cDNA sequence of two sheep mast cell tryptases and the differential expression of tryptase and sheep mast cell proteinase-1 in lung, dermis and gastrointestinal tract

• Published in: Clinical and experimental allergy 2000-06, Vol.30 (6), p.818-832
• Main Authors: PEMBERTON, A. D, MCALEESE, S. M, HUNTLEY, J. F, COLLIE, D. D. S, SCUDAMORE, C. L, MCEUEN, A. R, WALLS, A. F, MILLER, H. R. P
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antibodies, Monoclonal - immunology; Biological and medical sciences; Chymases; Dermis - enzymology; Digestive System - enzymology; Disease Models, Animal; DNA, Complementary - genetics; Dogs; Experimental and animal immunopathology. Animal models; Gene Expression; Humans; Immunohistochemistry; Immunopathology; Lung - enzymology; Mast Cells - enzymology; Medical sciences; Molecular Sequence Data; proteinase 1; Rabbits; Respiratory Hypersensitivity; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, DNA; Serine Endopeptidases - chemistry; Serine Endopeptidases - genetics; Serine Endopeptidases - immunology; Serine Endopeptidases - metabolism; Sheep; Tryptases
• ISSN: 0954-7894; 1365-2222
• DOI: 10.1046/j.1365-2222.2000.00831.x

Mast cell tryptases are a family of serine proteinases which are implicated in the proliferation of smooth muscle cells and fibroblasts, upregulation of interleukin-8 synthesis by endothelial cells, and recruitment of neutrophils and eosinophils. Trials in sheep showed that administration of a specific tryptase inhibitor reduced the late-phase response to inhaled allergen. The aim of this study was to characterize the sequence and distribution of sheep tryptase(s), to validate the sheep model of allergic lung disease. Reverse transcriptase PCR cloning was used to obtain cDNA sequences for two sheep tryptases. Lung and gut extracts were used as a source of tryptase for partial purification and characterization of the protein. The distribution of tryptase in skin, lung and gut was determined by immunohistochemistry, and compared with the distribution of sheep mast cell proteinase-1 (sMCP-1). Two highly similar cDNA sequences encoding sheep tryptase were found, indicating the presence of a 28 amino acid leader sequence, and a mature peptide of 245 amino acids. Partial purification of a putative sheep tryptase from lung and gut extracts was achieved using heparin-Sepharose affinity chromatography. Rabbit antihuman skin tryptase antiserum recognized the putative sheep tryptase on Western blot (approximate Mr 32-34 000) and paraformaldehyde-fixed tissue sections. Tryptase was detected in all lung, skin and gut mast cells by this antibody, and transcripts for tryptase were detected in all three tissues by RT PCR. Sheep mast cell proteinase-1, detected by a specific monoclonal antibody, was present in all intestinal and gastric mucosal mast cells, but was not found in mast cells of the muscularis, thus defining at least two mast cell phenotypes in the gut. Whereas all dermal and pulmonary mast cells were tryptase positive, only a low proportion in the lung, and almost none in the dermis, were positive for sMCP-1. In view of the structural and functional similarities of sheep and human tryptases, and their similarity in tissue distribution in normal sheep, the sheep lung appears to be a good model for in vivo studies relating to human tryptase.