Urinary trypsin inhibitor (UTI) is a small proteoglycan consistingof a 147 amino acids long polypeptide that carries an N-linked oligosaccharide at Asn45 and a O-linked low-charge chondroitinsulfate (CS) chain at Ser10. UTI levels are usually low inhealthy individuals but they increase up to 10 fold in both acuteand chronic inflammatory diseases. Recently, we set up a methodfor UTI purification and quantitation useful for its structuralcharacterization1. This report describes the structural characterizationof both protein and chondroitin sulfate moieties of UTI intype 1 and type 2 diabetic patients. UTI purification was performedby anion exchange chromatography starting from a volumeof urine corresponding to 2 mg of creatinine. The eluate wassplitted in two aliquots for structural and quantitative analyses.The first aliquot was subjected to chondroitin lyase ABC treatmentand the obtained disaccharide units were analysed byFluorophore-Assisted Carbohydrate Electrophoresis afterderivatization with 2-aminoacridone. The second one wasresolved by SDS-PAGE. A calibration curve for protein quantitationwas set up by using a highly purified UTI fraction.Furthermore, UTI band was subjected to trypsin digestion andstructural characterization by Nano-LC-MS/MS analysis. Themethod was applied to urine samples from 29 patients with type1 diabetes, 22 patients with type 2 diabetes and 42 healthy controls,matched for age and sex with patients, evidencing higherUTI 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 ofseveral sites prone to oxidative modifications. Interestingly, oneof them (residues 107-121) may represent a potential marker ofoxidation in both type 1 and type 2 diabetic patients. Preliminaryresults suggest that both UTI levels and structure could be modifiedrepresenting a useful marker of chronic inflammatory conditionin type 1 and 2 diabetes. The effects of such quantitativeand structural alterations on UTI localization, function andpathophysiological activities deserve further studies.
QUANTITATIVE AND STRUCTURAL ANALYSES OF URINARY TRYPSIN INHIBITOR IN TYPE 1 AND TYPE 2 DIABETES
NIEDDU, Gabriele;
2014-01-01
Abstract
Urinary trypsin inhibitor (UTI) is a small proteoglycan consistingof a 147 amino acids long polypeptide that carries an N-linked oligosaccharide at Asn45 and a O-linked low-charge chondroitinsulfate (CS) chain at Ser10. UTI levels are usually low inhealthy individuals but they increase up to 10 fold in both acuteand chronic inflammatory diseases. Recently, we set up a methodfor UTI purification and quantitation useful for its structuralcharacterization1. This report describes the structural characterizationof both protein and chondroitin sulfate moieties of UTI intype 1 and type 2 diabetic patients. UTI purification was performedby anion exchange chromatography starting from a volumeof urine corresponding to 2 mg of creatinine. The eluate wassplitted in two aliquots for structural and quantitative analyses.The first aliquot was subjected to chondroitin lyase ABC treatmentand the obtained disaccharide units were analysed byFluorophore-Assisted Carbohydrate Electrophoresis afterderivatization with 2-aminoacridone. The second one wasresolved by SDS-PAGE. A calibration curve for protein quantitationwas set up by using a highly purified UTI fraction.Furthermore, UTI band was subjected to trypsin digestion andstructural characterization by Nano-LC-MS/MS analysis. Themethod was applied to urine samples from 29 patients with type1 diabetes, 22 patients with type 2 diabetes and 42 healthy controls,matched for age and sex with patients, evidencing higherUTI 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 ofseveral sites prone to oxidative modifications. Interestingly, oneof them (residues 107-121) may represent a potential marker ofoxidation in both type 1 and type 2 diabetic patients. Preliminaryresults suggest that both UTI levels and structure could be modifiedrepresenting a useful marker of chronic inflammatory conditionin type 1 and 2 diabetes. The effects of such quantitativeand structural alterations on UTI localization, function andpathophysiological activities deserve further studies.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
