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.
QUANTITATIVE AND STRUCTURAL ANALYSES OF URINARY TRYPSIN INHIBITOR IN TYPE 1 AND TYPE 2 DIABETES / Nieddu, Gabriele; Lepedda, Antonio Junior; Rocchiccioli, S; Fresu, P; Lobina, O; Idini, M; DE MURO, Pierina; Formato, Marilena. - In: EUROPEAN JOURNAL OF HISTOCHEMISTRY. - ISSN 1121-760X. - volume 58/supplement 1:(2014). (Intervento presentato al convegno XXXIV National Meeting of the Italian Society for the study of connective tissues (SISC) tenutosi a Modena nel October 3-4, 2014).
QUANTITATIVE AND STRUCTURAL ANALYSES OF URINARY TRYPSIN INHIBITOR IN TYPE 1 AND TYPE 2 DIABETES
NIEDDU, Gabriele;LEPEDDA, Antonio Junior;DE MURO, Pierina;FORMATO, Marilena
2014-01-01
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
