DETERMINATION OF THE PRIMARY STRUCTURES OF HUMAN SKIN CHYMASE AND CATHEPSIN-G FROM CUTANEOUS MAST-CELLS OF URTICARIA PIGMENTOSA LESIONS

This study establishes the primary structure of human skin chymase and provides further evidence for the presence of a cathepsin G-like prot einase within human mast cells. The amino acid sequence of human skin chymase was established by protein methods and by analysis of PCR ampl ification products obtained with cDNA-derived from urticaria pigmentos a (UP) lesions. UP is a disease characterized by skin lesions containi ng high numbers of mast cells. Proteolytic digests of human chymase pu rified from normal skin yielded 10 resolvable peptides that were seque nced by automated Edman degradation. The amino acid sequences for thes e peptides combined with the sequence obtained for the protein's NH2-t erminal region (35 residues) accounted for 137 residues of the human s kin chymase sequence. This partial amino acid sequence corresponded to the sequence of human heart chymase, a proteinase isolated from heart tissue with immunologic and hydrolytic properties similar to skin chy mase. PCR amplification of UP-derived cDNA with primers based on the c DNA structure of heart chymase demonstrated a single amplification pro duct of expected size which was subcloned and sequenced. The amino aci d sequence (135 residues) deduced from this product was identical to t hat of heart chymase in the region between the primers. This sequence, along with that established for the purified protein, constituted 99% of the heart chymase primary structure, strongly indicating that huma n skin and heart chymases have identical primary structures. Amplifica tion of the same UP-cDNA with primers coding for the NH2- and COOH-ter minal sequences of human neutrophil cathepsin G also produced a specif ic amplification product which was sequenced. The deduced amino acid s equence between the primers was identical to that reported for neutrop hil cathepsin G, indicating that the protein of cutaneous mast cells p reviously shown to be immunologically cross-reactive with neutrophil c athepsin G has a comparable amino acid sequence. UP-cDNA demonstrating amplification products for cathepsin G did not demonstrate amplificat ion products for human neutrophil elastase, suggesting that the cathep sin G PCR amplification product was not derived from neutrophils or mo nocytes possibly contaminating the lesion. These studies provide furth er evidence that human skin mast cells contain two different chymotryp sin-like proteinases.