A saposin-like domain influences the intracellular localization, stability, and catalytic activity of human acyloxyacyl hydrolase.
Acyloxyacyl hydrolase, a leukocyte enzyme that acts on bacterial lipopolysaccharides (LPSs) and many glycerolipids, is synthesized as a precursor polypeptide that undergoes internal disulfide linkage before being proteolytically processed into two subunits. The larger subunit contains an amino acid sequence (Gly-X-Ser-X-Gly) that is found at the active sites of many lipases, while the smaller subunit has amino acid sequence similarity to saposins (sphingolipid activator proteins), cofactors for sphingolipid glycohydrolases. We show here that both acyloxyacyl hydrolase subunits are required for catalytic activity toward LPS and glycerophosphatidylcholine. In addition, mutations that truncate or delete the small subunit have profound effects on the intracellular localization, proteolytic processing, and stability of the enzyme in baby hamster kidney cells. Remarkably, proteolytic cleavage of the precursor protein increases the activity of the enzyme toward LPS by 10-20-fold without altering its activity toward glycerophosphatidylcholine. Proper orientation of the two subunits thus seems very important for the substrate specificity of this unusual enzyme.
Staab
J F
JF
Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas 75235-9113.
Ginkel
D L
DL
Rosenberg
G B
GB
Munford
R S
RS
D000818:Animals; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D002451:Cell Compartmentation; D002460:Cell Line; D002918:Chymotrypsin / Q000378:metabolism; D006224:Cricetinae; D017931:DNA Primers / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D013379:Substrate Specificity; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
D017931:DNA Primers; D006023:Glycoproteins; C483345:PSAP protein, human; D011498:Protein Precursors; D049231:Saposins; D049128:Sphingolipid Activator Proteins; D002265:Carboxylic Ester Hydrolases; C048964:acyloxyacyl hydrolase; D002918:Chymotrypsin; D014357:Trypsin