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Journal List > Biochem J > v.295(Pt 2); Oct 15, 1993

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Biochem J. 1993 October 15; 295(Pt 2): 367–378.
PMCID: PMC1134891
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The Alzheimer beta-amyloid protein precursor/protease nexin-II is cleaved by secretase in a trans-Golgi secretory compartment in human neuroglioma cells.
S L Kuentzel, S M Ali, R A Altman, B D Greenberg, and T J Raub
Upjohn Laboratories, Upjohn Company, Kalamazoo, MI 49001.
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Abstract
Alzheimer beta-amyloid protein precursor (beta APP) is expressed endogenously and abundantly by human neuroglioma (H4) cells. Its secretory processing has been shown to involve discrete proteolysis within the beta A4 region, thus preventing beta-amyloid formation, by an enzyme which has been referred to as 'beta APP secretase'. This cleavage results in secretion of a soluble N-terminal 135 kDa protein and retention of an integral membrane C-terminal fragment within the cell. The membrane-associated C-terminal fragment is sorted to lysosomes where it undergoes limited degradation. We show here that most newly synthesized beta APP is degraded via a non-lysosomal pathway before maturation in H4 cells, and most mature beta APP is processed predominantly by the so-called secretase. The rapid kinetics of appearance/disappearance of a cleaved 135 kDa protein within a microsomal fraction and the slow accumulation of this form in the extracellular medium indicated that secretase cleaves beta APP in an intracellular compartment. Low-temperature block (20 degrees C) was used to demonstrate that beta APP is cleaved within a late Golgi compartment after sulphation which occurs in the trans-Golgi network (TGN). This is consistent with (1) the immunolocalization of most of the beta APP within a Golgi compartment that reacts with wheat germ agglutinin, (2) the fact that less than 1.5% of the total mature full-length beta APP is present at the plasma membrane and (3) subcellular fractionation studies which showed that the mature full-length and intracellular cleaved beta APPs co-sediment with a membrane fraction that is slightly more dense than the plasma membrane. This study provides evidence that most of the beta APP secretase in H4 cells is intracellular, and confirms that the resulting C-terminal fragment is delivered to lysosomes immediately after cleavage. These results are discussed with regard to the possibility that mature full-length beta APP escapes secretase cleavage and is delivered directly from the TGN to the lysosome without passing through the plasma membrane. Either pathway will result in the generation of amyloidogenic fragments.
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Selected References
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  • Alcaraz G, Kinet JP, Kumar N, Wank SA, Metzger H. Phase separation of the receptor for immunoglobulin E and its subunits in Triton X-114. J Biol Chem. 1984 Dec 10;259(23):14922–14927. [PubMed]
  • Anderson JP, Esch FS, Keim PS, Sambamurti K, Lieberburg I, Robakis NK. Exact cleavage site of Alzheimer amyloid precursor in neuronal PC-12 cells. Neurosci Lett. 1991 Jul 8;128(1):126–128. [PubMed]
  • Aronson NN, Jr, Touster O. Isolation of rat liver plasma membrane fragments in isotonic sucrose. Methods Enzymol. 1974;31(Pt A):90–102. [PubMed]
