Ala-217A; Arg-65A/Gln-65A; and the presence of two new cysteine residues (Cys-98 and Cys-99B) which computer modeling studies predict could form a new disulfide bond, not previously observed for serine proteases. We also present evidence which suggests that human pancreas does not synthesize a basic, alanine-specific elastase similar to porcine elastase 1.

Localization and characterization of the glycosylation site of human pancreatic elastase 1

“…To gain insight into this function and to compare E1 with elastase isoenzymes, which have recently been sequenced after gene cloning [3,4], we are studying the structure of El, a glycoprotein [2,5]. We report here the partial sequence analysis of El, confirming the two potential Nglycosylation sites which are also present in the sequence of protease E [3] and elastase III [4], the assignment of the oligosaccharide to a specific peptide sequence and analysis of the components of the carbohydrate moiety.…”

Elastase in Intestinal Mucus Enhances the Cytotoxicity of Shiga Toxin Type 2d

“…These proteolytic enzymes are found in the intestinal mucus content of many species including humans (21,22,35,36), where they would presumably be available for the activation of Stx2d produced by infecting E. coli. We have shown that strains of E. coli that express activable Stx2d are exquisitely virulent in a streptomycin-treated mouse oral challenge model (8), and we now hypothesize that in that model mouse pancreatic elastase can activate Stx2d in vivo and increase the virulence of the infecting strain (5,6).…”

Identification of a procarboxypeptidase A‐truncated protease E binary complex in human pancreatic juice

“…The N-terminal sequence of G32 is reported in table 1 and compared to the sequences of human P 35 [15], protease E isoenzymes (HPEl , HPE2) [21,22] and to bovine subunit III [23]. Human G32, like bovine subunit III and human P 35, lacks the N-terminal dipeptide Val-Val.…”