The triplet of lysine residues (Lys724-Lys725-Lys726) of Alzheimer's amyloid precursor protein plays an important role in membrane anchorage and processing.
One of the pathological changes of Alzheimer's disease is the deposit of beta/A4 protein, which is derived from Alzheimer amyloid precursor protein (APP). In the secretory pathway, APP is cleaved at an internal region of beta/A4 protein by a hypothetical enzyme "secretase." Our previous study showed that the site of cleavage of APP by secretase is determined by the length from the membrane-spanning region. To investigate the role of the transmembrane region in APP secretion, we constructed the mutations of triplet lysine residues (Lys724-Lys725-Lys726), which are located just in the carboxyl region after the proposed membrane domain. The mutations were as follows: VVK, Val724-Val725-Lys726; LLI, Leu724-Leu725-Ile726; and EEE, Glu724-Glu725-Glu726. Wild-type APP and mutant APPs were expressed transiently in COS-1 cells by cDNA transfection. The hydrophobic mutant VVK and LLI were processed and secreted in a way similar to that of the wild-type APP, although the rate of secretion was decreased. The acidic mutant EEE was not secreted into medium. Proteinase K treatment and cell surface biotinylation of the COS-1 cells expressing APP revealed that APP was located in the plasma membrane with a short intracellular carboxyl region. However, EEE was completely digested by proteinase K treatment, which suggested that the whole residues of this mutant are located at the outer surface of the cell, including its proposed membrane domain and carboxyl region. This mutant was not cleaved at all by secretase. These findings suggested that the triplet lysine residues of AP after the predicted membrane spanning domain play an important role in the membrane anchorage.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Molecular Biology, Tokyo Institute of Psychiatry, Japan.
M
M
Usami
W
W
Yamao-Harigaya
K
K
Maruyama
D000544:Alzheimer Disease / Q000378:metabolism*; D000595:Amino Acid Sequence; D016564:Amyloid beta-Protein Precursor / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000235:genetics; D011499:Protein Processing, Post-Translational; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism; D014162:Transfection
D016564:Amyloid beta-Protein Precursor; D015345:Oligonucleotide Probes