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Keywords:

  • developing nervous system;
  • glycoconjugates;
  • glycolipids;
  • glycoproteins;
  • high mobility group protein 1 (HMG-1);
  • induction of neurite outgrowth

Abstract

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

HNK-1 antibody reactive sulfoglucuronyl carbohydrate (SGC) and SSEA-1 antibody reactive Lewis X (Lex) epitope are expressed on several glycolipids, glycoproteins, and proteoglycans of the nervous system and have been implicated in cell–cell recognition, neurite outgrowth, and/or neuronal migration during development. Interaction of SGC with its binding protein Amphoterin and interaction of Amphoterin with a cell-signaling molecule, receptor for advance glycation end product (RAGE) have been suggested to regulate neurite outgrowth and neuronal migration. The regulation of expression of SGC, Lex, Amphoterin, and RAGE was studied in embryonal carcinoma P19 cells after treatment with retinoic acid (RA). The untreated proliferating P19 cells strongly expressed the Lex epitope, which was mostly due to Lex-glycoproteins. P19 cells, when differentiated into neuron-like cells by RA, did not express the Lex epitope, but expressed increasing levels of SGC, with time in culture. Quantitative biochemical analyses showed that in the P19 cells after RA treatment, the amount of SGC-glycoproteins increased at a significantly higher level than sulfoglucuronyl glycolipid-1 (SGGL-1). The increase in the levels of SGGL-1 was due to 16-fold upregulation in the activity of lactosylceramide: N-acetylglucosaminyl-transferase (Lc3 synthase), which synthesizes the key intermediate lactotriosylceramide (Lc3Cer), for lacto- and neolacto-glycolipids. The large increase in the activity of Lc3 synthase appeared to regulate the levels of other neolacto glycolipids, such as Lc3Cer, nLc4Cer, nLc6Cer, disialosyl-nLc4Cer (LD1), and Lex-glycolipids. Strong upregulation of glucuronyl-transferase and modest twofold enhancement in the activity of the glucuronyl-sulfotransferase, which catalyze the final steps in the SGC synthesis, also would account for the large increase in the synthesis SGC-glycoproteins. RA also upregulated the synthesis of Amphoterin and RAGE in P19 cells. SGC, RAGE, and Amphoterin were co-localized in the RA-differentiated neurons. The initiation of neurite outgrowth along with co-ordinated upregulation of Amphoterin, RAGE, SGC-glycoproteins, and SGGLs in RA-treated P19 cells support the hypothesis that these molecules are involved in the neuronal process formation.

Abbreviations used
EC

embryonal carcinoma

FITC

fluorescein isothiocyanate

GFAP

glial fiber acid protein

GlcA-ST

glucuronyl:3 sulfotransferase

GlcA-Tr

neolactotetraosyl-ceramide:β1,3glucuronyltransferase

GlcNAc-Tr

or Lc3 synthase, β1,3-N-acetyl glucosaminyltransferase

HMG-1

high mobility group protein

Lc3 synthase

lactosylceramide: N-acetylglucosaminyl-transferase

Lc3Cer

lactotriosylceramide

LcOse2Cer

or Lc2Cer, lactosylceramide

LcOse3Cer

or Lc3Cer, lactotriosylceramide

LD1

disialosyl-nLc4Cer

Lex

Lewis X

nLcOse4Cer

or nLc4Cer, neo-lactotetraosyl-ceramide

PBS

phosphate-buffered saline

RA

retinoic acid

RAGE

receptor for advance glycation end product

SBP-1

sulfoglucuronyl carbohydrate binding protein 1 (same as Amphoterin)

SDS–PAGE

sodium dodecyl sulphate polyacrylamide gel electrophoresis

SGC

sulfoglycuronyl carbohydrate

SGGL-1

sulfoglucuronyl glycolipid-1

SSEA-1

stage specific embryonic antigen 1

Embryonal carcinoma (EC) P19 cells were first isolated from a murine teratocarcinoma (McBurney and Rogers 1982). These cells are pluripotent and differentiate into several cell lineages (Jones-Villeneuve et al. 1982). On treatment with retinoic acid (RA), P19 cells aggregate and differentiate into neurons, in a manner closely resembling that of neural cells in vivo (Jones-Villeneuve et al. 1982; McBurney et al. 1988). The RA-differentiated cells have many properties in common with in vivo embryonic neurons in the mammalian CNS, including elaboration of axons and dendrites, expression of multiple neuronal markers, neurotransmitters and receptors, and formation of functional synapses (Staines et al. 1994). For these reasons, P19 cells have been extensively utilized as an in vitro model system for early steps in neuronal development.

Sulfoglucuronyl carbohydrate (SGC), reactive with HNK-1 antibody, is expressed in several glycolipids, glycoproteins, and proteoglycans of the nervous system (Jungalwala 1994). SGC has been suggested to involve in cell–cell recognition, neurite outgrowth, and neuronal migration during development, through its interaction with SGC binding protein SBP-1 (Amphoterin; Kunemund et al. 1988; Chou et al. 2000; Zhao et al. 2000a, 2000b). Several cell adhesion molecules of the immunoglobulin super-family (such as NCAM, L1, integrins, Cytotactin, TAG-1, MAG, P0, and PMP-22) express SGC (Jungalwala 1994; Schachner and Martini 1995; Schachner et al. 1995). SGC has been implicated in migration of neural crest cells (Bronner-Fraser 1987), the adhesion of astrocytes and neurons to laminin (Kunemund et al. 1988; Hall et al. 1993, 1995), the outgrowth of neuritic and astrocytic processes (Chou and Jungalwala 1998; Kunemund et al. 1988), the preferential outgrowth of neurites from motor neurons (Martini et al. 1992), and in homophilic binding of neural cell adhesion molecules, such as P0 and NCAM (Cole et al. 1988; Schmitz et al. 1994). Recent studies have suggested a role for SGC in the alterations of synaptic efficacy, spatial learning, memory, and long-term potentiation in pyramidal cells (Pradel et al. 1999; Saghatelyan et al. 2000; Senn et al. 2002).

