Bovine tryptases. cDNA cloning, tissue specific expression and characterization of the lung isoform.
A complementary DNA encoding a new bovine tryptase isoform (here named BLT) was cloned and sequenced from lung tissue. Analysis of sequence indicates the presence of a 26-amino acid prepro-sequence and a 245 amino acid catalytic domain. It contains six different residues when compared with the previously characterized tryptase from bovine liver capsule (BLCT), with the most significant difference residing at the primary specificity S1 pocket. In BLT, the canonical residues Asp-Ser are present at positions 188-189, while in BLCT these positions are occupied by residues Asn-Phe. This finding was confirmed by mass fingerprinting of the peptide mixture obtained upon in-gel tryptic digestion of BLT. Analysis by gel filtration of the purified protein shows that BLT is probably tetrameric, similar to the previously identified tryptases from other species, with monomer migrating as 35-40 kDa multiple bands in SDS/PAGE. As expected, the catalytic abilities of the two bovine tryptases are different. The specificity constant values (kcat/Km) assayed with model substrates are 10- to 60-fold higher in the case of BLT. The tissue-specific expression of the two tryptases was evaluated at the RNA level by analysis of their different restriction patterns. In lung, only BLT was found to be expressed, while in liver capsule only BLCT is present. Both isoforms are distributed in similar amounts in heart and spleen. Analysis of the two gene sequences reveals the presence of several recognition sequences in the promoter regions and suggest a role for hormones in governing the mechanism of tissue expression of bovine tryptases.
Gambacurta
Alessandra
A
Department of Experimental Medicine and Biochemical Sciences, University of Rome Tor Vergata, Rome, Italy.
Fiorucci
Laura
L
Basili
Paolo
P
Erba
Fulvio
F
Amoresano
Angela
A
Ascoli
Franca
F
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000032:analysis*; D007527:Isoenzymes; D008168:Lung / Q000201:enzymology*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014018:Tissue Distribution; D053802:Tryptases
D018076:DNA, Complementary; D007527:Isoenzymes; D012333:RNA, Messenger; D012697:Serine Endopeptidases; D053802:Tryptases