Affinity Purification of Recombinant Trypsinogen Using Immobilized Ecotin

“…Upon self-splicing of the mini-intein moiety intracellularly, the liberated trypsinogen contains a homogeneous intact N terminus. In vitro refolding and purification of trypsinogen on immobilized ecotin was performed as described previously (21,22). Concentrations of trypsinogen preparations were determined from the UV absorbance at 280 nm using extinction coefficients 38,890 M Ϫ1 cm Ϫ1 (PRSS2) and 37,525 M Ϫ1 cm Ϫ1 (PRSS1).…”

Section: Methodsmentioning

confidence: 99%

Tighter Control by Chymotrypsin C (CTRC) Explains Lack of Association between Human Anionic Trypsinogen and Hereditary Pancreatitis

Jancsó

Sahin–Tóth

2016

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

The human pancreas expresses two major trypsinogen isoforms, cationic trypsinogen (PRSS1) and anionic trypsinogen (PRSS2). Mutations in PRSS1 cause hereditary pancreatitis by altering cleavage of regulatory nick sites by chymotrypsin C (CTRC) resulting in reduced trypsinogen degradation and increased autoactivation. Despite 90% identity with PRSS1 and a strong propensity for autoactivation, mutations in PRSS2 are not found in hereditary pancreatitis suggesting that activation of this isoform is more tightly regulated. Here, we demonstrated that CTRC promoted degradation and thereby markedly suppressed autoactivation of human anionic trypsinogen more effectively than previously observed with cationic trypsinogen. Increased sensitivity of anionic trypsinogen to CTRC-mediated degradation was due to an additional cleavage site at Leu-148 in the autolysis loop and the lack of the conserved Cys-139 -Cys-206 disulfide bond. Significant stabilization of anionic trypsinogen against degradation was achieved by simultaneous mutations of CTRC cleavage sites Leu-81 and Leu-148, autolytic cleavage site Arg-122, and restoration of the missing disulfide bridge. This stands in stark contrast to cationic trypsinogen where single mutations of either Leu-81 or Arg-122 resulted in almost complete resistance to CTRC-mediated degradation. Finally, processing of the trypsinogen activation peptide at Phe-18 by CTRC inhibited autoactivation of anionic trypsinogen, although cationic trypsinogen was strongly stimulated. Taken together, the observations indicate that human anionic trypsinogen is controlled by CTRC in a manner that individual natural mutations are unlikely to increase stability enough to promote intra-pancreatic activation. This unique biochemical property of anionic trypsinogen explains the lack of association of PRSS2 mutations with hereditary pancreatitis.

show abstract

“…After a 1-min (enteropeptidase) or 5-min (trypsin) incubation, the rate of p-nitroaniline release was measured again for 1 min. Zymogens were further purified from high activity fractions by ecotin affinity chromatography, as reported previously (18).…”

Section: Methodsmentioning

confidence: 99%

“…Expression 18 -. The method involves the use of a self-splicing miniintein fused in frame with the N terminus of human cationic trypsinogen and a newly engineered E. coli strain deficient in aminopeptidase P, designated E. coli LG-3.…”

Section: Methodsmentioning

confidence: 99%

Chymotrypsin C (Caldecrin) Stimulates Autoactivation of Human Cationic Trypsinogen

Nemoda

Sahin–Tóth

2006

Journal of Biological Chemistry

Self Cite

104

View full text Add to dashboard Cite

Affinity Purification of Recombinant Trypsinogen Using Immobilized Ecotin

Cited by 44 publications

References 5 publications

Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis

Pathogenic cellular role of the p.L104P human cationic trypsinogen variant in chronic pancreatitis

Tighter Control by Chymotrypsin C (CTRC) Explains Lack of Association between Human Anionic Trypsinogen and Hereditary Pancreatitis

Chymotrypsin C (Caldecrin) Stimulates Autoactivation of Human Cationic Trypsinogen

Contact Info

Product

Resources

About