N-linked oligosaccharides affect the enzymatic activity of CD39: diverse interactions between seven N-linked glycosylation sites

Abstract

Rat CD39, a membrane-bound ectonucleoside triphosphate diphosphohydrolase that hydrolyzes extracellular nucleoside tri- and diphosphates, has seven potential N-glycosylation sites at asparagine residues 73, 226, 291, 333, 375, 429, and 458. To determine their roles in the structure and function of CD39, we mutated these sites individually or in combination by replacing asparagine with serine or glutamine and analyzed the surface expression and the enzymatic activity of the mutants. The results indicate that rat CD39 can be glycosylated at all seven sites when expressed in COS7 cells. Glycosylation sites 73 at the N terminus, 333 in the middle, and 429 and 458 at the C terminus were principally required for cell surface appearance of enzymatically active CD39. Whereas deletion of these sites individually had modest effects on surface ATPase activity, some double deletions of these sites had major effects on both surface activity and expression. The importance of these N-glycosylation sites is recognizable in other members of the ectonucleoside triphosphate diphosphohydrolase family.

Figure 1.

Electrophoretic mobility of CD39 mutants with single and multiple N-glycosylation sites. (A) Schematic structure of rat CD39. The potential N-glycosylation sites are indicated above the protein. The transmembrane domains are shown as lined boxes. The apyrase conserved regions are shown as empty boxes. (B) Crude membranes (10 μg of protein) from COS7 cells expressing different CD39 mutants were examined by SDS-PAGE, and the gels were immunoblotted with an anti-HA antibody. The symbol “+” indicates the presence of the indicated N-glycosylation sites, whereas all the other sites have been removed. The numbering scheme is shown in A. (C) Crude membranes (10 μg of protein) from COS7 cells expressing mutant +2 containing only the second N-glycosylation site were treated with PNGase F, followed by SDS-PAGE and immunoblotting with an anti-HA antibody.

Figure 2.

Expression and enzymatic activity of CD39 mutants lacking a single N-glycosylation site. (A) Intact COS7 cells expressing CD39 mutants were incubated with ATP in the presence of calcium; the released phosphate was measured and compared with that of COS7 cells expressing wild-type CD39. (B) Crude membranes (10 μg of protein) of COS7 cells transfected with plasmids containing wild-type and mutant CD39 cDNA were analyzed with SDS-PAGE and immunoblotting with an anti-HA antibody. The symbol “Δ” indicates the mutated N-glycosylation site. Δ2 is the N226Q mutation. WT is wild-type CD39. (C) COS7 cells expressing wild-type, Δ2(N226S), ΔAll(N226S) (Δ1,2,3,4,5,6,7), and + 4,7 CD39 were solubilized with Laemmli buffer, and an aliquot of the solution was examined by SDS-PAGE. The proteins were then transferred to a nitrocellulose membrane and immunoblotted with an anti-HA antibody.

Figure 3.

Characterization of the glycans of wild-type and mutant CD39. COS7 cells expressing wild-type or mutant CD39 were solubilized with glycoprotein denaturing buffer (New England Biolabs) and incubated at 100°C for 10 min. Equal amounts of cell lysate were treated with Endo H, PNGase F, or without glycosidase at 37°C overnight. After heating at 95°C for 5 min, the lysates were subjected to electrophoresis and immunoblotting with anti-HA antibody. (A) Wild-type CD39 and single site mutants. (B) Double site mutants. (C) Multiple site mutants. C, control without glycosidase treatment; H, treated with Endo H; F, treated with PNGase F.

Figure 4.

Surface expression of the N-glycosylation site mutants of CD39. (A and B) COS7 cells expressing wild-type or mutant CD39 were biotinylated with EZ-link Sulfo-NHS-Biotin and then solubilized with RIPA buffer. Top, solutions were incubated with monoclonal anti-HA agarose to collect CD39 molecules. The beads were incubated with Laemmli buffer, and the released proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunoblotted with ECL streptavidin-HRP. Bottom, solutions were subjected to SDS-PAGE, and the proteins were transferred to a nitrocellulose membrane and immunoblotted with anti-HA antibody. Relative amounts of mutant proteins are determined in comparison with the amount of wild-type CD39 in each panel; comparisons between panels are not valid because of differences in exposure times.

Figure 5.

Immunofluorescence photographs of CHO cells expressing CD39 wild-type and CD39 mutants. CHO cells expressing wild-type CD39 and the Δ1, Δ4, and +2 mutants were grown for 48 h on coverslips. After fixation and permeabilization, the cells were stained with rabbit anti-HA antibody and FITC-conjugated anti-rabbit IgG secondary antibody. The nucleus was stained with DAPI. The cells were visualized with a Zeiss LSM510 confocal microscope.

Figure 6.

Ca²⁺ dependence of the ATPase activity of wild-type CD39 and N-glycosylation site mutants. Crude membranes (10 μg of protein) of COS7 cells expressing wild-type or mutant CD39 were incubated with ATP in the presence of different concentrations of free calcium as described in Grinthal and Guidotti (2000). Released phosphate was assayed according to Ames (1966).

Figure 7.

Sucrose density gradient sedimentation of solubilized wild-type CD39 and the Δ7 mutant. Crude membranes prepared from COS7 cells expressing CD39 wild-type or the Δ7 mutant were solubilized with 1% digitonin, layered on a linear gradient 5–20% (wt/vol) sucrose, and centrifuged for 14 h at 150,000 × g. Fractions of 200 μl were collected from the top of centrifuge tubes. The presence of CD39 was determined by measuring the ATPase activity of the fractions (see Materials and Methods).

Figure 8.

Effect of N-glycosylation on CD39 posttranslational processing. Crude membranes (10 μg of protein) of COS7 cells expressing wild-type or mutant CD39 were subjected to SDS-PAGE, and the proteins, after transfer to nitrocellulose, were immunoblotted with an anti-CD39 antibody.

Figure 9.

Schematic representation of the N-glycosylation sites and cysteine residues of NTPDases 1–6. The structures of NTPDases 1–6 are shown sequentially. The potential N-glycosylation sites are indicated by a black arrow on top of the rectangle; the cysteine residues by a line under the rectangle. NTPDases 1, 2, and 3 have similar arrangements of all 10 of the extracytoplasmic cysteine residues, whereas only two N-glycosylation sites are common to all three structures.