cDNA cloning and substrate specificity of equine tryptase, a possible mediator in equine heaves.
BACKGROUND
Mast cell mediators are believed to play a central role in inflammatory lung disorders such as human allergic and occupational asthma. Equine heaves is characterized by reversible neutrophilic airway inflammation and airway obstruction, primarily due to bronchospasm and mucus hypersecretion, following exposure of susceptible horses to organic stable dusts. As such, heaves shares many similarities with human occupational dust-induced asthma and therefore it is proposed that mast cells may also be implicated in the pathogenesis of heaves. Tryptase, a mast cell-specific proteinase, can be used as an indicator of biological mast cell activity.


OBJECTIVE
The aim of this study was to determine the cDNA sequence of equine tryptase and to investigate its substrate specificity in order to rationalize its enzymatic activity.


METHODS
RT-PCR cloning was used to sequence equine tryptase. Substrate specificity of equine tryptase was investigated using arginine and lysine containing substrates.


RESULTS
The cDNA and deduced amino acid (Aa) sequences for equine tryptase shared strong identity with other tryptases. Unusually for a trypsin-like proteinase however, equine tryptase has alanine at residue 216, rather than glycine, which confers increased arginine substrate specificity in vitro and may restrict fibrinogenolysis in vivo.


CONCLUSIONS
Cloning and sequencing of the mast cell proteinase equine tryptase will allow molecular probing of its expression in the lung of control and heaves-affected horses. Further work is warranted to determine the biological relevance of the unique alanine 216 substitution in the molecular sequence of the equine tryptase substrate-binding pocket.
Dacre
K J
KJ
Department of Veterinary Clinical Studies, Easter Bush Veterinary Centre, Royal Dick School of Veterinary Studies, Midlothian, UK. K.J.Dacre@massey.ac.nz
McAleese
S M
SM
Knight
P
P
McGorum
B C
BC
Pemberton
A D
AD
D000595:Amino Acid Sequence; D000818:Animals; D001249:Asthma / Q000201:enzymology* / Q000276:immunology / Q000662:veterinary*; D001483:Base Sequence; D015415:Biomarkers / Q000032:analysis; D004796:Clinical Enzyme Tests; D003001:Cloning, Molecular; D016172:DNA Fingerprinting; D018076:DNA, Complementary / Q000032:analysis*; D005340:Fibrinogen / Q000378:metabolism; D006734:Horse Diseases / Q000201:enzymology* / Q000276:immunology; D006736:Horses; D006801:Humans; D008407:Mast Cells / Q000276:immunology; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D013379:Substrate Specificity; D053802:Tryptases / Q000235:genetics* / Q000378:metabolism
D015415:Biomarkers; D018076:DNA, Complementary; D005340:Fibrinogen; D053802:Tryptases