Serum carboxypeptidase A activity as a biomarker for early-stage pancreatic carcinoma.

BACKGROUND: The early stage of pancreatic carcinoma lacks typical clinical signs and symptoms, and is difficult to diagnose. We developed an assay for active form of serum carboxypeptidase A (F-CPA) and its zymogen precursor pro CPA, and evaluated them as a marker for early-stage pancreatic carcinoma. METHODS: Serum CPA from 406 patients including 169 with pancreatic carcinoma, 53 with acute pancreatitis, 23 with chronic pancreatitis, and 161 with non-pancreatic diseases were assayed by a method with N-acetyl-phenylalanyl-l-3-thiaphenylalanine as substrate and dl-benzylsuccinic acid as specific inhibitor of CPA. Activation of pro CPA was carried out using trypsin. RESULTS: An established assay system was performed fully automatically and possesses enough performance for routine clinical assay. This system requires 31 minutes for CPA detection. No significant differences were detected between the F-CPA activity of patients with pancreatic carcinoma and that of patients with non-pancreatic diseases (p=0.168). F-CPA was mainly increased in patients with pancreatitis. Since total-CPA (T-CPA, T-CPA=pro CPA+F-CPA) was increased both in patients with pancreatic carcinoma and those with acute pancreatitis (p<0.0001, each case), the F-CPA/T-CPA ratio was low only in the patients with pancreatic carcinoma. The rate of positivity of T-CPA in the early stage of pancreatic carcinoma in which tumor was less than 2 cm was 77%, and higher than those of CA19-9 (31%), CEA (8%), and elastase 1 (46%). CONCLUSION: It was suggested that assays of both T-CPA and F-CPA in serum might be useful for the surveillance of early-stage pancreatic carcinoma.

A CLN2-related and thermostable serine-carboxyl proteinase, kumamolysin: cloning, expression, and identification of catalytic serine residue.

The gene encoding kumamolysin, a thermostable pepstatin-insensitive carboxyl proteinase, was cloned and expressed. (i) Kumamolysin was synthesized as a large precursor consisting of two regions: amino-terminal prepro (188 amino acids) and mature proteins (384 amino acids). (ii) The deduced amino acid sequence of the mature region exhibited high similarity to those of such bacterial pepstatin-insensitive enzymes as Pseudomonas carboxyl proteinase (PSCP; EC 3.4.23.37, identity = 37%), Xanthomonas carboxyl proteinase (XCP; EC 3.4.23.33, identity = 36%), and human CLN2 gene product (identity = 36%), which is related to a fatal neurodegenerative disease. (iii) The presumed catalytic triad, Glu78, Asp82, Ser278 [three-dimensional structure of PSCP: Wlodawer, A. et al. (2001) Nature Struct. Biol., 8, 442-446], was found to be conserved in the amino acid sequence of kumamolysin. (iv) Kumamolysin was inactivated by such aldehyde-type inhibitors as Ac-Ile-Pro-Phe-CHO (K(i) = 0.7 0.14 microM). In PSCP, it has been clarified that these inhibitors form a hemiacetal linkage with the catalytic serine residue and inactivate the enzyme. (v) Mutational analysis of the Ser278 residue revealed that the mutant lost both auto-processing activity and proteolytic activity. These results strongly suggest that kumamolysin has a unique catalytic triad consisting of Glu78, Asp82, and Ser278 residues, as previously observed for PSCP.