Research Article: A Ultrasensitive Near‐Infrared Fluorescent Probe Reveals Pyroglutamate Aminopeptidase 1 Can Be a New Inflammatory Cytokine

Date Published: January 22, 2018

Publisher: John Wiley and Sons Inc.

Author(s): Qiuyu Gong, Ruifen Zou, Jie Xing, Lingchao Xiang, Renshuai Zhang, Aiguo Wu.


Previous study showed that pyroglutamate aminopeptidase 1 (PGP‐1) has a relationship with the immune response in cells. However, whether PGP‐1 is involved in inflammatory response in vivo and can serve as a new inflammatory cytokine are still unclear. To address these issues, a new near‐infrared fluorescent probe, which exhibits high selectivity and super sensitivity, is developed. With this probe, the up‐regulation of PGP‐1 (evidenced by western blot) in BALB/c mice legs and livers under the stimulation of two main immunopotentiators is revealed for the first time. The occurrence of inflammatory process (including tissue necrosis) in mice is determined by up‐regulation of tumor necrosis factor‐α and hematoxylin‐eosin staining. Interestingly, it is revealed for the first time that knocking down PGP‐1 leads to the weakness of inflammatory process in RAW264.7 cells. These new findings suggest that PGP‐1 is indeed involved in inflammatory response in vivo and can be a new inflammatory cytokine.

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Reagents: N‐(tert‐butoxycarbonyl)‐L‐pyroglutamic acid (BOC‐L‐pyroglutamic acid) was obtained from Tokyo Chemical Industry Co., Ltd. N,N‐Diisopropylethylamine (DIPEA) and 3‐nitrophenol were purchased from Acros Organics. Stannous chloride (SnCl2) was obtained from J. K. Chemical Co., Ltd., Beijing, China. 2‐(7‐Aza‐1H‐benzotriazole‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate (HATU) and trifluoroacetic acid (CF3COOH, HPLC grade) were purchased from Alfa Aesar Chemicals. 11‐Chloro‐1,1′‐di‐n‐propyl‐3,3,3′,3′‐tetramethyl‐10,12‐trimethyleneindatricarbocyanine iodide (IR‐780 iodide), FCA, LPS, D‐Gal, dimethyl sulfoxide (DMSO), iodoacetamide, esterase, prolidase, trypsin, leucine aminopeptidase, dipeptidyl peptidase IV, and commercial PGP‐1 probe (L‐pyroglutamic acid 7‐amido‐4‐methylcoumarin) were purchased from Sigma‐Aldrich. Fibroblast activation protein was purchased from R&D Systems Inc. PGP‐1 (molecular weight, 23 kDa) was obtained from State Key Laboratory of Antibody Medicine and Targeted Therapy, Shanghai, China. Lipidosome 3000, Roswell Park Memorial Institute‐1640 medium (RPMI‐1640), new‐born calf serum (CS), and RAW264.7 cell lines were purchased from KeyGEN BioTECH Co., Ltd., Nanjing, China. Proteins were pure as judged by Coomassie‐stained sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis. Radio immunoprecipitation assay lysis buffer (CW2333) was purchased from CWbiotech Co. Ltd., Beijing, China. Agarose, poly(vinylidene fluoride) membranes, gel electrophoresis kits, WB kits, enhanced chemiluminescence kits (ECL), PGP‐1 siRNA (sequence: GGUUACAAAGGACUGGAUATTUAUCCAGUCCUUUGUAACCTT), and Braford protein assay kits were purchased from KeyGEN BioTECH Co. Ltd., Nanjing, China. Anti‐TNF‐α and Anti‐PGP‐1 antibody were purchased from Proteintech, USA. All other chemicals used were local products of analytical grade. Ultrapure water (over 18 MΩ cm) from a Milli‐Q reference system (Millipore) was used throughout. The stock solution (500 × 10−6m) of probe was prepared by dissolving requisite amount of it in DMSO. Stock solutions of other substances were prepared by dissolving in PBS or water.

The authors declare no conflict of interest.




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