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Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells.

Chyung AS, Greenberg BD, Cook DG, Doms RW, Lee VM - J. Cell Biol. (1997)

Bottom Line: Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments.In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates.Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type beta-amyloid precursor protein (APP) to amyloid beta peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by beta-secretase cleavage (APPbeta) is indeed produced from the endogenous full length APP (APPFL). Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments. In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates. Furthermore, the production of intracellular APPbeta from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPbeta was not detected in several non-neuronal cell lines. Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

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APPβ is produced intracellularly in NT2N neurons.  Culture dishes containing >99% pure NT2N cells were metabolically labeled with [35S]methionine for 16 h. Cells were rinsed  twice with PBS and then incubated on ice for 20 min with PBS  alone (lane 1), with 10 μg/ml trypsin (lane 2), or with 10 μg/ml  trypsin and 0.1% Triton X-100 (lane 3). The cells were processed  for immunoprecipitation with the anti-APPβ antibody 53, as described in Materials and Methods.
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Figure 3: APPβ is produced intracellularly in NT2N neurons. Culture dishes containing >99% pure NT2N cells were metabolically labeled with [35S]methionine for 16 h. Cells were rinsed twice with PBS and then incubated on ice for 20 min with PBS alone (lane 1), with 10 μg/ml trypsin (lane 2), or with 10 μg/ml trypsin and 0.1% Triton X-100 (lane 3). The cells were processed for immunoprecipitation with the anti-APPβ antibody 53, as described in Materials and Methods.

Mentions: To determine if the recovery of APPβ from the cell lysates reflects its intracellular origin or its association with the cell surface, we treated cultures of NT2N neurons with trypsin at 4°C. Under such conditions, cell surface-associated but not intracellular APPβ should be proteolyzed. Fig. 3 shows that a similar amount of APPβ was recovered from NT2N neurons regardless of trypsin treatment (Fig. 3, compare lanes 1 and 2). By contrast, when the NT2N neurons were treated with trypsin and 0.1% Triton X-100, intracellular APPβ was completely eliminated (Fig. 3, lane 3). This experiment provides evidence that the APPβ recovered from the NT2N cell lysate is indeed produced in an intracellular compartment.


Novel beta-secretase cleavage of beta-amyloid precursor protein in the endoplasmic reticulum/intermediate compartment of NT2N cells.

Chyung AS, Greenberg BD, Cook DG, Doms RW, Lee VM - J. Cell Biol. (1997)

APPβ is produced intracellularly in NT2N neurons.  Culture dishes containing >99% pure NT2N cells were metabolically labeled with [35S]methionine for 16 h. Cells were rinsed  twice with PBS and then incubated on ice for 20 min with PBS  alone (lane 1), with 10 μg/ml trypsin (lane 2), or with 10 μg/ml  trypsin and 0.1% Triton X-100 (lane 3). The cells were processed  for immunoprecipitation with the anti-APPβ antibody 53, as described in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2141643&req=5

Figure 3: APPβ is produced intracellularly in NT2N neurons. Culture dishes containing >99% pure NT2N cells were metabolically labeled with [35S]methionine for 16 h. Cells were rinsed twice with PBS and then incubated on ice for 20 min with PBS alone (lane 1), with 10 μg/ml trypsin (lane 2), or with 10 μg/ml trypsin and 0.1% Triton X-100 (lane 3). The cells were processed for immunoprecipitation with the anti-APPβ antibody 53, as described in Materials and Methods.
Mentions: To determine if the recovery of APPβ from the cell lysates reflects its intracellular origin or its association with the cell surface, we treated cultures of NT2N neurons with trypsin at 4°C. Under such conditions, cell surface-associated but not intracellular APPβ should be proteolyzed. Fig. 3 shows that a similar amount of APPβ was recovered from NT2N neurons regardless of trypsin treatment (Fig. 3, compare lanes 1 and 2). By contrast, when the NT2N neurons were treated with trypsin and 0.1% Triton X-100, intracellular APPβ was completely eliminated (Fig. 3, lane 3). This experiment provides evidence that the APPβ recovered from the NT2N cell lysate is indeed produced in an intracellular compartment.

Bottom Line: Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments.In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates.Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
Previous studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type beta-amyloid precursor protein (APP) to amyloid beta peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by beta-secretase cleavage (APPbeta) is indeed produced from the endogenous full length APP (APPFL). Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments. In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates. Furthermore, the production of intracellular APPbeta from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPbeta was not detected in several non-neuronal cell lines. Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

Show MeSH
Related in: MedlinePlus