Our previous studies have shown that the expression of SGC was developmentally and spatially regulated both in the cerebral cortex and cerebellum (Schwarting et al. 1987; Prasadarao et al. 1990; Chou et al. 1991, 2000; Zhao et al. 2000a, 2000b). SGC interacted with its binding protein, SBP-1 (Nair and Jungalwala 1997), whose expression was chronologically co-ordinated with that of SGC (Schwarting et al. 1987; Prasadarao et al. 1990; Chou et al. 1991, 2000; Zhao et al. 2000a, 2000b). Recently, we have shown the identity of SBP-1 to neurite outgrowth promoting protein Amphoterin and to a nuclear binding protein, called high mobility group protein, HMG-1 (Chou et al. 2001). Amphoterin was strongly expressed specifically in the proliferating and migrating cortical neurons of the cerebral cortex and in granule neurons of the cerebellum (Zhao et al. 2000a, 2000b). Once these neurons reached their expected location, i.e. cortical plate of the cerebral cortex and internal granule cell layer of the cerebellum, Amphoterin declined and faded away from these cells. SGC was expressed in neuronal and/or glial processes surrounding the Amphoterin-expressing neurons. Studies with in vitro explant cultures of cerebellum and cerebral cortex showed that SGC–Amphoterin interaction was important for cell–cell recognition and cell migration (Chou et al. 2000).

Previously, McBurney et al. (1988) have shown that P19 cells, after treatment with RA, express the HNK-1 epitope (i.e. SGC) on virtually all cells with neuronal-like processes along with the neurofilament protein. To define which form of SGC is synthesized after RA treatment and to identify cell-specific expression and function of SGC, we have initiated studies of SGC synthesis on glycoproteins and glycolipids in the pluripotent embryonal carcinoma P19 cells following differentiation with RA.

More recently, the interaction of Amphoterin (SBP-1) with a cell signaling molecule, receptor for advance glycation end product (RAGE) and its downstream signaling of GTPases, Rac, and cdc42, which promote actin polymerization, has been implicated in the regulation of neurite outgrowth and neuronal migration (Hori et al. 1995; Huttunen et al. 1999, 2000; Fages et al. 2000; Srikrishna et al. 2002). Therefore, the expression of Amphoterin and RAGE was simultaneously investigated, along with that of SGC, in the RA-treated P19 cells.

Antibody SSEA-1 (stage-specific embryonic antigen), reactive with Lewis X (Lex) carbohydrate epitope, is expressed on embryonic pluripotent stem cells and on adult CNS stem cells. It has recently been used as a marker for stem cell isolation (Capela and Temple 2002). In addition, RA-treated P19 cells enhance the synthesis of Lex glycolipids, which have been suggested to be involved in several cell–cell interaction systems, such as in early neural development of the mouse and chick embryos (Boubelik et al. 1996; Osanai et al. 2001). In the present study, we have also analyzed the expression of Lex glycolipids and glycoproteins in RA-treated P19 cells. Part of this work was previously reported in abstract forms (Henion et al. 2000; Chou et al. 2002).

P19 EC stem cell culture and differentiation with retinoic acid

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

P19 EC cells were grown according to Bain and Gottlieb (1998) and Bain et al. (1995, 1998). Frozen cells were seeded into a T25 flask with 5 mL of P19G [P19-growth medium: α-MEM (Gibco no. 12571–014, Gibco, Rockville, MD, USA) 90%, newborn calf serum 7.5%, and fetal calf serum 2.5%]. Cells were allowed to reach near confluent, then passed to new flasks or 150-mm Falcon culture dishes (Falcon Plastics, Los Angeles, CA, USA), until enough cells were grown. Cells grown on these dishes for 2 days were then harvested by scrapping as undifferentiated cells. Cells grown in flasks were trypsinized gently with trypsin (0.025%) and then seeded into 150-mm non-adhesive culture dishes, containing P19IM (P19-induction medium: α-MEM 95%, fetal calf serum 5% and 5 × 10−7 M all trans RA), where cells form aggregates. P19IM was replaced with new P19IM in 2 days. After total of 4 days in P19IM, cells were trypsinized gently and plated into polylysine-coated (100 µg/mL) or commercially treated 150-mm culture dishes and grown in P19G. Cells were harvested at different days as described by scrapping.

Immunocytochemistry and fluorescent microscopy of cells

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min when necessary. To remove non-specific antibody binding, cells were blocked with 5% normal goat serum for 10 min. Cells were double-stained sequentially with appropriate primary and secondary antibodies. The cells were immunoreacted with antibodies HNK-1 for the SGC and with antibody7A (for SSEA-1 Lex epitope) without detergent, followed by anti-mouse IgM-Cy3-conjugated secondary antibodies (Zhao et al. 2000a, 2000b). The expression of Amphoterin (SBP-1) in the cells was detected by polyclonal anti-Amphoterin antibodies in the presence of 0.2% Triton X-100 for 10 min, followed by anti-rabbit IgG–fluorescein isothiocyanate (FITC)-conjugated secondary antibodies for 20 min (Zhao et al. 2000a, 2000b). The expression of RAGE in the cells was detected by monoclonal anti-RAGE peptide antibodies (mAb 5328,Chemicon International, Temecula, CA, USA). Cells were also immunoreacted with anti-GFAP, anti-MAP-2, and anti-TUJ1, followed by anti-mouse IgG(γ)–FITC-conjugated secondary antibodies, to identify cells of glial and neuronal origin. The details of the staining procedures and viewing of the fluorescence by epifluorescence and laser scanning confocal microscopes have been previously reported (Zhao et al. 2000a, 2000b).

Immunocytochemistry of cells with anti-Amphoterin and HNK-1 antibodies under non-penetrating conditions – RA-differentiated P19 cells, grown in the serum-free medium, were fixed with 0.5% paraformaldehyde for 10 min. After blocking the cells with 5% normal goat serum for 10 min for non-specific staining, the cells were first immunostained with anti-Amphoterin antibodies (from B. D. Pharmingen, San Diego, CA, USA) for 30 min without any detergent, followed by goat anti-rabbit IgG-Cy2-conjugated secondary antibodies for 20 . Cells were then double immunostained with HNK-1 antibodies followed by anti-mouse IgM-Cy3-conjugated secondary antibodies.

