Abstract
We have characterized the molecular species and subcellular distribution of Alzheimer beta/A4 amyloid precursor protein (APP) in neutrophilic granulocytes purified from human peripheral blood. APP was readily detectable in these cells. Immunochemical analysis with a panel of antibodies revealed that this APP species lacked the transmembrane and cytoplasmic domains previously demonstrated in cell-associated APP. However, it contained a protease inhibitor domain of the Kunitz type, indicating that neutrophil APP is a potent inhibitor of certain serine proteases. Upon subcellular fractionation, APP was primarily localized to azurophilic granules, which are neutrophil-specific phagocytic organelles assigned to enzymatic digestion of invading microbes and dead or injured tissue. Apparently, in the neutrophil, a nonsecretory organelle stores truncated, soluble APP, a species previously found only in blood plasma and cerebrospinal fluid or in conditioned medium of cultured cells. Soluble APP in neutrophils may therefore have intracellular functions in addition to its previously described extracellular functions. These findings also indicate that there are previously uncharacterized cell-specific differences in processing, trafficking, and storage of the APP molecule. Finally, the precise subcellular localization of APP to neutrophil-specific phagocytic organelles is suggestive of a role for APP in the nonimmunological host defense.
Highlights
From the $Department of Clinical Neuroscience, Section of Drug Addiction Research, The Karolinska Institute, S-171 Stockholm, Sweden, the Illepartment of Cell and Molecular Biology, The Medical Nobel Institute, The Karolinska Institute, S-171 Stockholm, Sweden, the §Department of Geriatric Medicine, Huddinge University Hospital, The Karolinska Institute, S-141 86 Huddinge, Sweden, and the **Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021
Proteins from neutrophils and plate- dase activity in the gradient, which suggests that amyloid precursor protein (APP) and lets,extracted by this procedure,were separated by SDS- myeloperoxidase were present in the samoreganelle
Tfrhaections with the highest myeoloperoxidase activity were found approximately in the middle of the 40%10%to 60%/100%sucroseldeuterium oxide gradient (Fig. 4A).APP content in the fractions was analyzed by immunoblotting (Fig.4B).By comparing the data presented in Fig. 4, A and B, it can be concluded that myeloperoxidase activity and APP immunoreactivitywerecodistributed and probably were present in the same structure
Summary
From the $Department of Clinical Neuroscience, Section of Drug Addiction Research, The Karolinska Institute, S-171 Stockholm, Sweden, the Illepartment of Cell and Molecular Biology, The Medical Nobel Institute, The Karolinska Institute, S-171 Stockholm, Sweden, the §Department of Geriatric Medicine, Huddinge University Hospital, The Karolinska Institute, S-141 86 Huddinge, Sweden, and the **Department of Neurology and Neuroscience, Cornell University Medical College, New York, New York 10021. En of antibodies revealed thatthis APP species lacked the route to, or at, the plasma membrane some of the APP moltransmembraneandcytoplasmicdomainspreviously ecules are cleaved between amino acid residues 15 and 17 in demonstrated in cell-associatedAPP. It con- the region corresponding to the PIA4 peptide, yielding a 100-. Fore decided to investigateif the human neutrophilic granulocyte, a cell type which plays an important role in the nonimmunological host defense, contains APP. The neutrophilic granulocyte represents the main cell type found in early inflammatory lesions It has a characteristic morphology witha polymorphic nucleus anda large number of cytoplasmic granules. In the present study we show that neutrophils contain significant amounts of truncated soluble APP, and we characterizethis AF’P speciesimmunochemicallyandsurveyits unusual subcellular localization
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