Identification and Expression of the cDNA-encoding Human Mesotrypsin(ogen), an Isoform of Trypsin with Inhibitor Resistance

Cornelion.M Nyaruhucha,Makoto Kito,Shin-Ichi Fukuoka

doi:10.1074/jbc.272.16.10573

Abstract

The cDNA encoding a novel isoform of human trypsinogen was identified. The isoelectric points of the proenzyme and active forms calculated from the deduced amino acid sequence are consistent with those of mesotrypsin(ogen), known to be an inhibitor-resistant trypsin isoform. The cDNA attached with a bacterial signal peptide sequence was expressed in Escherichia coli. The recombinant proenzyme purified from periplasm showed enterokinase-dependent activation similar to a major isoform of human trypsinogen. The enzyme was far less inhibited by trypsin inhibitors such as soybean trypsin inhibitor, aprotinin, or pancreatic secretory trypsin inhibitor than the control trypsin. A gel filtration assay showed that the enzyme and aprotinin did not form a stable complex. It is noteworthy that the amino acid at position 198, which is in close vicinity to the active Ser, is Arg while those of other major trypsins are all Gly. It is concluded that the cloned cDNA encodes human mesotrypsinogen, a unique isoform of trypsinogen with inhibitor resistance.

Highlights

Trypsinogen 1 and 3 represent 23.1 and 16.0%, respectively, of total pancreatic secretory proteins in humans and play a major role in the proteolytic digestion [1]
At present, little is known of trypsinogen III, and the estimated pI based on the deduced amino acid sequence is different from those experimentally obtained in native mesotrypsinogen [1, 2]
CDNA Screening—-A human pancreatic cDNA library constructed in ␭-gt10 was screened with a rat trypsinogen cDNA fragment as a probe

Summary

EXPERIMENTAL PROCEDURES

General Recombinant DNA Methods—-DNA modification enzymes and restriction enzymes were purchased from standard commercial sources and used according to the manufacturers’ instructions. CDNA of Human Mesotrypsin expression vector was constructed in the same manner (with a cDNA-encoding human trypsinogen 1 (anionic trypsinogen, pI ϭ 4.9)) and utilized as a control This vector is referred to as pFlag-T1. Enzyme Assay—The recombinant mesotrypsinogen or trypsinogen 1 (0.1 mg/ml) was activated with enterokinase (1 ␮g/ml) (New England Biolabs) at 37 °C for 40 min, and benzoyl arginine p-nitroanilide (BApNA, Wako Pure Chemicals, 2 mM) was added as a substrate according to the method described [10]. The reaction mixtures were incubated at 22 °C for 15 min to allow inhibitor/enzyme interaction, after which BApNA was added. Activated recombinant mesotrypsin or trypsin 1 (0.1 mg/ml) was incubated with BPTI (250 ␮g/ml) (about equal molecular ratio) at 22 °C for 30 min, and the mixture was loaded onto the column. Elution profiles were monitored with a spectrophotometer (Beckman DU-650) at 280 nm

RESULTS

This study

DISCUSSION