Assay of glycosyltransferases

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Lactosylceramide:N-acetylglucosaminyltransferase (Lc2Cer-GlcNAc-Tr), neo-lactotetraosylceramide:glucuronyltransferase (nLc4Cer-GlcA-Tr) and glucuronylglycolipid:sulfotransferase (GGL-S04-Tr) enzyme activities, before and after RA treatment, in the cell cultures were assayed according Chou and Jungalwala (1993a, 1993b) and Chou et al. (1991).

Cells homogenates

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Cells washed with PBS and scraped from plates were collected by centrifugation. Collected cells were stored frozen at −80°C till use. Cells were homogenized in 10 mm Tris–HCl buffer, pH 7.4, along with protease inhibitor cocktail (Sigma, St Louis, MO, USA). A portion was used for quantitative analysis of proteins using the Bicinchoninic acid reagent (Pierce Chemical Co, Rockford, IL, USA).

Glycolipids extraction and analysis

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Cell homogenates equivalent to 10–20 mg protein were extracted with chloroform, methanol and water (10 : 10 : 1, by volume) overnight at 37°C. After extraction, the solvent was adjusted to chloroform, methanol and water (30 : 60 : 8) and the extracts were applied to 2 mL of DEAE–Sephadex column as previously described (Chou et al. 1986). The column was eluted with 5 bed volumes of the loading solvent, followed by further elution with 5 volumes of methanol to remove neutral lipids. Acidic lipids were eluted sequentially from the column with 10 volumes of 0.02 m, 0.08 m, 0.2 m, and 0.5 m ammonium acetate in methanol. Neutral glycolipids were recovered in run through and methanol wash (without salt). Solvents in this fraction were evaporated and the lipids were hydrolyzed with 0.2 N NaOH in methanol, overnight at 37°C. The mixture was then neutralized with 0.1 N HCl. Salt and polar glycolipids were partitioned into upper phase according to Folch partition (Folch et al. 1957) without additional salt. Both upper and lower phases were checked for the neutral glycolipids. Disialosyl glycolipids were recovered in 0.08 m and 0.2 m ammonium acetate fractions, whereas SGGLs were recovered in the 0.5 m fraction. After desalting the fractions using a C18-SepPak cartridge, the lipids from each fraction were analyzed by HPTLC-immuno-overlay described as follows, with known amount standard as references (Chou et al. 1986).

Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Glycolipids in the fractions equivalent to 1 mg protein, except for the 0.5-m fraction with SGGLs 2 mg protein, were applied on aluminum backed HPTLC plate. The lipids were resolved by using chloroform : methanol : aqueous 0.25% CaCl2 (5 : 4 : 1, by volume). The plates were dried, coated with 0.05% polyisobutylmethacrylate in hexane, dried and then soaked in 1% bovine serum albumin solution in PBS. The plates were exposed to monoclonal antibodies TE5 (Holmes and Green 1991) and 1B2 (Young et al. 1981) together. TE5 recognizes glycolipids with terminal GlcNAcß1, 3Gal-structure, such as in Lc3Cer and Lc5Cer, whereas 1B2 recognizes glycolipids with terminal lactosamine, such as in nLc4Cer and nLc6Cer. Lex glycolipids were identified with antibody 7A (Yamamoto et al. 1985) and disialosyl gangliosides GD3 and LD1 were identified with antibody 7C7 (Yamamoto et al. 1994). SGGLs were identified with antibody HNK-1 (Chou et al. 1986). After washing the plates with PBS, they were exposed to anti-mouse–IgM–horseradish-peroxide-conjugated secondary antibody. The immunoreactive bands were developed with 4-chloronaphthol and hydrogen peroxide and the intensities of the immunoreactive bands were compared with known amounts of standards, which are within the range of the test glycolipids (Chou et al. 1986).

Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Aliquots of cell homogenates (equivalent to: 20 µg protein for Amphoterin and RAGE, 30 µg for SGC-glycoproteins and 10 µg for Lex-glycoproteins) were suspended in 8 mm Tris–HCl buffer, pH 6.8 containing 4% sodium dodecylsulfate (SDS) and 10% 2-mercaptoethanol and heated at 100°C for 5 min. The samples were subjected to 4–15% gradient SDS–polyacrylamide gel electrophoresis (SDS–PAGE) according to the procedure of Laemmli (1970). The resolved proteins were transblotted electrophoretically onto nitrocellulose according to the method of Bjerrum and Schafer-Nielsen (1986). The blots were blocked with 4% skimmed milk (Carnation, Glendale, CA, USA) in PBS overnight at 4°C, and incubated with the corresponding primary antibodies for 2 h at room temperature, followed by IgG- or IgM-peroxidase-conjugated secondary antibodies (1 : 10 000 diluted) for 2 h at room temperature. The reactive protein bands were detected with chemiluminescent substrate according to manufacturer's suggestion (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA).

RA-differentiated P19 cells express SGC and Amphoterin

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

P19 cells were grown as dispersed cell cultures on cover slips or as cell aggregates with RA (0.5 µm) in the culture medium for 4 days (Bain et al. 1995, 1998; Yao et al. 1995; Bain and Gottlieb 1998). The cells were then dispersed and replated for additional 1–5 days without RA. The expression of SGC and Amphoterin in these cells was visualized with HNK-1 and anti-Amphoterin antibodies (Fig. 1). The untreated cells did not express any detectable SGC (Fig. 1b) and minimal level of Amphoterin (Fig. 1c). Some of the RA-treated P19 cells, on the sixth day in culture (i.e. after treatment with RA for 4 days and without RA for 2 days) began to differentiate in to neuron-like cells and expressed SGC both on the plasma membrane and newly developed processes (Fig. 1e). The same cells with processes also began to strongly express Amphoterin in the cell soma (Fig. 1f). By 7 and 8 days in culture, increasingly more number of P19 cells treated with RA differentiated into neuron-like cells, having per cell more number of SGC-bearing processes which were longer than those at previous stages (Figs 1h and k). The cells bearing SGC also simultaneously and increasingly expressed Amphoterin, with days in culture (Figs 1i and l).

image

Figure 1. Expression of SGC and Amphoterin in P19 cells, before and after treatment with retinoic acid (RA). P19 embryonic carcinoma cells in culture were induced to differentiate into neurons after treatment with 0.5 µm all trans-RA, for 4 days. The cell aggregates were dispersed with trypsin and replated on polylysine-coated chamber slides. After 6, 7, and 8 days in vitro culture, cells were immunostained with HNK-1 and anti-Amphoterin antibodies, followed by goat anti-mouse IgM-Cy3 (red fluorescence) and goat anti-rabbit IgG-FITC (green fluorescence), respectively. (a–c) cells without RA; (d–l) cells after RA treatment in vitro: (d–f) 6 days; (g and h) 7 days; (j–l) 8 days. (a, d, g, and j) are bright field microphotographs. The scale bar in (l) applies to all panels. Untreated proliferating cells did not express SGC and minimal amount of Amphoterin (a–c). Upon treatment with RA, the cells began to express SGC and Amphoterin and their expressions increased with time in culture.

