Abstract
We previously reported that CR-Fc, an Fc chimeric protein containing the cysteine-rich (CR) domain of the mannose receptor, binds to marginal zone metallophilic macrophages (Mo) and B cell areas in the spleen and to subcapsular sinus Mo in lymph nodes of naive mice (CR-Fc(+) cells). Several CR-Fc ligands were found in spleen and lymph node tissue lysates using ligand blots. In this paper we report the identification of two of these ligands as sialoadhesin (Sn), an Mo-specific membrane molecule, and the leukocyte common antigen, CD45. CR-Fc bound selectively to Sn purified from spleen and lymph nodes and to two low molecular weight isoforms of CD45 in a sugar-dependent manner. CR-Fc binding and non-binding forms of Sn, probably derived from CR-Fc(+) and CR-Fc(-) cells respectively, were selected from spleen lysates. Analysis of the glycan pool associated with the CR-Fc-binding form revealed the presence of charged structures resistant to sialidase, absent in the non-binding form, that could correspond to sulfated structures. These results confirm the identification of the CR region of the mannose receptor as a lectin. We also demonstrate that the same glycoprotein expressed in different cells of the same organ can display distinct sugar epitopes that determine its binding properties.
Highlights
The mannose receptor (MR)1 is a type I membrane glycoprotein that mediates the uptake of mannosylated glycoconjugates by macrophages (Mø), dendritic cells (DCs), and hepatic endothelium [1,2,3,4,5]
This study presents the identification of two of these ligands as sialoadhesin (Sn), an Mø-restricted membrane molecule [12, 13], and CD45 [14, 15]. Biochemical characterization of these interactions showed that CR-Fc binding to both molecules is sugar-dependent. These results indicate that the CR domain of the MR itself has lectin-like activity and that the different ligands recognized in tissue lysates could be different proteins expressed by CR-Fcϩ cells sharing the same sugar structure
By using the chimeric protein CR-Fc, several putative ligands for the CR domain of the MR were detected in spleen and peripheral (p) lymph nodes (LN) lysates by ligand blot analysis [10]
Summary
Balb/c mice were bred at the Sir William Dunn School of Pathology and were used at 8 –10 weeks of age. CR-Fc [10], Sn 1-Fc [22], and MAG 1–5-Fc [23] were prepared as described. The lysate was clarified by centrifugation at 35,000 rpm for 60 min at 4 °C in a 60.Ti rotor and precleared using human IgG (Sigma) coupled to protein A-Sepharose (10 ml, 2 mg/ml) (Amersham Pharmacia Biotech) prepared as described elsewhere [25]. After extensive washing in 10 mM Tris-HCl, pH 8, 2% (v/v) Triton X-100, 150 mM NaCl (450 ml), bound material was eluted using 0.5% (v/v) diethylamine in 2% (v/v) Triton X-100 into tubes containing 1 M TrisHCl, pH 7 (1/10 of final volume). After extensive washing in 10 mM Tris-HCl, pH 8, 2% (v/v) Triton X-100, 150 mM NaCl (450 ml), bound material was eluted using 0.5% (v/v) diethylamine in 2% (v/v) Triton X-100 into tubes containing 1 M TrisHCl, pH 7 (1/10 of final volume). 2-ml fractions were collected and 10-l aliquots were tested for CR-Fc binding activity by dot blot
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