Activation of transglutaminase during embryonic development

Area of Science:

• Developmental Biology
• Biochemistry
• Molecular Biology

Background:

• Protein synthesis and modification are crucial during early embryonic development.
• The role of specific enzymes like transglutaminase in fertilization and early development is not fully understood.
• Putrescine incorporation into proteins is observed in sea urchin eggs post-fertilization.

Purpose of the Study:

• To investigate the mechanism of [3H]putrescine incorporation into proteins in sea urchin eggs after fertilization.
• To confirm whether transglutaminase is responsible for this incorporation.
• To quantify the activity and product of transglutaminase in early sea urchin embryos.

Main Methods:

• Fertilization of sea urchin eggs and incubation with [3H]putrescine.
• Inhibition studies using emetine (protein synthesis inhibitor), transglutaminase inhibitors (2-[3-(diallylamino)-propionyl]benzothiophene, dansylcadaverine), and an inert analogue.
• Proteolytic digestion of labeled proteins and isolation/quantification of reaction products (gamma-glutamyl[3H]putrescine, epsilon-(gamma-glutamyl)lysine).

Main Results:

• Fertilization markedly increased [3H]putrescine incorporation into proteins.
• Emetine did not affect labeling, while transglutaminase inhibitors blocked it.
• High yields of gamma-glutamyl[3H]putrescine and epsilon-(gamma-glutamyl)lysine confirmed transglutaminase activity and cross-linking in vivo.

Conclusions:

• Transglutaminase activity significantly increases upon fertilization in sea urchin eggs.
• This enzyme plays a role in post-fertilization protein modification and cross-linking.
• The study provides strong evidence for a transglutaminase-mediated mechanism of putrescine incorporation.