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SGC and Amphoterin are co-localized in RA-differentiated neurons

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

RA-treated P19 cells, after 8 days in culture, were double immunostained with HNK-1, and anti-MAP2 and TUJ-1 antibodies. The differentiated neuron-like cells, which expressed neuronal marker proteins MAP2 (Figs 2b and c) and TUJ-1 (not shown), also expressed SGC (Figs 2a and c), indicating that the SGC was primarily in the differentiated neurons. Double-labeling of these cells with HNK-1 and antiglial fiber acid protein (GFAP) antibodies showed that SGC was not localized in the GFAP-expressing glial cells (Figs 2d–f). The differentiated neuron-like cells expressing neuronal marker protein MAP2 also expressed Amphoterin, indicating that the Amphoterin-like SGC was primarily in the differentiated neurons (Figs 2g–i). Further, SGC was co-localized in the same cells which expressed Amphoterin (Figs 2j–l); however, Amphoterin was mostly restricted to cell soma and not in the processes.

image

Figure 2. The expression of SGC and Amphoterin is primarily on neuronal cells, which are differentiated after RA treatment of P19 cells. P19 cells, after treatment with RA and in culture for 8 days, were doubled immunostained with either HNK-1 or anti-Amphoterin antibodies, along with a neuronal marker, anti-MAP2 or with a glial marker anti-GFAP antibodies. The fluorescent labeling of cells is given in each panel. (a–l) Fluorescent microphotographs viewed with an epi-fluorescence microscope. The scale bar in (l) applies to panels (a–l).

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Laser scanning confocal microscopy at high magnification showed that the expression of SGC was restricted to cell membranes and processes of the differentiated neurons (Figs 3a and c), whereas Amphoterin was highly expressed in the nucleus and cell cytoplasm (Fig. 3b). The cytosolic and plasma membrane localization of Amphoterin was recognized when the RA-differentiated neurons were immunostained with antibodies under ‘non-penetrating conditions’ (i.e. mild fixation of cells with 0.5% paraformaldehyde and without the use of detergent; Fig. 3d). Under these conditions, strong nuclear staining of Amphoterin was avoided.

image

Figure 3. P19 cells, after treatment with RA and in culture for 8 days and fixation with 4% paraformaldehyde were doubled immunostained with HNK-1 and with anti-Amphoterin antibodies in the presence of a detergent (a and b) or after mild fixation with 0.5% paraformaldehyde and immunostained with the same antibodies without any detergent (c and d). The cells were viewed with a laser-scanning confocal fluorescence microscope. The scale bar in (b) applies to (a) and (b); the scale bar in (d) applies to (c) and (d). Note the absence of nuclear staining of Amphoterin in (d), which allows the recognition of cytosolic and plasma membrane staining of Amphoterin. The strong nuclear staining of a cell in (d) appears to be due to a damaged cell.

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Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

It was of interest to determine if the immunocytochemical expression of SGC in the differentiated P19 cells after RA treatment was due to synthesis of SGGLs or to SGC-glycoproteins. To evaluate how the synthesis of SGGLs was upregulated in the neurons, the activities of three key enzymes involved in the biosynthesis of SGGLs were measured in the homogenates of the untreated and RA-treated P19 cells.

The pathway for the biosynthesis of SGGL-1 and other neolacto glycolipids is shown in Fig. 4. The activity of lactosylceramide:β1,3 N-acetyglucosaminyltransferase (Lc2Cer-GlcNAc-Tr) was measured in the cell homogenates of P19 cells with Lc2Cer as the acceptor and UDP[14C]-GlcNAc as the donor, to synthesize radioactive Lc3Cer. Lc3Cer formed after the reaction was purified, resolved by HPTLC and detected by autoradiography (Chou and Jungalwala 1993b). The homogenates of P19 cells without the RA treatment catalyzed the formation of a minimal amount of Lc3Cer (Fig. 4a, lane 1). However, homogenates of P19 cells treated with RA after 2, 4 and 9 days synthesized increasing amounts of Lc3Cer (Fig. 4a, lanes 2–4, respectively). At 9 days after the RA treatment, the activity of GlcNAc-Tr was elevated 16-fold relative to untreated cells. The 9-day RA-treated cell homogenates also synthesized neolactotetraosylceramide (nLc4Cer) from the radioactive Lc3Cer formed, as catalyzed by the endogenous Lc3Cer: β1,4 galactoysltransferase (Chou and Jungalwala 1994) (Fig. 4a, lane 4, lower band).

image

Figure 4. Expression of enzyme activities involved in the biosynthesis of SGGL-1 in RA-treated P19 cells. At various times after treatment with RA, P19 cell homogenates were assayed for Lc2Cer-GlcNAc-Tr (a), nLc4Cer-GlcA-Tr (b), and GGL-1-sulfo-Tr (c) activities, using radiolabeled donor substrates UDP-[14C]-GlcNAc, UDP-[14C]-GlcA, and PAP35S, respectively. The labeled products were purified on reverse phase C18-cartridge and analyzed by HPTLC. The HPTLC-autoradiograms of the labeled products are shown. The migration of standard lipids, viewed with orcinol spray, is shown near each panel and in (d). In (a) and (b), the enzyme activities were measured: in lane 1, untreated P19 cells; lanes 2–4, RA-treated cells at 2, 4, and 9 days in culture, respectively. (b) Lane 5 represents enzyme activity measured in RA-treated cells at 9 days in culture, but without the nLc4Cer as an acceptor. (c) The enzyme activities were measured: in lane 1, untreated P19 cells; lane 2, RA-treated cells at 8 days in culture.

