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Aims: 1) Identify the presence and characterize the plasmatic isoforms of Receptor Type Protein Tyrosine Phosphatase Gamma (PTPRG) 2) Characterization of new monoclonal antibodies specific for the extracellular domain of the protein. Background: PTPRG is a broadly expressed enzyme mainly expressed in lung, stomach, esophagus, colon, liver, spleen and kidney. PTPRG maps to chr 3p14.2/21 and has been implicated as a candidate tumor suppressor gene. Loss of eterozigosity, gene deletion, point mutation and promoter hypermethylation has been reported. The majority of the receptor PTP share similar intracellular domains while the extracellular portion exhibits broad structural variation. Rat brain has been reported to express various PTPRG isoforms, including the isolated extracellular region 1. Previous work from our group suggested the production on an extracellular soluble form of PTPRG in thyroid 2 . Methods: immunoprecipitation, western blotting, flow cytometry. Results: We have identified, in human and murine plasma, new PTPRG isoforms, one of which previously characterized by cDNA cloning only in rat 1. The polypeptides identified have an apparent MW of about 120 kDa (major band) and 90 kDa (minor band) respectively. Surprisingly, also the full length protein, that include a transmembrane region, was identified in the serum and was present in the fraction precipitating at 100000 ×g, suggesting the presence of an exosome-associated form. The major band (120 kDa) was also identified in mouse liver (isolated after saline perfusion in order to remove plasma) and in serum-free conditioned medium derived from HepG2, an hepatocellular carcinoma cell line commonly used for the study of the regulation of hepatic protein synthesis. The 120 kDa isoform was up-regulated by treatment with ethanol and potassium dichromate, two compounds known to be cytotoxic for liver 3-5 . With the future goal to set up an ELISA-based assay for the rapid screening of soluble PTPRG expression in various physiopathological conditions, we have developed and characterized two new monoclonal antibodies raised against the extracellular domain of PTPRG expressed in eukariotic cells (HEK 293F). Conclusion: We have described for the first time in human and mouse the presence of soluble, plasmatic forms of PTPRG that appear to be released primarily by liver. The major 120 kDa band appear to be glycosylated and its expression is modulated by stimuli known to produce liver injury. The production and initial characterization of new monoclonal antibodies capable to specifically recognize this protein here described will permit the set up of new immunoassays and will pave the way to a better characterization of this proteins and its role in health and disease.
IDENTIFICATION AND CHARACTERIZATION OF NEW PLASMATIC ISOFORMS OF THE TUMOR SUPPRESSOR GENE PTPRG AND DEVELOPMENT OF NEW SPECIFIC MONOCLONAL ANTIBODIES
MORATTI, Elisabetta
2010-01-01
Abstract
Aims: 1) Identify the presence and characterize the plasmatic isoforms of Receptor Type Protein Tyrosine Phosphatase Gamma (PTPRG) 2) Characterization of new monoclonal antibodies specific for the extracellular domain of the protein. Background: PTPRG is a broadly expressed enzyme mainly expressed in lung, stomach, esophagus, colon, liver, spleen and kidney. PTPRG maps to chr 3p14.2/21 and has been implicated as a candidate tumor suppressor gene. Loss of eterozigosity, gene deletion, point mutation and promoter hypermethylation has been reported. The majority of the receptor PTP share similar intracellular domains while the extracellular portion exhibits broad structural variation. Rat brain has been reported to express various PTPRG isoforms, including the isolated extracellular region 1. Previous work from our group suggested the production on an extracellular soluble form of PTPRG in thyroid 2 . Methods: immunoprecipitation, western blotting, flow cytometry. Results: We have identified, in human and murine plasma, new PTPRG isoforms, one of which previously characterized by cDNA cloning only in rat 1. The polypeptides identified have an apparent MW of about 120 kDa (major band) and 90 kDa (minor band) respectively. Surprisingly, also the full length protein, that include a transmembrane region, was identified in the serum and was present in the fraction precipitating at 100000 ×g, suggesting the presence of an exosome-associated form. The major band (120 kDa) was also identified in mouse liver (isolated after saline perfusion in order to remove plasma) and in serum-free conditioned medium derived from HepG2, an hepatocellular carcinoma cell line commonly used for the study of the regulation of hepatic protein synthesis. The 120 kDa isoform was up-regulated by treatment with ethanol and potassium dichromate, two compounds known to be cytotoxic for liver 3-5 . With the future goal to set up an ELISA-based assay for the rapid screening of soluble PTPRG expression in various physiopathological conditions, we have developed and characterized two new monoclonal antibodies raised against the extracellular domain of PTPRG expressed in eukariotic cells (HEK 293F). Conclusion: We have described for the first time in human and mouse the presence of soluble, plasmatic forms of PTPRG that appear to be released primarily by liver. The major 120 kDa band appear to be glycosylated and its expression is modulated by stimuli known to produce liver injury. The production and initial characterization of new monoclonal antibodies capable to specifically recognize this protein here described will permit the set up of new immunoassays and will pave the way to a better characterization of this proteins and its role in health and disease.
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