Human cationic trypsinogen is sulfated on Tyr154.

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Previous studies identified Tyr154 modification in human cationic trypsinogen as phosphorylation.
• This modification was initially reported based on crystal structure analysis.

Purpose of the Study:

• To re-evaluate the chemical modification of Tyr154 in human trypsinogen.
• To determine if Tyr154 is phosphorylated or sulfated.
• To investigate the functional and evolutionary implications of Tyr154 modification.

Main Methods:

• Purification and alkaline hydrolysis of human cationic and anionic trypsinogens.
• Separation and analysis of modified tyrosine amino acids using Dowex cation-exchange chromatography and thin-layer chromatography.
• Expression of human cationic trypsinogen in HEK293T cells and assessment of [(35)S]SO(4) incorporation.
• Site-directed mutagenesis of Tyr154 to Phenylalanine (Phe).
• Comparison of autoactivation rates between sulfated and non-sulfated trypsinogen forms.
• Sequence alignment of trypsinogen from various species.

Main Results:

• Human cationic and anionic trypsinogens contain tyrosine sulfate, but not tyrosine phosphate.
• Bovine trypsinogen lacks both tyrosine sulfate and phosphate.
• Radioactive sulfate incorporation into human cationic trypsinogen was confirmed and localized to Tyr154.
• Sulfated human cationic trypsinogen showed faster autoactivation compared to its non-sulfated counterpart.
• The tyrosine sulfation motif is conserved only in primate trypsinogens.

Conclusions:

• Tyr154 in human trypsinogen is sulfated, not phosphorylated.
• Tyrosine sulfation enhances the autoactivation of human cationic trypsinogen.
• Tyrosine sulfation of trypsinogen is a primate-specific modification.