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. 1992 Dec 1;119(5):1137–1150. doi: 10.1083/jcb.119.5.1137

Overexpression of human alpha-galactosidase A results in its intracellular aggregation, crystallization in lysosomes, and selective secretion

PMCID: PMC2289730  PMID: 1332979

Abstract

Human lysosomal alpha-galactosidase A (alpha-Gal A) was stably overexpressed in CHO cells and its biosynthesis and targeting were investigated. Clone AGA5.3-1000Mx, which was the highest enzyme overexpressor, produced intracellular alpha-Gal A levels of 20,900 U/mg (approximately 100 micrograms of enzyme/10(7) cells) and secreted approximately 13,000 U (or 75 micrograms/10(7) cells) per day. Ultrastructural examination of these cells revealed numerous 0.25-1.5 microns crystalline structures in dilated trans-Golgi network (TGN) and in lysosomes which stained with immunogold particles using affinity- purified anti-human alpha-Gal A antibodies. Pulse-chase studies revealed that approximately 65% of the total enzyme synthesized was secreted, while endogenous CHO lysosomal enzymes were not, indicating that the alpha-Gal A secretion was specific. The recombinant intracellular and secreted enzyme forms were normally processed and phosphorylated; the secreted enzyme had mannose-6-phosphate moieties and bound the immobilized 215-kD mannose-6-phosphate receptor (M6PR). Thus, the overexpressed enzyme's selective secretion did not result from oversaturation of the M6PR-mediated pathway or abnormal binding to the M6PR. Of note, the secreted alpha-Gal A was sulfated and the percent of enzyme sulfation decreased with increasing amplification, presumably due to the inaccessibility of the enzyme's tyrosine residues for the sulfotransferase in the TGN. Overexpression of human lysosomal alpha-N-acetylgalactosaminidase and acid sphingomyelinase in CHO cell lines also resulted in their respective selective secretion. In vitro studies revealed that purified secreted alpha-Gal A was precipitated as a function of enzyme concentration and pH, with 30% of the soluble enzyme being precipitated when 10 mg/ml of enzyme was incubated at pH 5.0. Thus, it is hypothesized that these overexpressed lysosomal enzymes are normally modified until they reach the TGN where the more acidic environment of this compartment causes the formation of soluble and particulate enzyme aggregates. A significant proportion of these enzyme aggregates are unable to bind the M6PR and are selectively secreted via the constitutive secretory pathway, while endogenous lysosomal enzymes bind the M6PRs and are transported to lysosomes.