  • Baeuerle PA, Huttner WB. Tyrosine sulfation is a trans-Golgi-specific protein modification. J Cell Biol. 1987 Dec;105(6 Pt 1):2655–2664. [PubMed]
  • Benowitz LI, Rodriguez W, Paskevich P, Mufson EJ, Schenk D, Neve RL. The amyloid precursor protein is concentrated in neuronal lysosomes in normal and Alzheimer disease subjects. Exp Neurol. 1989 Dec;106(3):237–250. [PubMed]
  • Berkenbosch F, Refolo LM, Friedrich VL, Jr, Casper D, Blum M, Robakis NK. The Alzheimer's amyloid precursor protein is produced by type I astrocytes in primary cultures of rat neuroglia. J Neurosci Res. 1990 Mar;25(3):431–440. [PubMed]
  • Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem. 1981 Feb 25;256(4):1604–1607. [PubMed]
  • Buxbaum JD, Gandy SE, Cicchetti P, Ehrlich ME, Czernik AJ, Fracasso RP, Ramabhadran TV, Unterbeck AJ, Greengard P. Processing of Alzheimer beta/A4 amyloid precursor protein: modulation by agents that regulate protein phosphorylation. Proc Natl Acad Sci U S A. 1990 Aug;87(15):6003–6006. [PubMed]
  • Caporaso GL, Gandy SE, Buxbaum JD, Greengard P. Chloroquine inhibits intracellular degradation but not secretion of Alzheimer beta/A4 amyloid precursor protein. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2252–2256. [PubMed]
  • Carlsson SR, Fukuda M. The lysosomal membrane glycoprotein lamp-1 is transported to lysosomes by two alternative pathways. Arch Biochem Biophys. 1992 Aug 1;296(2):630–639. [PubMed]
  • Catteruccia N, Willingale-Theune J, Bunke D, Prior R, Masters CL, Crisanti A, Beyreuther K. Ultrastructural localization of the putative precursors of the A4 amyloid protein associated with Alzheimer's disease. Am J Pathol. 1990 Jul;137(1):19–26. [PubMed]
  • Chen WJ, Goldstein JL, Brown MS. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J Biol Chem. 1990 Feb 25;265(6):3116–3123. [PubMed]
  • Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. [PubMed]
  • de Curtis I, Howell KE, Simons K. Isolation of a fraction enriched in the trans-Golgi network from baby hamster kidney cells. Exp Cell Res. 1988 Apr;175(2):248–265. [PubMed]
  • De Strooper B, Van Leuven F, Van den Berghe H. Alpha 2-macroglobulin and other proteinase inhibitors do not interfere with the secretion of amyloid precursor protein in mouse neuroblastoma cells. FEBS Lett. 1992 Aug 10;308(1):50–53. [PubMed]
  • Esch FS, Keim PS, Beattie EC, Blacher RW, Culwell AR, Oltersdorf T, McClure D, Ward PJ. Cleavage of amyloid beta peptide during constitutive processing of its precursor. Science. 1990 Jun 1;248(4959):1122–1124. [PubMed]
  • Estus S, Golde TE, Kunishita T, Blades D, Lowery D, Eisen M, Usiak M, Qu XM, Tabira T, Greenberg BD, et al. Potentially amyloidogenic, carboxyl-terminal derivatives of the amyloid protein precursor. Science. 1992 Feb 7;255(5045):726–728. [PubMed]
  • Fujiki Y, Hubbard AL, Fowler S, Lazarow PB. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. [PubMed]
  • Fukuchi K, Kamino K, Deeb SS, Smith AC, Dang T, Martin GM. Overexpression of amyloid precursor protein alters its normal processing and is associated with neurotoxicity. Biochem Biophys Res Commun. 1992 Jan 15;182(1):165–173. [PubMed]
  • Fuller SD, Bravo R, Simons K. An enzymatic assay reveals that proteins destined for the apical or basolateral domains of an epithelial cell line share the same late Golgi compartments. EMBO J. 1985 Feb;4(2):297–307. [PubMed]
