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 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.
Natsuaki
M
M
Department of Dermatology, University of California, San Francisco 94143-0536.
Stewart
C B
CB
Vanderslice
P
P
Schwartz
L B
LB
Natsuaki
M
M
Wintroub
B U
BU
Rutter
W J
WJ
Goldstein
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics*; D002417:Cattle; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D008168:Lung / Q000166:cytology; D008407:Mast Cells / Q000201:enzymology*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism; D010179:Pancreas / Q000201:enzymology; D012867:Skin / Q000166:cytology* / Q000201:enzymology; D013045:Species Specificity; D013379:Substrate Specificity
D006023:Glycoproteins; D009842:Oligopeptides; D002268:Carboxypeptidases