figure1.gif (8177 bytes)
Figure 1
Overview of the construction of a glypican-AP fusion plasmid.  In its native state (A), glypican consists of an extracellular domain attached to the cell-surface through a glycosylphosphotidyl (GPI) anchor (not shown).  From its DNA sequence data (notably the presence of 14 cysteine residues) and anomalously fast migration on non-reducing gels, glypican’s extracellular domain is thought to consist of a compact globular core and a short linear segment that contains varying number of glycosaminoglycan (GAG) attachment sites near the C-terminus. In the fusion protein (B), the GPI anchor is removed and replaced with heat-stable alkaline phosphatase.  To generate the fusion plasmid (C), the appropriate glypican DNA sequence (nucleotides 1 to 1641) is inserted into APtag-2 immediately upstream of the AP-coding sequence.  APtag-2 contains an SV40 origin of replication that selectively enhances the production of the plasmid in certain mammalian cell lines as well as a CMV promoter that promotes the expression of the plasmid.   The expressed fusion protein, lacking a GPI anchor, is secreted into the extracellular medium and can be conveniently harvested.

Assaying for the presence of glycanated glypican-AP fusion protein in the cell media:
AP activity in the media was assayed by adding an equal volume of the phosphatase substrate p-nitrophenyl phosphate (PNP) from Sigma Inc.  (St. Louis, MO; made in 2M diethanolamine and 1 mM MgCl
2) to 50 ”l of cell medium and then, incubated at 37șC for 5 min.  AP activity is signaled by a change in the color of the solution. The type of GAG chains present­HS or chondroitin sulfate­was determined by treating the cell media with heparitinase, chondroitinase ABC, or both, and separating the digested products on a 1% low melting point agarose gel made in 50 mM Tris-Cl (pH 8) and 1 mM MgCl2.  The AP-containing proteins on the gel were detected with the addition of an AP substrate (NBT/BCIP), made from Sigma Fast tablets (Sigma).  The solution turns blue in the presence of AP.  The cos-7 cell medium was also analyzed by Western blotting, where the protein was probed with a mouse anti-AP antibody according to the ECL Western blotting protocol (Life Science).

Purifying the fusion proteins with diethylaminoethyl (DEAE) chromatography:
A binding column, with a volume equal to 30% of that of the protein sample to be purified, was made using DEAE Sephacel beads (Pharmacia Biotech Inc., Piscataway, NJ) and equilibrated with 10 column volumes of 50 mM Tris-Cl buffer (pH 8) containing 150 mM NaCl.  After 30 to 40 ml of cell medium passed through, the column was washed with two column volumes of 50 mM Tris-Cl (pH 8) containing 150 mM NaCl, followed with the same buffer containing 250 mM NaCl.  The column was eluted with two column volumes of 750 mM NaCl made in the same buffer, and collected in 1.5 to 1.75 ml fractions.  The fractions with the highest amount of AP activity, as determined by a visual inspection of their color (using AP substrate PNP) after 5 to 10 min of incubation at 37șC, were pooled. Protease inhibitors (250 ”g/ml NEM, 1 ”g/ml Pepstatin A, and 0.5 mM PMSF) were added to the combined fractions, which were then dialyzed against ACE buffer (see below) and stored at -80șC.

Measuring binding by affinity co-electrophoresis:

The binding affinities of the glypican fusion protein were determined by ACE (Herndon and Lander 1997; Figure 2). ACE gels were developed by incubation with the AP substrate NBT/BCIP at 37șC for several hours or overnight, analyzed using the software ImageQuant (Molecular Dynamics, Inc. Sunnyvale, CA) and quantified essentially as described (San Antonio et al. 1993), except that a colorimetric rather than radioactivity-based method was used to determine mobility.   For each lane, relative AP-staining intensity as a function of the distance from the top of the lane was measured

figure2.gif (9378 bytes)
Figure 2
Schematic diagram of ACE.  The illustrations represent an ACE gel before and after electrophoresis.  A labeled PG, loaded into the top horizontal slot, migrates through lanes containing the test ligand at various concentrations, embedded in 1% low melting point agarose.  The extent to which the PG migration is retarded, as a function of ligand
concentration, can be used to calculate the dissociation constant Kd for the interaction.  If the concentration of labeled PG is much less than Kd, the value of Kd may be calculated from the equation R = R/(1+ Kd/[P]) where R is the retardation coefficient, calculated as m, the amount of retardation, divided by n, the distance that unimpeded PG travels, R is the value of R approached at saturating ligand concentrations.  [P] is the concentration of the ligand embedded in each lane.   Reprinted with the permission of author (Herndon and Lander 1997).

back.gif (287 bytes) back.gif (221 bytes)

Page 3


Huy Pham - Determination of Functionally Relevant Cell-Type... [1] [2] [3] [4] [5] [6] [7]