  • Gandy SE, Bhasin R, Ramabhadran TV, Koo EH, Price DL, Goldgaber D, Greengard P. Alzheimer beta/A4-amyloid precursor protein: evidence for putative amyloidogenic fragment. J Neurochem. 1992 Jan;58(1):383–386. [PubMed]
  • Golde TE, Estus S, Younkin LH, Selkoe DJ, Younkin SG. Processing of the amyloid protein precursor to potentially amyloidogenic derivatives. Science. 1992 Feb 7;255(5045):728–730. [PubMed]
  • Green SA, Zimmer KP, Griffiths G, Mellman I. Kinetics of intracellular transport and sorting of lysosomal membrane and plasma membrane proteins. J Cell Biol. 1987 Sep;105(3):1227–1240. [PubMed]
  • Griffiths G, Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. [PubMed]
  • Griffiths G, Pfeiffer S, Simons K, Matlin K. Exit of newly synthesized membrane proteins from the trans cisterna of the Golgi complex to the plasma membrane. J Cell Biol. 1985 Sep;101(3):949–964. [PubMed]
  • Haass C, Hung AY, Selkoe DJ. Processing of beta-amyloid precursor protein in microglia and astrocytes favors an internal localization over constitutive secretion. J Neurosci. 1991 Dec;11(12):3783–3793. [PubMed]
  • Haass C, Koo EH, Mellon A, Hung AY, Selkoe DJ. Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments. Nature. 1992 Jun 11;357(6378):500–503. [PubMed]
  • Hall CW, Liebaers I, Di Natale P, Neufeld EF. Enzymic diagnosis of the genetic mucopolysaccharide storage disorders. Methods Enzymol. 1978;50:439–456. [PubMed]
  • Harter C, Mellman I. Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane. J Cell Biol. 1992 Apr;117(2):311–325. [PubMed]
  • Hayashi Y, Kashiwagi K, Yoshikawa K. Protease inhibitors generate cytotoxic fragments from Alzheimer amyloid protein precursor in cDNA-transfected glioma cells. Biochem Biophys Res Commun. 1992 Sep 30;187(3):1249–1255. [PubMed]
  • Hubbard AL, Cohn ZA. Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells. J Cell Biol. 1975 Feb;64(2):438–460. [PubMed]
  • Joachim CL, Selkoe DJ. The seminal role of beta-amyloid in the pathogenesis of Alzheimer disease. Alzheimer Dis Assoc Disord. 1992 Spring;6(1):7–34. [PubMed]
  • Johnson KF, Kornfeld S. A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function. J Biol Chem. 1992 Aug 25;267(24):17110–17115. [PubMed]
  • Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, Multhaup G, Beyreuther K, Müller-Hill B. The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature. 325(6106):733–736. [PubMed]
  • Kitaguchi N, Takahashi Y, Tokushima Y, Shiojiri S, Ito H. Novel precursor of Alzheimer's disease amyloid protein shows protease inhibitory activity. Nature. 1988 Feb 11;331(6156):530–532. [PubMed]
  • Klausner RD, Sitia R. Protein degradation in the endoplasmic reticulum. Cell. 1990 Aug 24;62(4):611–614. [PubMed]
  • Knops J, Lieberburg I, Sinha S. Evidence for a nonsecretory, acidic degradation pathway for amyloid precursor protein in 293 cells. Identification of a novel, 22-kDa, beta-peptide-containing intermediate. J Biol Chem. 1992 Aug 15;267(23):16022–16024. [PubMed]
  • Kuismanen E, Saraste J. Low temperature-induced transport blocks as tools to manipulate membrane traffic. Methods Cell Biol. 1989;32:257–274. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Letourneur F, Klausner RD. A novel di-leucine motif and a tyrosine-based motif independently mediate lysosomal targeting and endocytosis of CD3 chains. Cell. 1992 Jun 26;69(7):1143–1157. [PubMed]
  • Lisanti MP, Sargiacomo M, Graeve L, Saltiel AR, Rodriguez-Boulan E. Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9557–9561. [PubMed]
