Identification of Cross-linked 46 KDa Protein in Experimentally Induced Silicotic Nodule in Rat Lung.
- Author:
You Mie KIM
1
;
Young Jin KIM
;
Soo Young LEE
Author Information
1. Department of Natural Sciences, College of Medicine, The Catholic University, Korea. lsyng@catholic.ac.kr
- Publication Type:Original Article
- Keywords:
Silicotic nodule;
N epsilon-(gamma-glutamyl) lysine dipeptide;
cross-linked protein
- MeSH:
Alanine;
Animals;
Chromatography;
Chromatography, Affinity;
Chromatography, High Pressure Liquid;
Electrophoresis, Polyacrylamide Gel;
Extracellular Matrix Proteins;
Fibrosis;
Glycine;
Immunoglobulin G;
Leucine;
Lung*;
Lysine;
Plasma;
Rats*;
Rats, Sprague-Dawley;
Silicon Dioxide;
Silicosis;
Sodium Chloride;
Sulfhydryl Reagents;
Urea
- From:Korean Journal of Occupational and Environmental Medicine
2003;15(2):181-187
- CountryRepublic of Korea
- Language:Korean
-
Abstract:
OBJECTIVES: This study was conducted in order to understand the cellular events associated with silica-induced pathogenesis of the rat lung. METHODS: Silicosis was induced by an intratracheal instillation of 50 mg of silica (SiO2, 0.15 - 10 micrometer) suspended in 500 microliter of a sterile saline solution in Sprague-Dawley rats weighing 200g. Silicotic nodules were excised from the rat lungs 4 weeks after silica instillation, then boiled for 4 days at 110 degrees in solution containing 2% SDS, 10 M urea and 40 mM DTT. The insoluble cellular encapsulates were electrophoresed on 4-12 % gradient SDS-PAGE, and the amino acid composition was analyzed. Affinity chromatographies of the homogenate supernatants of the control lung, silicotic nodule, and normal rat plasma were performed using rabbit IgG, anti-rat, cross-linked protein from the silicotic nodule. The amounts of N epsilon-(gamma-glutamyl) lysine cross-linked in the control lungs and silicotic nodules were determined using HPLC analysis. RESULTS: The remaining cross-linked protein was insoluble in the 10 M urea and 40 mM sulfhydryl reagents even under prolonged boiling conditions. The encapsulate revealed the retention of silica particles within the protein whose amino acid composition showed a high percentage of alanine, leucine and glycine. A 46 KDa protein was identified as a cross-linked protein in the silicotic nodule by affinity chromatography. The level of N epsilon-(gamma-glutamyl) lysine dipeptide in the nodule digest was prominently increased compared with that in the control lung. CONCLUSIONS: Transglutaminase (TGase)-catalyzed cross-linking appears to be involved in the silicotic nodule formation, and the 46 KDa protein may be cross-linked to itself and other extracellular matrix proteins during fibrosis and the formation of eventually insoluble nodule.
