Biosynthesis of phosphatidylinositol glycan-anchored membrane proteins. Design of a simple protein substrate to characterize the enzyme that cleaves the COOH-terminal signal peptide.
Many nascent proteins that are destined to be anchored to plasma membranes by a phosphatidylinositol glycan (PI-G) are in the range of 50-70 kDa so that changes of 2-3 kDa between precursors and products during processing are not easily detected. Furthermore, PI-G-anchored proteins are generally glycosylated so that changes between the nascent (prepro) proteins and the mature products are not due simply to the loss of signal peptides. These problems have made it difficult to monitor the processing of the prepro form of wild type human placental alkaline phosphatase (PLAP) in a cell-free system. We have designed a smaller and simpler substrate of PI-G "transamidase" derived by deletion of approximately 60% of the internal sequence of preproPLAP 513. This engineered protein, preprominiPLAP 208, retains the NH2- and COOH-terminal signal peptides of PLAP as well as all the epitopes for site-directed antibodies of the latter, but is devoid of glycosylation sites, the active site, and most of the cysteine residues. With preprominiPLAP, it has been possible to demonstrate, in a cell-free system, step by step conversion to the pro form and then to the mature form, with the concomitant loss of the appropriate signal peptides. These changes were shown to be time- and enzyme concentration-dependent. Studies with Asp-179 site-directed mutants of preprominiPLAP showed the same specificity for amino acids with a monosubstituted beta carbon at the cleavage/attachment site that were found previously with wild type PLAP.
Kodukula
K
K
Department of Neurosciences, Roche Research Center, Nutley, New Jersey 07110.
Micanovic
R
R
Gerber
L
L
Tamburrini
M
M
Brink
L
L
Udenfriend
S
S
D000217:Acyltransferases / Q000378:metabolism; D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D019881:Aminoacyltransferases; D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D002522:Chlorocebus aethiops; D010450:Endopeptidases / Q000378:metabolism; D017261:Glycosylphosphatidylinositols; D007158:Immunologic Techniques; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D010920:Placenta / Q000201:enzymology; D011134:Polysaccharides / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D012697:Serine Endopeptidases; D014162:Transfection
D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D010716:Phosphatidylinositols; D011134:Polysaccharides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D000217:Acyltransferases; D019881:Aminoacyltransferases; C023175:transamidases; D000469:Alkaline Phosphatase; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase