New findings on epidermal transglutaminase substrates

Area of Science:

• Biochemistry
• Dermatology
• Protein Chemistry

Background:

• Epidermal transglutaminase (eTG) plays a role in skin structure.
• Natural substrates for eTG are present in epidermal extracts.
• The specific function and nature of these substrates require further investigation.

Purpose of the Study:

• To investigate a soluble 36,000 molecular weight substrate for epidermal transglutaminase.
• To characterize the cross-linking activity of eTG on this substrate.
• To elucidate the potential in vivo pathway of substrate modification.

Main Methods:

• Purification of a 36,000 molecular weight protein from cow snout epidermis.
• In vitro cross-linking assays using purified epidermal transglutaminase.
• Analysis of cross-linked products by SDS-PAGE and identification of epsilon-(gamma-glutamyl) lysine bonds.
• Inhibition studies using EDTA, iodoacetamide, and putrescine.

Main Results:

• Purified 36,000 molecular weight substrate was efficiently cross-linked by eTG into soluble polymers and an insoluble aggregate.
• The epsilon-(gamma-glutamyl) lysine bond was formed during cross-linking.
• Cross-linking was inhibited by EDTA, iodoacetamide, and putrescine.
• SDS-PAGE revealed that 90% of the substrate was reduced to 8,000-10,000 molecular weight subunits.

Conclusions:

• A specific 36,000 molecular weight protein acts as a substrate for epidermal transglutaminase.
• eTG-mediated cross-linking results in both soluble polymers and insoluble aggregates via epsilon-(gamma-glutamyl) lysine bonds.
• A model is proposed involving intracellular synthesis of the substrate, followed by insolubilization and cross-linking at the cell membrane.