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First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01386

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N-Glycans, not the GPI anchor, mediate the apical targeting of a naturally glycosylated, GPI-anchored protein in polarised epithelial cells

Susan Pang^*, Paula Urquhart^* and Nigel M. Hooper

School of Biochemistry and Microbiology, University of Leeds, Leeds, LS2 9JT, UK

View larger version (14K): [in a new window]   Fig. 1. Schematic of the membrane dipeptidase (MDP) constructs used. Wild-type porcine MDP (wtMDP) contains an N-terminal 16 amino acid signal sequence (diagonally hatched box), two N-linked glycosylation sites (N41 and N263, lollipops) and a C-terminal GPI anchor addition signal (chequered box). The GPI anchor is attached to S368. In MDPGPI and MDPGPIgly, A369 was mutated to a stop codon, resulting in secreted proteins. In MDPgly and MDPGPIgly, N41 and N263 were mutated to Gln to prevent N-glycosylation.

View larger version (31K): [in a new window]   Fig. 2. Expression and enzymatic activity of the MDP constructs in MDCK cells. Confluent monolayers of MDCK cells stably expressing wtMDP (wt), MDPGPI (GPI), MDPgly (gly), MDPGPIgly (GPIgly) or empty vector (pCI) were incubated in OptiMEM for 7 hours. The conditioned medium was harvested and membranes prepared from the cells as described in Materials and Methods. (A) Membranes (10 µg protein per lane) and medium (40 µg protein per lane) were subjected to western blot analysis with an antibody against porcine MDP. Results are representative of three independent experiments. (B) MDP activity was quantified using the selective substrate Gly-D-Phe with the product D-Phe being separated and quantified by reverse phase HPLC (Hooper et al., 1987). The data are the mean±s.e.m. of three determinations.

View larger version (39K): [in a new window]   Fig. 3. Glycosylation and glycosylphosphatidylinositol (GPI) anchorage of the MDP constructs. (A) Membranes (10 µg of protein) from MDCK cells stably expressing either wtMDP (wt) or MDPgly (gly) or medium (40 µg of protein) from cells expressing MDPGPI (GPI) or MDPGPIgly (GPIgly) were incubated in the absence or presence of peptide N-glycosidase F (PNGase F) for 16 hours. The samples were then subjected to western blot analysis with anti-MDP antibody. The results are representative of three independent experiments. (B-E) Cells transfected with either wtMDP (B,D) or MDPgly (C,E) were incubated in the absence (B,C) or presence (D,E) of 150 mU/ml B. thuringiensis PI-PLC, then fixed in paraformaldehyde and subjected to immunocytochemistry using the polyclonal anti-MDP antibody. The images are representative of three independent experiments. Bar, 20 µm.

View larger version (91K): [in a new window]   Fig. 4. Polarised distribution of the GPI-anchored MDP constructs in MDCK cells. (A,B) MDCK cells stably expressing either wtMDP (A) or MDPgly (B) were seeded on polycarbonate filters at confluent levels and left for 3 days to polarise with feeding every other day. Cells were fixed with methanol for 5 minutes prior to labelling with (i) anti-MDP antibody and (ii) anti-Na+/K+ ATPase antibody; (v) anti-MDP antibody; and (vi) apical marker gp135 antibody; which were visualised using Alexa488- and Alexa594-conjugated secondary antibodies (green and red, respectively). (iii) and (vii) Merged images with colocalisation of antibodies appearing yellow. (iv) and (viii) Z-axis images of cells in (iii) and (vii) are also shown. Bar, 10 µm. The images are representative of five independent experiments.

View larger version (71K): [in a new window]   Fig. 5. Polarised distribution of the GPI-anchored MDP constructs in CaCo-2 cells. (A,B) CaCo-2 cells stably expressing either wtMDP (A) or MDPgly (B) were seeded on polycarbonate filters at confluent levels and grown for 14 days to allow them to polarise with feeding every other day. (i) Anti-MDP was visualised with Alexa488 (green) and (ii) anti-Na+/K+ ATPase antibody with Alexa594-conjugated secondary antibody (red). (iii) Merged images with colocalisation of the two antibodies appearing yellow. (iv) Z-axis images of cells in (iii). Bar, 10 µm. The images are representative of three independent experiments.

View larger version (24K): [in a new window]   Fig. 6. Lipid raft association of the GPI-anchored constructs. (A-F) MDCK cells stably expressing either wtMDP or MDPgly were lysed in MBS containing 0.5% Lubrol WX at 4°C and lipid rafts isolated by buoyant sucrose density gradient centrifugation. The sucrose gradients were fractionated in 0.5 ml aliquots (P, insoluble pellet; 1, bottom of tube; 9 top of tube). Total protein in each fraction was measured (A) and activity assays for dipeptidyl peptidase IV (D) and MDP (F) were carried out. Fractions were also analysed by immunoblotting with (B) caveolin-1, (C) clathrin or (E) MDP. The results are representative of three experiments.

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