Korean J Occup Environ Med.  2003 Jun;15(2):181-187.

Identification of Cross-linked 46 KDa Protein in Experimentally Induced Silicotic Nodule in Rat Lung

• Kim YM
• Kim YJ
• Lee SY

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.

Keyword

Silicotic nodule; N epsilon-(gamma-glutamyl) lysine dipeptide; cross-linked protein

MeSH Terms

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
Alanine
Extracellular Matrix Proteins
Glycine
Immunoglobulin G
Leucine
Lysine
Silicon Dioxide
Sodium Chloride
Sulfhydryl Reagents
Urea