Figure 2.

The Cys-Rich region of the Man/S4GGnM-Fc chimera accounts for binding of oligosaccharides with terminal GalNAc-4-SO₄. Each of the Man/S4GGnM-Fc constructs shown in Fig. 1 was expressed in CHO-Tag 30A cells and isolated from the medium by affinity chromatography on protein A-Sepharose (Pharmacia) as described (4). The purified chimeras were incubated with S4GGnM-[¹²⁵I]BSA (striped bars) or Man-[¹²⁵I]BSA (solid bars) for 30 min at room temperature. The complexes formed were precipitated by addition of polyethylene glycol and were collected by filtration on Whatman GF/C filter discs as described (3). The amount of radiolabel precipitated was determined using a γ-counter. Because the efficiency of secretion differed significantly among the Fc chimeras, the amount of protein secreted during metabolic labeling with Tran³⁵S-label (ICN) as compared with the wild-type chimera was used to estimate the relative amount of each construct being produced.