Human Skin Mast Cell Carboxypeptidase: Functional Characterization, cDNA Cloning, and Genealogy

Human Skin Mast Cell Carboxypeptidase: Functional Characterization, cDNA Cloning, and Genealogy

We functionally characterized human skin mast cell carboxypeptidase A (MC-CPA), and explored its evolutionary relationship to other carboxypeptidases to understand further the structural basis for the substrate preferences of this enzyme. Purified human skin MC-CPA displayed more activity than did b...

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Bibliographic Details
Published in:Journal of investigative dermatology Vol. 99; no. 2; pp. 138 - 145
Main Authors: Natsuaki, Masaru, Stewart, Caro-Beth, Vanderslice, Peter, Schwartz, Lawrence B., Natsuaki, Megumi, Wintroub, Bruce U., Rutter, William J., Goldstein, Sanford M.
Format: Journal Article
Language:English
Published: Danvers, MA Elsevier Inc 01-08-1992
Nature Publishing
Subjects:
Alanine
Amino Acid Sequence
Animals
Base Sequence
Biological and medical sciences
Carboxypeptidases - chemistry
Carboxypeptidases - genetics
Cattle
Fundamental and applied biological sciences. Psychology
Glycoproteins - chemistry
Humans
Lung - cytology
Mast Cells - enzymology
Molecular and cellular biology
Molecular Sequence Data
Oligopeptides - metabolism
Pancreas - enzymology
Skin - cytology
Skin - enzymology
Species Specificity
Substrate Specificity
Characterization
Human
Nucleotide sequence
Enzyme
Carboxypeptidase
Mast cell
Skin
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Summary:We functionally characterized human skin mast cell carboxypeptidase A (MC-CPA), and explored its evolutionary relationship to other carboxypeptidases to understand further the structural basis for the substrate preferences of this enzyme. Purified human skin MC-CPA displayed more activity than did bovine pancreatic carboxypeptidase A (CPA) against carboxyl-terminal leucine residues, about equal activity with phenylalanine and tyrosine residues, and no activity with tryptophan or alanine. To correlate kinetic data with structure, we isolated and sequenced a cDNA encoding MC-CPA from human skin, and directly sequenced 30% of the purified protein. These sequences agreed with that of human lung MC-CPA, and further support the evidence for a single MC-CPA gene in humans. Four amino acid replacements, resulting in a net positive change in non-hydrogen atoms in the S1' subsite of MC-CPA, were associated with less alteration in substrate specificity, relative to bovine CPA, than might be expected from studies using rat CPA1 and CPA2. We noted two consensus N-linked glycosylation sites in human MC-CPA that are not found in rat and mouse MC-CPA, or in bovine CPA; that at least one of these sites is glycosylated in vivo was verified by N-glycosidase F treatment, lentil lectin binding, and Concanavalin A–Sepharose chromatography. Evolutionary trees constructed from the known carboxypeptidase sequences suggested that MC-CPA most likely evolved from a carboxypeptidase B-like enzyme, independent of the pancreatic CPA. Thus, in the carboxypeptidase gene family, MC-CPA displays a unique genealogy and several amino acid replacements in its S1' binding pocket that result in substrate specificity quite similar to bovine CPA.
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ISSN:0022-202X
1523-1747
DOI:10.1111/1523-1747.ep12616776