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The activity of the enzyme nLc4Cer:β1,3 glucuronyltransferase (nLc4Cer-GlcA-Tr) was measured in the cell homogenates of P19 cells with nLc4Cer as the acceptor and UDP[14C]-GlcA as the donor, to synthesize radioactive GlcA-nLc4Cer (glucuronyl glycolipid, GGL-1). The autoradiogram of the products separated on HPTLC showed that the homogenates of untreated P19 cells and 2-day RA-treated P19 cells did not catalyze the formation of GGL-1 (Fig. 4b, lanes 1 and 2, respectively). However, 4- and 9-day RA-treated P19 cell homogenates catalyzed the formation of increasing amounts of GGL-1 (Fig. 4b, lanes 3 and 4, respectively). In the absence of the nLc4Cer acceptor the 9-day RA-treated P19 cell homogenate did not synthesize any detectable amount of GGL-1 (Fig. 4b, lane 5).

The terminal step in the biosynthesis of SSGL-1 is catalyzed by the enzyme GGL-1:3-sulfotransferase (Chou and Jungalwala 1993a). The activity of this enzyme was measured using GGL-1 as the acceptor and nucleotide phosphoadenosine phospho[35S]- sulfate as the sulfate donor. The autoradiogram of the products formed showed that although untreated P19 cells had some constitutive activity for this enzyme (Fig. 4c, lane 1), the 8-day RA-treated P19 cells had 2.5-fold elevated activity.

Levels of SGGL-1 increased in P19 cells after RA treatment

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Because the activities of the two glycosyltransferases and the sulfotransferase for the synthesis of SGGL-1 were upregulated on RA treatment of the P19 cells, the levels of SGGLs in the P19 cells were analyzed (Figs 5a and 6a). The amount of SGGL-1 in the untreated P19 cells was extremely low (Fig. 5a, lane 0), but was significantly increased on RA treatment after 4 and 9 days (Fig. 5a, lanes 4 and 9). SGGL-2 in these cells was not detectable. Quantitative analyses showed that the amount of SGGL-1 increased seven- and 14-fold after 4 and 9 days of RA treatment, respectively (Fig. 6a).

image

Figure 5. Analysis of neolactoglycolipids in P19 cells after RA treatment by HPTLC-immuno-overlays. Neolactoglycolipids, eluted in various fractions from DEAE–Sephadex column, were analyzed in P19 cells by HPTLC-immunostaining using appropriate antibodies, as described in the methods section. In all panels, lane 0, represent untreated cells, and lanes 4, 6, and 9, represent RA-treated cells for 4, , and 9 days in culture, respectively. (a) SGGLs were analyzed in the 0.5-m ammonium acetate fraction equivalent to 2.0 mg protein applied to HPTLC plate and the plate immunostained with HNK-1 antibody. Lanes S1, S2, and S3, standard SGGLs prepared from bovine peripheral nervous system, 1, 2, and 4 ng, respectively. (The strong fast migrating upper band in the PNS standard represents SGGL-1 with long chain fatty acids.) (b) Lc3Cer, nLc4Cer, and Lc5Cer were analyzed in the neutral glycolipid fraction and partitioned in the Folch lower phase. Fractions equivalent to 1.0 mg protein were applied to HPTLC plate and the plate immunostained with antibodies TE5 and 1B2, simultaneously. Lane S1, standard Lc3Cer, 20 ng; lane S2, standard nLc4Cer, 50 ng. (c) Glycolipid nLc6Cer, in the neutral glycolipid fraction, which partitioned mainly in the Folch upper phase, was analyzed in each fraction equivalent to 1.0 mg protein and immunostained with antibody TE5 and1B2. Lane S, standard neolactoglycolipids as shown. (d) Disailosyl-gangliosides LD1 and GD3 were recovered in 0.08 and 0.2 m ammonium acetate fractions. Fractions equivalent to 0.75 mg protein were applied to HPTLC plate and the plate immunostained with antibody 7C7. Lane S1, standards GD3, 8 ng and LD1, 16 ng. S2, standard gangliosides visualized with orcinol. (e) Fuco-neolactoglycolipids, Fuc-nLc6Cer, and (Fuc)2-nLc6Cer, which were eluted in the neutral glycolipid fraction and partitioned mainly in the Folch upper phase, were analyzed in each fraction, equivalent to 1.0 mg protein, by HPTLC and immunostained with 7A (Lex, SSEA-1) antibody. Lane S1, standard kidney Lex-glycolipid, Galß1,4 (Fucα1,3)GlcNAcß1,6IV globoside (Williams et al. 1988), 10 ng; S2 standard gangliosides visualized with orcinol.

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image

Figure 6. Levels of various neolactoglycolipids and ganglioside GD3 in untreated P19 cells (0 days) and after treatment with RA. Each point represents average of two determinations.

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Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

The levels of SGGL-1 were exceedingly low (∼ 0.1–1.4 ng/mg protein) in the untreated and RA-treated P19 cells, despite robust levels of the upregulated activities of the enzymes, which synthesized SGGL-1. Therefore, the levels of precursors Lc3Cer and nLc4Cer and their higher analog nLc6Cer were analyzed in these cells (Figs 5b and c and 6a). In the untreated control cells, the level of Lc3Cer (Fig. 5b, lane 0, upper band doublet) was about 10-fold higher than SGGL-1 (Fig. 5a, lane 0; and Fig. 6A). It increased threefold after RA treatment for 4 days (Fig. 5b, lane 4; and Fig. 6a) and then declined on days 6 and 9 (Fig. 5b, lanes 6 and 9; and Fig. 6a). The level of nLc4Cer was fourfold higher than Lc3Cer in the untreated cells (Fig. 5b, lane 0, lower band; and Fig. 6a), which followed similar trend as Lc3Cer after treatment with RA (Fig. 5b, lanes 4, 6, and 9; and Fig. 6a). The levels of nLc6Cer, which is synthesized from nLc4Cer, increased significantly after treatment with RA (Figs 5c and 6a).