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Selected References

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  1. Alksnis M., Barkhem T., Strömstedt P. E., Ahola H., Kutoh E., Gustafsson J. A., Poellinger L., Nilsson S. High level expression of functional full length and truncated glucocorticoid receptor in Chinese hamster ovary cells. Demonstration of ligand-induced down-regulation of expressed receptor mRNA and protein. J Biol Chem. 1991 Jun 5;266(16):10078–10085. [PubMed] [Google Scholar]
  2. Andrews G. K., Adamson E. D. Butyrate selectively activates the metallothionein gene in teratocarcinoma cells and induces hypersensitivity to metal induction. Nucleic Acids Res. 1987 Jul 10;15(13):5461–5475. doi: 10.1093/nar/15.13.5461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bishop D. F., Desnick R. J. Affinity purification of alpha-galactosidase A from human spleen, placenta, and plasma with elimination of pyrogen contamination. Properties of the purified splenic enzyme compared to other forms. J Biol Chem. 1981 Feb 10;256(3):1307–1316. [PubMed] [Google Scholar]
  4. Bishop D. F., Kornreich R., Desnick R. J. Structural organization of the human alpha-galactosidase A gene: further evidence for the absence of a 3' untranslated region. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3903–3907. doi: 10.1073/pnas.85.11.3903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bishop D. F., Wampler D. E., Sgouris J. T., Bonefeld R. J., Anderson D. K., Hawley M. C., Sweeley C. C. Pilot scale purification of alpha-galactosidase A from Cohn fraction IV-1 of human plasma. Biochim Biophys Acta. 1978 May 11;524(1):109–120. doi: 10.1016/0005-2744(78)90109-2. [DOI] [PubMed] [Google Scholar]
  6. Böhlen P., Stein S., Dairman W., Udenfriend S. Fluorometric assay of proteins in the nanogram range. Arch Biochem Biophys. 1973 Mar;155(1):213–220. doi: 10.1016/s0003-9861(73)80023-2. [DOI] [PubMed] [Google Scholar]
  7. Calos M. P., Lebkowski J. S., Botchan M. R. High mutation frequency in DNA transfected into mammalian cells. Proc Natl Acad Sci U S A. 1983 May;80(10):3015–3019. doi: 10.1073/pnas.80.10.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Capony F., Rougeot C., Montcourrier P., Cavailles V., Salazar G., Rochefort H. Increased secretion, altered processing, and glycosylation of pro-cathepsin D in human mammary cancer cells. Cancer Res. 1989 Jul 15;49(14):3904–3909. [PubMed] [Google Scholar]
  9. Davis S. J., Ward H. A., Puklavec M. J., Willis A. C., Williams A. F., Barclay A. N. High level expression in Chinese hamster ovary cells of soluble forms of CD4 T lymphocyte glycoprotein including glycosylation variants. J Biol Chem. 1990 Jun 25;265(18):10410–10418. [PubMed] [Google Scholar]
  10. Desnick R. J., Bernstein H. S., Astrin K. H., Bishop D. F. Fabry disease: molecular diagnosis of hemizygotes and heterozygotes. Enzyme. 1987;38(1-4):54–64. doi: 10.1159/000469190. [DOI] [PubMed] [Google Scholar]
  11. Dong J. M., Prence E. M., Sahagian G. G. Mechanism for selective secretion of a lysosomal protease by transformed mouse fibroblasts. J Biol Chem. 1989 May 5;264(13):7377–7383. [PubMed] [Google Scholar]
  12. Dong J. M., Sahagian G. G. Basis for low affinity binding of a lysosomal cysteine protease to the cation-independent mannose 6-phosphate receptor. J Biol Chem. 1990 Mar 15;265(8):4210–4217. [PubMed] [Google Scholar]
  13. Dorner A. J., Wasley L. C., Kaufman R. J. Increased synthesis of secreted proteins induces expression of glucose-regulated proteins in butyrate-treated Chinese hamster ovary cells. J Biol Chem. 1989 Dec 5;264(34):20602–20607. [PubMed] [Google Scholar]
  14. Dorner A. J., Wasley L. C., Raney P., Haugejorden S., Green M., Kaufman R. J. The stress response in Chinese hamster ovary cells. Regulation of ERp72 and protein disulfide isomerase expression and secretion. J Biol Chem. 1990 Dec 15;265(35):22029–22034. [PubMed] [Google Scholar]
