Determination of the primary structures of human skin chymase and cathepsin G from cutaneous mast cells of urticaria pigmentosa lesions

Norman M. Schechter, Z. Mei Wang, Russell W. Blacher, Stuart R. Lessin, Gerald Sylvan Lazarus, Harvey Rubin

Research output: Contribution to journalArticle

Abstract

This study establishes the primary structure of human skin chymase and provides further evidence for the presence of a cathepsin G-like proteinase within human mast cells. The amino acid sequence of human skin chymase was established by protein methods and by analysis of PCR amplification products obtained with cDNA-derived from urticaria pigmentosa (UP) lesions. UP is a disease characterized by skin lesions containing high numbers of mast cells. Proteolytic digests of human chymase purified from normal skin yielded 10 resolvable peptides that were sequenced by automated Edman degradation. The amino acid sequences for these peptides combined with the sequence obtained for the protein's NH2-terminal region (35 residues) accounted for 137 residues of the human skin 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 chymase. PCR amplification of UP-derived cDNA with primers based on the cDNA structure of heart chymase demonstrated a single amplification product of expected size which was subcloned and sequenced. The amino acid sequence (135 residues) deduced from this product was identical to that 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 human skin and heart chymases have identical primary structures. Amplification of the same UP-cDNA with primers coding for the NH2 and COOH-terminal sequences of human neutrophil cathepsin G also produced a specific amplification product which was sequenced. The deduced amino acid sequence between the primers was identical to that reported for neutrophil cathepsin G, indicating that the protein of cutaneous mast cells previously shown to be immunologically cross-reactive with neutrophil cathepsin G has a comparable amino acid sequence. UP-cDNA demonstrating amplification products for cathepsin G did not demonstrate amplification products for human neutrophil elastase, suggesting that the cathepsin G PCR amplification product was not derived from neutrophils or monocytes possibly contaminating the lesion. These studies provide further evidence that human skin mast cells contain two different chymotrypsin-like proteinases.

Original languageEnglish (US)
Pages (from-to)4062-4069
Number of pages8
JournalJournal of Immunology
Volume152
Issue number8
StatePublished - Apr 15 1994
Externally publishedYes

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Urticaria Pigmentosa
Cathepsin G
Chymases
Mast Cells
Skin
Amino Acid Sequence
Complementary DNA
Neutrophils
Peptide Hydrolases
Polymerase Chain Reaction
Proteins
Leukocyte Elastase
Peptides
Chymotrypsin
Skin Diseases
Monocytes

ASJC Scopus subject areas

  • Immunology

Cite this

Determination of the primary structures of human skin chymase and cathepsin G from cutaneous mast cells of urticaria pigmentosa lesions. / Schechter, Norman M.; Wang, Z. Mei; Blacher, Russell W.; Lessin, Stuart R.; Lazarus, Gerald Sylvan; Rubin, Harvey.

In: Journal of Immunology, Vol. 152, No. 8, 15.04.1994, p. 4062-4069.

Research output: Contribution to journalArticle

Schechter, Norman M. ; Wang, Z. Mei ; Blacher, Russell W. ; Lessin, Stuart R. ; Lazarus, Gerald Sylvan ; Rubin, Harvey. / Determination of the primary structures of human skin chymase and cathepsin G from cutaneous mast cells of urticaria pigmentosa lesions. In: Journal of Immunology. 1994 ; Vol. 152, No. 8. pp. 4062-4069.
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