The macrophage/endothelial cell mannose receptor cDNA encodes a protein that binds oligosaccharides terminating with SO4-4-GalNAcbeta1,4GlcNAcbeta or Man at independent sites

Abstract

Lutropin (LH) and other glycoproteins bearing oligosaccharides with the terminal sequence SO4-4-GalNAcbeta1,4GlcNAcbeta1,4Man- (S4GGnM) are rapidly removed from the circulation by an S4GGnM-specific receptor (S4GGnM-R) expressed at the surface of hepatic endothelial cells. The S4GGnM-R isolated from rat liver is closely related to the macrophage mannose-specific receptor (Man-R) isolated from rat lung both antigenically and structurally. The S4GGnM-R and Man-R isolated from these tissues nonetheless differ in their ability to bind ligands bearing terminal GalNAc-4-SO4 or Man. In this paper, we have explored the structural relationship between the Man-R and the S4GGnM-R by examining the properties of the recombinant Man-R in the form of a transmembrane protein and a soluble chimeric fusion protein in which the transmembrane and cytosolic domains have been replaced by the Fc region of human IgG1. Like the S4GGnM-R isolated from liver, the chimeric fusion protein is able to bind ligands terminating with GalNAc-4-SO4 and Man at independent sites. When expressed in CHO cells the recombinant Man-R is able to mediate the uptake of ligands bearing either terminal GalNAc-4-SO4 or terminal Man. We propose that the Man-R be renamed the Man/S4GGnM receptor on the basis of its multiple and independent specificities.

Figure 1

Man/S4GGnM-Fc(Wt) binds to Man-Sepharose and LH-Sepharose. Man/S4GGnM-Fc(Wt) metabolically labeled with [³⁵S]cysteine/[³⁵S]methionine was purified from the medium of CHO-Tag 30A cells by affinity chromatography on protein-A-Sepharose. Equal amounts of the purified chimera were incubated with LH-Sepharose or Man-Sepharose. The supernatant was removed and contained unbound Man/S4GGnM-Fc(Wt). After washing with incubation buffer Man/S4GGnM-Fc(Wt) that remained bound to the affinity matrix was released by boiling in PAGE loading buffer containing 2-mercaptoethanol (A) or elution with 200 mM Man (B). The unbound and bound fractions were then analyzed by SDS/PAGE and visualized by autoradiography. (A) Lane 1, Man/S4GGnM-Fc(Wt) not exposed to affinity matrix; lane 2, unbound and lane 3, and bound fractions from incubation with LH-Sepharose in the presence of EDTA; lane 4, unbound and lane 5, bound fractions from incubation with LH-Sepharose in the presence of Ca²⁺; lane 6, unbound and lane 7, bound fractions from incubation with Man-Sepharose in the presence of EDTA; lane 8, unbound and lane 9 bound fractions from incubation with Man-Sepharose in the presence of Ca²⁺. (B) Man/S4GGnM-Fc(Wt) was incubated with Man-Sepharose in the presence of 2 mM Ca²⁺. After unbound chimera had been removed (lanes 1 and 4), bound Man/S4GGnM-Fc(Wt) was eluted with either 200 mM Man in the presence of EDTA (lane 2) or 200 mM Man without EDTA (lane 5). After elution with Man, residual Man/S4GGnM-Fc(Wt) was released by boiling in SDS/PAGE loading buffer (lanes 3 and 6) as above.

Figure 2

Characteristics of S4GGnM-BSA and Man-BSA binding by the Man/S4GGnM-Fc(Wt) chimera. The affinity-purified Man/S4GGnM-Fc(Wt) chimera was incubated with either Man-¹²⁵I-BSA (2 × 10⁵ dpm) (hatched bars) or S4GGnM-¹²⁵I-BSA (2 × 10⁵ dpm) (solid bars) with no other additions, 10 μg of Man-BSA, 10 μg of S4GGnM-BSA, 40 mM monosaccharide, or 200 mM monosaccharide. Fuc, fucose. Man-BSA and S4GGnM-BSA bound by the Man/S4GGnM-Fc(Wt) chimera were precipitated by addition of PEG and collected by filtration on GF/C filters, and the amount of radiolabel was determined in a γ counter. Values are expressed as a percent of the amount bound in the absence of added inhibitor (None). The asterisks indicate the locations of bars where inhibition was complete.

Figure 3

Recombinant Man/S4GGnM-R mediates uptake of Man-BSA and S4GGnM-BSA when expressed in CHO cells. CHO-Tag 30A cells transfected with pcDNAI-Man/S4GGnM-R(Wt) were released from culture plates and incubated with either Man-¹²⁵I-BSA (□) or S4GGnM-¹²⁵I-BSA (○) at 37°C in αMEM. Nonspecific binding of Man-BSA and S4GGnM-BSA were determined by incubation in the presence of 1.25 mg/mannan (◊) and 0.1 mg/ml fucoidin (▵), respectively.