Levels of other neolactoglycolipids in P19 cells after treatment with RA

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

The levels of Lc3Cer and nLc4Cer were 10- and 40-fold higher than SGGL-1 in the untreated cells and after treatment with RA for 4 days they were 45- and 95-fold higher than SGGL-1 (Fig. 6a), suggesting that Lc3Cer and nLc4Cer could be directed for the synthesis of other neolactoglycolipids in addition to SGGL-1. It was therefore of interest to determine if other neolactoglycolipids derived from these precursors were upregulated in the RA-treated P19 cells. The levels of disialosylneolactotetraosyl ceramide, LD1 (NeuAcα2,8VNeuAcα2,3IV-nLc4Cer) in untreated (Fig. 5d, lane 0) and RA-treated P19 cells after 4, 6, and 9 days are shown in Fig. 5(d), lanes 4, 6, and 9, respectively. LD1 was sharply upregulated sevenfold after treatment with RA for 6 days (Fig. 5d, lane 6; and Fig. 6b). Antibody 7A (SSEA-1) reactive Lex fucosylglycolipids, Fucα1,3V-nLc6Cer and (Fuc)2α1,3III,3V-nLc6Cer (Chou et al. 1996) were present in the untreated P19 cells (Fig. 4e, lane 0; and Fig. 6b). The monofucosyl-nLc6Cer was fivefold higher than the difucosyl-nLc6Cer. Both these glycolipids increased moderately (1.6- and 3.5-fold) on treatment with RA after 6 days (Fig. 6b).

Previously it was reported that ganglioside GD3 was significantly upregulated in RA-treated P19 cells (Liour et al. 2000). This was confirmed in the present experiments (Fig. 5d, lanes 0, 4, 6, and 9, upper band doublet; and Fig. 6b).

SGC-glycoproteins are highly expressed in P19 cells after RA treatment

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Western blot analysis with HNK-1 antibody showed that SGC-glycoproteins were undetectable in untreated and 4-day RA-treated P19 cells (Fig. 7a, lanes 1 and 2). However, the expression of SGC-glycoproteins was highly upregulated at 6 and 9 days after RA treatment (Fig. 7a, lanes 3 and 4). Quantitative analysis of the two major SGC-glycoprotein bands showed that they contained 60–80 picomole of SGC/mg of cell homogenate protein, compared to 1.0 picomole of SGGL-1/mg protein.

image

Figure 7. Western blot analysis SGC-proteins, Amphoterin, Lex-proteins and RAGE in P19 cells, before and after treatment with RA. Proteins in the homogenates of P19 cells, before and after treatment with RA, were separated on a 4–15% SDS–PAGE gradient gel, transferred to nitrocellulose, and immunostained with either HNK-1, anti-Amphoterin anti-Lex (7A) or anti-RAGE antibodies. (a) SGC-proteins; (b) Amphoterin; (c) Lex proteins; and (d) RAGE. (a–c) Lane 1, untreated P19 cells; lanes 2–4, RA-treated P19 cells 4, 6, and 9 days in culture. (d) Lane 1, standard RAGE from rat lung; lanes 2–5, RA-treated P19 cells at day 4, 6, 7, and 9, respectively.

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Amphoterin is moderately upregulated in P19 cells after RA treatment

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Western blot analysis with anti-Amphoterin antibody showed low but detectable level of Amphoterin in untreated P19 cells (Fig. 7b, lane 1). The expression increased after 4 days of RA treatment (Fig. 7b, lane 2), which essentially remained the same after 7 and 9 days (Fig. 7b, lanes 3 and 4).

Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

Immunocytochemical analysis showed that SSEA-1-reactive Lex antigens are strongly expressed in untreated P19 cells (Figs 8a and b). Most of the cells expressed Lex antigen. However, on treatment with RA, the number of cells expressing the Lex antigen declined with time in culture (Figs 8c–h). By 7-day post-treatment, cells expressing Lex were almost undetectable.

image

Figure 8. Expression of Lex antigen in P19 cells before and after treatment with RA. P19 cells, before and after treatment with RA, were immunostained with anti-Lex antibodies, followed by goat anti-mouse IgM-Cy3 and viewed by bright field (a, c, e and g) and fluorescent (b, d, f and h) microscopy. (a and b) Untreated P19 cells. RA-treated cells, after 5 (c and d), 6 (e and f), and 7 (g and h) days in vitro.

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Western blot analysis of Lex-expressing proteins also showed that these proteins were highly expressed in the untreated P19 cells but severely declined after treatment with RA (Fig. 7c).

RAGE is upregulated in RA-differentiated P19 cells

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

P19 cells were grown as dispersed cell cultures on coverslips or as cell aggregates with RA (0.5 µm) in the induction medium for 4 days. The cells were then dispersed and replated for additional 1–5 days without RA. The expression of RAGE and SGC in these cells was visualized with anti-RAGE and HNK-1 antibodies (Fig. 9). The untreated cells did not express any detectable RAGE or SGC (Figs 9a–c). The RA-treated P19 cells, on the seventh day in culture (i.e. after treatment with RA for 4 days and without RA for 3 days) began to express RAGE (Fig. 9e) along with SGC (Fig. 9f), both on cell soma and newly developed processes (Figs 9e and f). By 9 days in culture, increasingly more number of P19 cells treated with RA differentiated into neuron-like cells, having per cell more number of RAGE- and SGC-bearing processes, which were longer than those at previous stages (Figs 9h and i, respectively). Western blot analysis of the cells with anti-RAGE antibodies is shown in Fig. 7(d). Two bands closely migrating near 48 kDa, similar to rat lung RAGE (Fig. 7d, lane 1), were present in P19 cells (Fig. 7d, lane 2–5). Quantitative analysis showed that the level of RAGE in P19 cells after RA treatment increased from 4 to 9 days in vitro by fivefold.

image

Figure 9. RA-treated P19 cells express RAGE and SGC. P19 embryonic carcinoma cells in culture were induced to differentiate into neurons on treatment with 0.5 µm all trans-RA, for 4 days. The cell aggregates were dispersed with trypsin and replated on polylysine coated chamber slides. After 7 and 9 days in vitro culture, cells were immunostained with anti-RAGE and HNK-1 antibodies, followed by goat anti-mouse IgG-Cy3 (red fluorescence) and goat anti-mouse IgM-Cy2 (green fluorescence), respectively. (a–c) Cells without RA; (d–f) cells after RA treatment in vitro for 7 days; (g–I) 9 days. (a, d and g) Bright field microphotographs. The scale bar in (c) applies to all panels. Untreated proliferating cells did not express RAGE or SGC (a–c). Upon treatment with RA, the cells began to express RAGE and SGC and their expressions increased with time in culture.