  15. Gal A. E., Brady R. O., Hibbert S. R., Pentchev P. G. A practical chromogenic procedure for the detection of homozygotes and heterozygous carriers of Niemann-Pick disease. N Engl J Med. 1975 Sep 25;293(13):632–636. doi: 10.1056/NEJM197509252931304. [DOI] [PubMed] [Google Scholar]
  16. 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. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  17. Halper L. A., Srere P. A. Interaction between citrate synthase and mitochondrial malate dehydrogenase in the presence of polyethylene glycol. Arch Biochem Biophys. 1977 Dec;184(2):529–534. doi: 10.1016/0003-9861(77)90462-3. [DOI] [PubMed] [Google Scholar]
  18. Hand A. R., Oliver C. Effects of secretory stimulation on the Golgi apparatus and GERL of rat parotid acinar cells. J Histochem Cytochem. 1984 Apr;32(4):403–412. doi: 10.1177/32.4.6142913. [DOI] [PubMed] [Google Scholar]
  19. Helms J. B., Karrenbauer A., Wirtz K. W., Rothman J. E., Wieland F. T. Reconstitution of steps in the constitutive secretory pathway in permeabilized cells. Secretion of glycosylated tripeptide and truncated sphingomyelin. J Biol Chem. 1990 Nov 15;265(32):20027–20032. [PubMed] [Google Scholar]
  20. Horst M., Hasilik A. Expression and maturation of human cathepsin D in baby-hamster kidney cells. Biochem J. 1991 Jan 15;273(Pt 2):355–361. doi: 10.1042/bj2730355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Israel D. I., Kaufman R. J. Highly inducible expression from vectors containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor. Nucleic Acids Res. 1989 Jun 26;17(12):4589–4604. doi: 10.1093/nar/17.12.4589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kane S. E., Troen B. R., Gal S., Ueda K., Pastan I., Gottesman M. M. Use of a cloned multidrug resistance gene for coamplification and overproduction of major excreted protein, a transformation-regulated secreted acid protease. Mol Cell Biol. 1988 Aug;8(8):3316–3321. doi: 10.1128/mcb.8.8.3316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kaufman R. J. Selection and coamplification of heterologous genes in mammalian cells. Methods Enzymol. 1990;185:537–566. doi: 10.1016/0076-6879(90)85044-o. [DOI] [PubMed] [Google Scholar]
  24. Kaufman R. J. Vectors used for expression in mammalian cells. Methods Enzymol. 1990;185:487–511. doi: 10.1016/0076-6879(90)85041-l. [DOI] [PubMed] [Google Scholar]
  25. Kaufman R. J., Wasley L. C., Dorner A. J. Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells. J Biol Chem. 1988 May 5;263(13):6352–6362. [PubMed] [Google Scholar]
  26. Kaufman R. J., Wasley L. C., Spiliotes A. J., Gossels S. D., Latt S. A., Larsen G. R., Kay R. M. Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells. Mol Cell Biol. 1985 Jul;5(7):1750–1759. doi: 10.1128/mcb.5.7.1750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Lazzarino D., Gabel C. A. Protein determinants impair recognition of procathepsin L phosphorylated oligosaccharides by the cation-independent mannose 6-phosphate receptor. J Biol Chem. 1990 Jul 15;265(20):11864–11871. [PubMed] [Google Scholar]
  29. Lemansky P., Bishop D. F., Desnick R. J., Hasilik A., von Figura K. Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease. J Biol Chem. 1987 Feb 15;262(5):2062–2065. [PubMed] [Google Scholar]
  30. McCormick F., Trahey M., Innis M., Dieckmann B., Ringold G. Inducible expression of amplified human beta interferon genes in CHO cells. Mol Cell Biol. 1984 Jan;4(1):166–172. doi: 10.1128/mcb.4.1.166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pallavicini M. G., DeTeresa P. S., Rosette C., Gray J. W., Wurm F. M. Effects of methotrexate on transfected DNA stability in mammalian cells. Mol Cell Biol. 1990 Jan;10(1):401–404. doi: 10.1128/mcb.10.1.401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Papkoff J. Inducible overexpression and secretion of int-1 protein. Mol Cell Biol. 1989 Aug;9(8):3377–3384. doi: 10.1128/mcb.9.8.3377. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rochefort H., Capony F., Garcia M. Cathepsin D in breast cancer: from molecular and cellular biology to clinical applications. Cancer Cells. 1990 Dec;2(12):383–388. [PubMed] [Google Scholar]
