The heterogeneity of mast cell tryptase from human lung and skin.
There has long been conjecture over the degree to which there may be structural and functional heterogeneity in the tetrameric serine protease tryptase (EC 3.4.21.59), a major mediator of allergic inflammation. We have applied 2D gel electrophoresis to analyze the extent, nature, and variability of this heterogeneity in lysates of mast cells isolated from lung and skin, and in preparations of purified tryptase. Gels were silver stained, or the proteins transferred to nitrocellulose blots and probed with either tryptase-specific monoclonal antibodies or various lectins. Tryptase was the major protein constituent in mast cell lysates, and presented as an array of 9-12 diffuse immunoreactive spots with molecular masses ranging from 29 to 40 kDa, and pI values from 5.1 to 6.3. Although the patterns obtained for lung and skin tryptase were broadly similar, differences were observed between tissues and between individual donors. Lectin binding studies indicated the presence of mono-antennary or bi-antennary complex-type oligosaccharide with varying degrees of sialylation. Deglycosylation with protein-N-glycosidase F (PNGase F) reduced the size of both lung and skin tryptase, while incubation with PNGase F or neuraminidase narrowed the pI range, indicating variable degrees of glycosylation as a major contributor to the size and charge heterogeneity. Comparison of different purified preparations of lung and skin tryptase revealed no significant difference in pH profiles, but differences were seen in reactivity towards a range of chromogenic substrates, with substantial differences in Km, kcat and degree of cooperativity. Mathematical modeling indicated that the variety in kinetics parameters could not result solely from the sum of varying amounts of isoforms obeying Michaelis-Menten kinetics but with different values of Km and kcat. The heterogeneity demonstrated for tryptase in these studies suggests that there are important differences in tryptase function in different tissues.
Peng
Qi
Q
Immunopharmacology Group and Tissue Remodelling and Repair, University of Southampton School of Medicine, Southampton General Hospital, Southampton, UK.
McEuen
Alan R
AR
Benyon
R Christopher
RC
Walls
Andrew F
AF
D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008407:Mast Cells / Q000201:enzymology*; D008958:Models, Molecular; D020033:Protein Isoforms; D012697:Serine Endopeptidases / Q000235:genetics*; D012867:Skin / Q000201:enzymology*; D013379:Substrate Specificity; D053802:Tryptases
D020033:Protein Isoforms; D012697:Serine Endopeptidases; D053802:Tryptases