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RAGE and SGC are co-localized in RA-differentiated neurons

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

RA-treated P19 cells, after 8 days in culture, were double immunostained with HNK-1 and anti-RAGE antibodies. Laser scanning confocal microscopy at high magnification showed that the differentiated neuron-like cells, which expressed SGC, also expressed RAGE (Fig. 10), which showed that RAGE and SGC were co-localized in the same cells.

image

Figure 10. Co-expression of RAGE and SGC in neuronal cells differentiated after RA treatment of P19 cells. P19 cells, after treatment with RA and in culture for 8 days, were doubled immunostained with anti-RAGE and HNK-1 antibodies, followed by goat anti-mouse IgG-Cy3 (red fluorescence) and anti-mouse IgM-Cy2 (green fluorescence), respectively. The fluorescent labeling of cells was viewed with a laser-scanning fluorescence microscope.

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Discussion

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References

P19 EC is a pluripotent stem cell line, which after treatment with RA reflects the early events of neuronal differentiation in vivo. It has been widely used as an in vitro model system for analyzing the mechanisms of mammalian neuronal differentiation (Kotani et al. 2002). Thus P19 cells after treatment with RA, express basic helix–loop–helix (bHLH) genes such as the Mash-1 (Johnson et al. 1992; Itoh et al. 1997), a mammalian homolog of achaete-scute, Math-1, and NeuroD as well as atonal and Nscl-2 (Itoh et al. 1997). The products of these bHLH genes have been well known to function as positive regulatory molecules on the neural fate determination and differentiation (Kotani et al. 2002). Sulfoglucuronyl and Lex carbohydrate epitopes, expressed in several membrane-associated glycolipids, glycoproteins, and proteoglycans of the nervous system, are known to be involved in many aspects of cell–cell and cell–extracellular matrix interactions during development. We have initiated studies of these carbohydrates in RA-treated P19 cells to define their specific molecular role and interactions with other molecules.

SGC epitope is stage specifically expressed strongly in initial cortical development (Zhao et al. 2000b). Differentiated P19 cells show neuron-like characteristics and express SGC, similar to migrating cortical neurons in vivo. Here, we have investigated the biochemical basis and regulation of expression of SGC in P19 cells after treatment with RA. We show that in the RA-differentiated neurons SGC was expressed both on glycolipids (i.e. SGGL-1) and glycoproteins of the cells. To understand the enzymatic basis of SGC expression, analysis of activities of glycosyltransferases involved in the synthesis of SGGL showed that the increase in the synthesis of SGGL-1 was due to upregulation of the synthetic enzymes, particularly enzymesLc2Cer-GlcNAc-Tr and nLc4Cer-GlcA-Tr, after RA treatment. Previously, we have shown that Lc2Cer-GlcNAc-Tr is the key regulatory enzyme for the expression of SGGLs in the cerebral cortex and cerebellum in vivo (Chou and Jungalwala 1993b, 1996). Previous studies have shown that the activities of several glycosyltransferases, such as GalNAc-Tr, sialyltransferase I, II, and III involved in the biosynthesis of gangliosides, were upregulated in RA-treated P19 cells (Osanai et al. 1997; Liour et al. 2000). Similarly, Osanai et al. (2001) have reported substantial increase in the activities of fucosyltransferases for glycolipids, glycoproteins, and oligosaccharide in RA-differentiated P19 cells. Analyses of levels of mRNA of these enzymes showed that the increases were regulated at the transcriptional level (Liour et al. 2000; Osanai et al. 2001). However, it is not clear if RA directly induces the glycosyltransferase activity by modulating the gene transcription or by initiating a program of neural differentiation in which glycosyltransferases are indirectly upregulated.

Although the activities of the glycosyltransferases after RA treatment for the synthesis of SGGL-1 were high, the amount of SGGL-1 expressed per mg protein of P19 cells was exceeding low (1.5 ng) compared to the key precursors Lc3Cer (∼30 ng) and nLc4Cer (∼60 ng) from which it is synthesized. This indicated that these precursors were diverted and possibly utilized more for other neolactoglycolipids. This notion was confirmed as the amounts of Lex-fuco-neolactoglycolipids as well as disialosyl-nLc4Cer (LD1) were expressed at comparable levels as the precursors after RA treatment. Previously it was shown that Lex-glycolipids were markedly increased in RA-treated P19 cells and their increase was attributed to concomitant elevation of Fuc-T gene transcripts (Osanai et al. 2001). Here, we have shown that elevation of these lipids could also be due to 16-fold elevation of the enzyme Lc3 synthase which synthesizes the precursor Lc3Cer, the precursor of nLc4Cer and nLc6Cer, which were significantly elevated in the RA-treated P19 cells.

Our results also show that the expression of SGC epitope as observed by immunocytochemistry after RA treatment was not only due to SGGL-1, but SGC-bearing glycoproteins were also upregulated. Our analysis showed that the mole amounts of SGC-glycoproteins per mg of P19 total proteins were more than 100-fold higher than that of SGGL-1. Thus the levels SGC on glycoproteins were upregulated at significantly higher level than SGGL-1 after RA treatment. Result of Tajima et al. (2000) also suggested that the glycoprotein glucuronyltransferase-P (GlcA-Tr-P) was dramatically increased in RA-treated P19 cells, which was a potential mechanism for the upregulation of SGC-protein expression.

