Dominant chymotrypsin-like esterase activity in human lymphocyte granules is mediated by the serine carboxypeptidase called cathepsin A-like protective protein.
We identified a chymotrypsin-like activity in the granules of IL-2 lymphokine-activated killer (LAK) cells and a NK cell line (YT) that reacted preferentially with the oligopeptide substrate succinyl-Phe-Leu-Phe-thiobenzyl ester (Suc-Phe-Leu-Phe-SBzl). The enzyme was isolated by detergent extraction of sedimented cytotoxic granules and then by a sequence of sieve, hydrophobic, and anion exchange chromatography. On SDS-PAGE, the protein migrated at 42 kDa in nonreduced form and became two bands (31 and 19 kDa, respectively) after reduction. Amino-terminal sequencing of the reduced protein bands revealed 100% homology with cathepsin A-like protective protein (CAPP), a lysosomal enzyme that expresses serine carboxypeptidase and deamidase activities. The carboxypeptidase activity of lymphocyte CAPP was verified by showing that the protease preferred hydrophobic amino acids in the penultimate position of the C terminus (i.e., cleaved arginine from dansyl-Phe-Leu-Arg). The presence of lymphocyte CAPP in secretory lysosomes was demonstrated by showing that Suc-Phe-Leu-Phe-SBzl activity co-migrated with tryptase and Asp-ase activities on Percoll density gradients and that 95% of the Suc-Phe-Leu-Phe-SBzl activity in granule fractions of cavitated YT cells could be immunoprecipitated with an anti-CAPP antiserum. In addition, calcium ionophore-stimulated YT cells were shown to secrete immunoprecipitable CAPP. As proposed for platelets, lymphocyte CAPP may be secreted to function extracellularly by inactivating bioactive peptides.
Hanna
W L
WL
Section of Rheumatology, Evanston Hospital, Northwestern University Medical School, IL 60201.
Turbov
J M
JM
Jackman
H L
HL
Tan
F
F
Froelich
C J
CJ
D000595:Amino Acid Sequence; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002268:Carboxypeptidases / Q000502:physiology*; D043402:Cathepsin A; D002403:Cathepsins / Q000502:physiology; D002499:Centrifugation, Density Gradient; D002918:Chymotrypsin / Q000502:physiology; D003594:Cytoplasmic Granules / Q000201:enzymology*; D004950:Esterases / Q000502:physiology*; D006023:Glycoproteins / Q000502:physiology*; D006801:Humans; D015979:Killer Cells, Lymphokine-Activated / Q000201:enzymology; D008214:Lymphocytes / Q000201:enzymology*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D000001:Calcimycin; D004950:Esterases; D002268:Carboxypeptidases; D002403:Cathepsins; C468629:CTSA protein, human; D043402:Cathepsin A; D002918:Chymotrypsin; D002118:Calcium