  • Lowery DE, Pasternack JM, Gonzalez-DeWhitt PA, Zürcher-Neely H, Tomich CC, Altman RA, Fairbanks MB, Heinrikson RL, Younkin SG, Greenberg BD. Alzheimer's amyloid precursor protein produced by recombinant baculovirus expression. Proteolytic processing and protease inhibitory properties. J Biol Chem. 1991 Oct 15;266(29):19842–19850. [PubMed]
  • Luo LQ, Martin-Morris LE, White K. Identification, secretion, and neural expression of APPL, a Drosophila protein similar to human amyloid protein precursor. J Neurosci. 1990 Dec;10(12):3849–3861. [PubMed]
  • Maxfield FR. Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts. J Cell Biol. 1982 Nov;95(2 Pt 1):676–681. [PubMed]
  • Nordstedt C, Gandy SE, Alafuzoff I, Caporaso GL, Iverfeldt K, Grebb JA, Winblad B, Greengard P. Alzheimer beta/A4 amyloid precursor protein in human brain: aging-associated increases in holoprotein and in a proteolytic fragment. Proc Natl Acad Sci U S A. 1991 Oct 15;88(20):8910–8914. [PubMed]
  • Nordstedt C, Caporaso GL, Thyberg J, Gandy SE, Greengard P. Identification of the Alzheimer beta/A4 amyloid precursor protein in clathrin-coated vesicles purified from PC12 cells. J Biol Chem. 1993 Jan 5;268(1):608–612. [PubMed]
  • Oltersdorf T, Fritz LC, Schenk DB, Lieberburg I, Johnson-Wood KL, Beattie EC, Ward PJ, Blacher RW, Dovey HF, Sinha S. The secreted form of the Alzheimer's amyloid precursor protein with the Kunitz domain is protease nexin-II. Nature. 1989 Sep 14;341(6238):144–147. [PubMed]
  • Oltersdorf T, Ward PJ, Henriksson T, Beattie EC, Neve R, Lieberburg I, Fritz LC. The Alzheimer amyloid precursor protein. Identification of a stable intermediate in the biosynthetic/degradative pathway. J Biol Chem. 1990 Mar 15;265(8):4492–4497. [PubMed]
  • Overly CC, Fritz LC, Lieberburg I, McConlogue L. The beta-amyloid precursor protein is not processed by the regulated secretory pathway. Biochem Biophys Res Commun. 1991 Dec 16;181(2):513–519. [PubMed]
  • Palacios G, Palacios JM, Mengod G, Frey P. Beta-amyloid precursor protein localization in the Golgi apparatus in neurons and oligodendrocytes. An immunocytochemical structural and ultrastructural study in normal and axotomized neurons. Brain Res Mol Brain Res. 1992 Oct;15(3-4):195–206. [PubMed]
  • Palmert MR, Podlisny MB, Witker DS, Oltersdorf T, Younkin LH, Selkoe DJ, Younkin SG. The beta-amyloid protein precursor of Alzheimer disease has soluble derivatives found in human brain and cerebrospinal fluid. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6338–6342. [PubMed]
  • Ponte P, Gonzalez-DeWhitt P, Schilling J, Miller J, Hsu D, Greenberg B, Davis K, Wallace W, Lieberburg I, Fuller F. A new A4 amyloid mRNA contains a domain homologous to serine proteinase inhibitors. Nature. 1988 Feb 11;331(6156):525–527. [PubMed]
  • Refolo LM, Wittenberg IS, Friedrich VL, Jr, Robakis NK. The Alzheimer amyloid precursor is associated with the detergent-insoluble cytoskeleton. J Neurosci. 1991 Dec;11(12):3888–3897. [PubMed]
  • Rosa P, Mantovani S, Rosboch R, Huttner WB. Monensin and brefeldin A differentially affect the phosphorylation and sulfation of secretory proteins. J Biol Chem. 1992 Jun 15;267(17):12227–12232. [PubMed]
  • Sahasrabudhe SR, Spruyt MA, Muenkel HA, Blume AJ, Vitek MP, Jacobsen JS. Release of amino-terminal fragments from amyloid precursor protein reporter and mutated derivatives in cultured cells. J Biol Chem. 1992 Dec 15;267(35):25602–25608. [PubMed]