Although SGC-glycoproteins were not expressed in undifferentiated P19 cells and were only expressed after RA treatment, SSEA-1-reactive Lex-glycoproteins were expressed in a large amount in the undifferentiated P19 cells. The expression of Lex epitope in the undifferentiated P19 cells appeared to be mostly due to Lex-glycoproteins. Previously, other murine and human EC cells also have been reported to express Lex epitope abundantly (Solter and Knowles 1978; Levine and Flynn 1986). Further, on RA treatment the differentiated P19 cells downregulated the expression of Lex-glycoproteins, despite moderate upregulation of Lex-glycolipids. These results suggested that loss of Lex-glycoproteins is a marker of differentiation from progenitor cells to neurons. Previously, it was reported that Lex antigen was expressed almost exclusively in germinal layers of the cerebral cortex beginning as early as day 11 of mouse gestation, and becomes undetectable by birth (Yamamoto et al. 1985). Similarly, Allendoerfer et al. (1995) have reported that the expression of Lex antigen was restricted to subpopulations of progenitor cells in the early embryonic CNS. Later, up to embryonic day 17.5, the antigen was specifically restricted to the telencephalon, where a correlation with mitotic activity was apparent. The expression was strong in the ventricular zone, while the cortical plate was negative (Tole et al. 1995). Thus the expressions of Lex proteins in the undifferentiated P19 cells and their downregulation in the differentiated neuron-like cells correlated well with the differentiation stages of cortical neurons in vivo. It should be also noted that despite the presence of Lex-glycolipids in the differentiated P19 cells, their expression was not visualized by immunocytochemistry. This could be due to low level of the glycolipids in these cells. Recent report on the Lex expression in identifying embryonic and adult stem cells in CNS raises new questions on the role of this carbohydrate in stem cell biology and function (Capela and Temple 2002).

Previously, we isolated and purified a ∼30 kDa SGC-binding protein (SBP-1) from neonatal rat brain and showed that SBP-1 was identical in sequence to adhesive neurite outgrowth promoting protein Amphoterin and HMG-1 (Rauvala and Pihlaskari 1987; Nair and Jungalwala 1997; Chou et al. 2001). In the rat cerebral cortex, the level of Amphoterin decreased after embryonic day 18 to almost undetectable level by post-natal day 10. The specific interaction and co-expression of Amphoterin with SGGLs and SGC proteins suggested a functional role for Amphoterin as a receptor for SGC ligand (Nair and Jungalwala 1997). Amphoterin is a non-histone chromosomal protein, which binds to DNA and has been implicated in multiple aspects of gene regulation and cellular differentiation (Bustin 1999). In the developing nervous system we have shown that Amphoterin is particularly overexpressed in the dividing and migrating cortical neurons of the cerebral cortex and in granule neurons of the cerebellum (Zhao et al. 2000a, 2000b). In these neurons it localizes to nucleus as well as to plasma membrane and to filopodia of advancing plasma membrane of neurites (Rauvala et al. 1988; Zhao et al. 2000a, 2000b). Experiments with in vitro explant cultures of cerebellum and cerebral cortex showed that Amphoterin was required for neurite outgrowth and SGC–Amphoterin interaction was important for cell–cell recognition and cell migration (Chou et al. 2000). It was reported that in transformed neuroblastoma cells Amphoterin localized to plasma membrane where it promoted cell differentiation (Rauvala et al. 1988). Amphoterin also binds to the extracellular moiety of RAGE, possibly through its N-glycans (Hori et al. 1995; Huttunen et al. 1999; Fages et al. 2000). RAGE is a cell surface molecule of the Ig super family and shares closest homology with NCAM (Huttunen et al. 1999). Amphoterin and RAGE have a similar spatial and temporal expression pattern in the developing rat nervous system (Hori et al. 1995). Amphoterin substratum in vitro promotes neurite outgrowth of cortical neurons, which is inhibited by anti-RAGE Fab antibodies and by soluble ectodomain of RAGE (sRAGE) itself, suggesting that Amphoterin is a physiological ligand of RAGE (Huttunen et al. 1999; Fages et al. 2000). The effect of Amphoterin on neurite outgrowth is mediated through cell surface RAGE signaling, involving GTPases, Rac, and Cdc42 (Huttunen et al. 1999; Fages et al. 2000).

We have shown here that after RA treatment of P19 cells, the differentiated neuron-like cells having extensive neurite outgrowth-expressed SGC, Amphoterin, and RAGE on their cell membranes. Further, in recent unpublished experiments, we showed that anti-RAGE antibodies immunoprecipitated RAGE along with Amphoterin from homogenates of RA-treated P19 cell, indicating an interaction between these proteins. Their co-ordinated expression and interaction in the differentiated P19 cells, similar to that reported in cortical neurons and granule neurons of the cerebellum during development, support the hypothesis that the interaction of these proteins with one another is important in neurite outgrowth. This cell line thus provides a good model for studies on the role of SGC in modulating the RAGE mediated signaling by Amphoterin in promoting neurite outgrowth.

References

  1. Top of page
  2. Abstract
  3. Experimental procedures
  4. P19 EC stem cell culture and differentiation with retinoic acid
  5. Immunocytochemistry and fluorescent microscopy of cells
  6. Assay of glycosyltransferases
  7. Cells homogenates
  8. Glycolipids extraction and analysis
  9. Analysis of levels of Lc3Cer, nLc4Cer, nLc6Cer, Lex-glycolipids, LD1, GD3, and SGGLs
  10. Western blot analysis of Amphoterin, RAGE, SGC-, and Lex-glycoproteins
  11. Results
  12. RA-differentiated P19 cells express SGC and Amphoterin
  13. SGC and Amphoterin are co-localized in RA-differentiated neurons
  14. Activities of glycosyltransferases and a sulfotransferase involved in the synthesis of SGGL-1 are upregulated in the RA-treated P19 cells
  15. Levels of SGGL-1 increased in P19 cells after RA treatment
  16. Analysis of precursors Lc3Cer, nLc4Cer, and nLc6Cer of SGGLs in P19 cells after treatment with RA
  17. Levels of other neolactoglycolipids in P19 cells after treatment with RA
  18. SGC-glycoproteins are highly expressed in P19 cells after RA treatment
  19. Amphoterin is moderately upregulated in P19 cells after RA treatment
  20. Lex epitope and Lex-glycoproteins are severely downregulated in P19 cells after RA treatment
  21. RAGE is upregulated in RA-differentiated P19 cells
  22. RAGE and SGC are co-localized in RA-differentiated neurons
  23. Discussion
  24. Acknowledgements
  25. References
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