cDNA cloning and substrate specificity of equine tryptase, a possible mediator in equine heaves.

The research attempts to establish the role of a proteinase called equine tryptase, found in mast cells, in causing a condition similar to human occupational dust-induced asthma in horses. By determining the cDNA sequence and investigating the substrate specificity of the enzyme, the researchers aim to evaluate its enzymatic activity.

Detailed Explanation of the Research Paper

• The paper begins by highlighting the speculative role of mast cell mediators in respiratory illnesses such as allergic and occupational asthma in humans. Equine heaves, a condition in horses that causes airway inflammation and obstruction, is compared to these illnesses. The researchers posit that mast cells – like in human conditions – might also be responsible for equine heaves.
• The study’s aim is to examine the cDNA sequence of equine tryptase – a proteinase produced mainly by mast cells – and to scrutinize its substrate specificity. The cDNA sequence is explored to map out its genetic coding, while substrate specificity helps determine its enzymatic activities, i.e., the types of molecules it can actively interact with.
• The researchers utilized the technique of RT-PCR cloning to sequence equine tryptase. After cloning, they assessed its substrate specificity using substrates containing arginine and lysine.
• On sequencing, it was found that the cDNA and deduced amino acid sequences of equine tryptase showed significant similarity with other tryptases. A unique discovery was the presence of an alanine at the 216th residue, usually expected to be glycine in a trypsin-like proteinase. The presence of alanine enhances arginine substrate specificity in vitro, which is hypothesized to limit fibrinogenolysis, the breakdown of fibrinogen – a blood protein essential for clotting – in living organisms.
• The paper concludes with the inference that determining the cDNA sequence of equine tryptase will assist in assessing its expression in both unaffected and heaves-affected horses. They also emphasize the necessity of future research to understand the biological consequences of the unique alanine substitution in the equine tryptase substrate-binding pocket.