QUANTITATIVE AND STRUCTURAL ANALYSES OF URINARY TRYPSIN INHIBITOR IN TYPE 1 AND TYPE 2 DIABETES

Urinary trypsin inhibitor (UTI) is a small proteoglycan consisting
of a 147 amino acids long polypeptide that carries an N-linked oligosaccharide at Asn45 and a O-linked low-charge chondroitin
sulfate (CS) chain at Ser10. UTI levels are usually low in
healthy individuals but they increase up to 10 fold in both acute
and chronic inflammatory diseases. Recently, we set up a method
for UTI purification and quantitation useful for its structural
characterization1. This report describes the structural characterization
of both protein and chondroitin sulfate moieties of UTI in
type 1 and type 2 diabetic patients. UTI purification was performed
by anion exchange chromatography starting from a volume
of urine corresponding to 2 mg of creatinine. The eluate was
splitted in two aliquots for structural and quantitative analyses.
The first aliquot was subjected to chondroitin lyase ABC treatment
and the obtained disaccharide units were analysed by
Fluorophore-Assisted Carbohydrate Electrophoresis after
derivatization with 2-aminoacridone. The second one was
resolved by SDS-PAGE. A calibration curve for protein quantitation
was set up by using a highly purified UTI fraction.
Furthermore, UTI band was subjected to trypsin digestion and
structural characterization by Nano-LC-MS/MS analysis. The
method was applied to urine samples from 29 patients with type
1 diabetes, 22 patients with type 2 diabetes and 42 healthy controls,
matched for age and sex with patients, evidencing higher
UTI levels in both groups of patients (2.5 and 1.8 fold increase,
respectively) in respect to controls (p<0.0001 and p<0.05,
respectively). UTI structural analysis evidenced the presence of
several sites prone to oxidative modifications. Interestingly, one
of them (residues 107-121) may represent a potential marker of
oxidation in both type 1 and type 2 diabetic patients. Preliminary
results suggest that both UTI levels and structure could be modified
representing a useful marker of chronic inflammatory condition
in type 1 and 2 diabetes. The effects of such quantitative
and structural alterations on UTI localization, function and
pathophysiological activities deserve further studies.