  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Sambamurti K, Shioi J, Anderson JP, Pappolla MA, Robakis NK. Evidence for intracellular cleavage of the Alzheimer's amyloid precursor in PC12 cells. J Neurosci Res. 1992 Oct;33(2):319–329. [PubMed]
  • Selkoe DJ. Amyloid beta protein precursor and the pathogenesis of Alzheimer's disease. Cell. 1989 Aug 25;58(4):611–612. [PubMed]
  • Selkoe DJ, Podlisny MB, Joachim CL, Vickers EA, Lee G, Fritz LC, Oltersdorf T. Beta-amyloid precursor protein of Alzheimer disease occurs as 110- to 135-kilodalton membrane-associated proteins in neural and nonneural tissues. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7341–7345. [PubMed]
  • Shelton ER, Cohn R, Fish L, Obernolte R, Tahilramani R, Nestor JJ, Chan HW. Characterization of beta-amyloid precursor proteins with or without the protease-inhibitor domain using anti-peptide antibodies. J Neurochem. 1990 Jul;55(1):60–69. [PubMed]
  • Shivers BD, Hilbich C, Multhaup G, Salbaum M, Beyreuther K, Seeburg PH. Alzheimer's disease amyloidogenic glycoprotein: expression pattern in rat brain suggests a role in cell contact. EMBO J. 1988 May;7(5):1365–1370. [PubMed]
  • Sisodia SS. Beta-amyloid precursor protein cleavage by a membrane-bound protease. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6075–6079. [PubMed]
  • Sisodia SS, Koo EH, Beyreuther K, Unterbeck A, Price DL. Evidence that beta-amyloid protein in Alzheimer's disease is not derived by normal processing. Science. 1990 Apr 27;248(4954):492–495. [PubMed]
  • Swanson JA, Yirinec BD, Silverstein SC. Phorbol esters and horseradish peroxidase stimulate pinocytosis and redirect the flow of pinocytosed fluid in macrophages. J Cell Biol. 1985 Mar;100(3):851–859. [PubMed]
  • Tamaoka A, Kalaria RN, Lieberburg I, Selkoe DJ. Identification of a stable fragment of the Alzheimer amyloid precursor containing the beta-protein in brain microvessels. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1345–1349. [PubMed]
  • Tanzi RE, McClatchey AI, Lamperti ED, Villa-Komaroff L, Gusella JF, Neve RL. Protease inhibitor domain encoded by an amyloid protein precursor mRNA associated with Alzheimer's disease. Nature. 1988 Feb 11;331(6156):528–530. [PubMed]
  • Tartakoff AM. Perturbation of vesicular traffic with the carboxylic ionophore monensin. Cell. 1983 Apr;32(4):1026–1028. [PubMed]
  • Tartakoff AM, Vassalli P. Lectin-binding sites as markers of Golgi subcompartments: proximal-to-distal maturation of oligosaccharides. J Cell Biol. 1983 Oct;97(4):1243–1248. [PubMed]
  • Trowbridge IS. Endocytosis and signals for internalization. Curr Opin Cell Biol. 1991 Aug;3(4):634–641. [PubMed]
  • Van Nostrand WE, Schmaier AH, Farrow JS, Cunningham DD. Protease nexin-II (amyloid beta-protein precursor): a platelet alpha-granule protein. Science. 1990 May 11;248(4956):745–748. [PubMed]
  • Weidemann A, König G, Bunke D, Fischer P, Salbaum JM, Masters CL, Beyreuther K. Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein. Cell. 1989 Apr 7;57(1):115–126. [PubMed]
  • Wolozin B, Bacic M, Merrill MJ, Lesch KP, Chen C, Lebovics RS, Sunderland T. Differential expression of carboxyl terminal derivatives of amyloid precursor protein among cell lines. J Neurosci Res. 1992 Sep;33(1):163–169. [PubMed]
  • Yamaoka K, Tanigawara Y, Nakagawa T, Uno T. A pharmacokinetic analysis program (multi) for microcomputer. J Pharmacobiodyn. 1981 Nov;4(11):879–885. [PubMed]
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