  34. Rochefort H., Cavailles V., Augereau P., Capony F., Maudelonde T., Touitou I., Garcia M. Overexpression and hormonal regulation of pro-cathepsin D in mammary and endometrial cancer. J Steroid Biochem. 1989;34(1-6):177–182. doi: 10.1016/0022-4731(89)90080-0. [DOI] [PubMed] [Google Scholar]
  35. Rothman J. H., Hunter C. P., Valls L. A., Stevens T. H. Overproduction-induced mislocalization of a yeast vacuolar protein allows isolation of its structural gene. Proc Natl Acad Sci U S A. 1986 May;83(10):3248–3252. doi: 10.1073/pnas.83.10.3248. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sahagian G. G., Gottesman M. M. The predominant secreted protein of transformed murine fibroblasts carries the lysosomal mannose 6-phosphate recognition marker. J Biol Chem. 1982 Sep 25;257(18):11145–11150. [PubMed] [Google Scholar]
  37. Sakuraba H., Eng C. M., Desnick R. J., Bishop D. F. Invariant exon skipping in the human alpha-galactosidase A pre-mRNA: Ag+1 to t substitution in a 5'-splice site causing Fabry disease. Genomics. 1992 Apr;12(4):643–650. doi: 10.1016/0888-7543(92)90288-4. [DOI] [PubMed] [Google Scholar]
  38. Sakuraba H., Oshima A., Fukuhara Y., Shimmoto M., Nagao Y., Bishop D. F., Desnick R. J., Suzuki Y. Identification of point mutations in the alpha-galactosidase A gene in classical and atypical hemizygotes with Fabry disease. Am J Hum Genet. 1990 Nov;47(5):784–789. [PMC free article] [PubMed] [Google Scholar]
  39. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schuchman E. H., Suchi M., Takahashi T., Sandhoff K., Desnick R. J. Human acid sphingomyelinase. Isolation, nucleotide sequence and expression of the full-length and alternatively spliced cDNAs. J Biol Chem. 1991 May 5;266(13):8531–8539. [PubMed] [Google Scholar]
  41. Srere P. A. Complexes of sequential metabolic enzymes. Annu Rev Biochem. 1987;56:89–124. doi: 10.1146/annurev.bi.56.070187.000513. [DOI] [PubMed] [Google Scholar]
  42. Stevens T. H., Rothman J. H., Payne G. S., Schekman R. Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y. J Cell Biol. 1986 May;102(5):1551–1557. doi: 10.1083/jcb.102.5.1551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Troen B. R., Gal S., Gottesman M. M. Sequence and expression of the cDNA for MEP (major excreted protein), a transformation-regulated secreted cathepsin. Biochem J. 1987 Sep 15;246(3):731–735. doi: 10.1042/bj2460731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Varki A., Kornfeld S. The spectrum of anionic oligosaccharides released by endo-beta-N-acetylglucosaminidase H from glycoproteins. Structural studies and interactions with the phosphomannosyl receptor. J Biol Chem. 1983 Mar 10;258(5):2808–2818. [PubMed] [Google Scholar]
  45. Walls J. D., Berg D. T., Yan S. B., Grinnell B. W. Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C. Gene. 1989 Sep 1;81(1):139–149. doi: 10.1016/0378-1119(89)90344-2. [DOI] [PubMed] [Google Scholar]
  46. Wang A. M., Bishop D. F., Desnick R. J. Human alpha-N-acetylgalactosaminidase-molecular cloning, nucleotide sequence, and expression of a full-length cDNA. Homology with human alpha-galactosidase A suggests evolution from a common ancestral gene. J Biol Chem. 1990 Dec 15;265(35):21859–21866. [PubMed] [Google Scholar]
  47. Wasley L. C., Atha D. H., Bauer K. A., Kaufman R. J. Expression and characterization of human antithrombin III synthesized in mammalian cells. J Biol Chem. 1987 Oct 25;262(30):14766–14772. [PubMed] [Google Scholar]
  48. Wong G. G., Witek J. S., Temple P. A., Wilkens K. M., Leary A. C., Luxenberg D. P., Jones S. S., Brown E. L., Kay R. M., Orr E. C. Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins. Science. 1985 May 17;228(4701):810–815. doi: 10.1126/science.3923623. [DOI] [PubMed] [Google Scholar]

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