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. 2010 Jun 1;110(3):645-59.
doi: 10.1002/jcb.22575.

Carboxylated N-glycans on RAGE promote S100A12 binding and signaling

Geetha Srikrishna  1 , Jonamani Nayak, Bernd Weigle, Achim Temme, Dirk Foell, Larnele Hazelwood, Anna Olsson, Niels Volkmann, Dorit Hanein, Hudson H Freeze
Affiliations

Carboxylated N-glycans on RAGE promote S100A12 binding and signaling

Geetha Srikrishna et al. J Cell Biochem. .

Erratum in

  • J Cell Biochem. 2010 Sep 1;111(1):248

Abstract

The receptor for advanced glycation end products (RAGE) is a signaling receptor protein of the immunoglobulin superfamily implicated in multiple pathologies. It binds a diverse repertoire of ligands, but the structural basis for the interaction of different ligands is not well understood. We earlier showed that carboxylated glycans on the V-domain of RAGE promote the binding of HMGB1 and S100A8/A9. Here we study the role of these glycans on the binding and intracellular signaling mediated by another RAGE ligand, S100A12. S100A12 binds carboxylated glycans, and a subpopulation of RAGE enriched for carboxylated glycans shows more than 10-fold higher binding potential for S100A12 than total RAGE. When expressed in mammalian cells, RAGE is modified by complex glycans predominantly at the first glycosylation site (N25IT) that retains S100A12 binding. Glycosylation of RAGE and maximum binding sites for S100A12 on RAGE are also cell type dependent. Carboxylated glycan-enriched population of RAGE forms higher order multimeric complexes with S100A12, and this ability to multimerize is reduced upon deglycosylation or by using non-glycosylated sRAGE expressed in E. coli. mAbGB3.1, an antibody against carboxylated glycans, blocks S100A12-mediated NF-kappaB signaling in HeLa cells expressing full-length RAGE. These results demonstrate that carboxylated N-glycans on RAGE enhance binding potential and promote receptor clustering and subsequent signaling events following oligomeric S100A12 binding.

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Figures

Figure 1
Figure 1. Carboxylated glycans on RAGE mediate binding to S100A12, but not to S100A11
To determine binding equilibrium of purified human S100A12 (A) or S100A11 (B) to purified bovine RAGE, increasing amounts of the ligands were added to total RAGE, mAbGB3.1 enriched RAGE, or RAGE deglycosylated using PNGaseF under non-denaturing conditions that removed both N-glycans. RAGE on plate was quantified using anti-RAGE. Bound ligands were quantified using specific antibodies. Data were fitted to non-linear regression analysis using GraphPad Prism. Each point is the mean ± SD of two determinations.
Figure 2
Figure 2. S100A12 shows specific binding to carboxylated glycans
Purified recombinant human S100A12 (A) was incubated with BSA-conjugated carboxylate-enriched bovine lung glycopeptides coated onto microtiter plates and binding was detected using anti-S100A12. Non-linear regression transforms of binding data using GraphPad Prism are presented. (B) Inhibitions of binding were carried out in presence of increasing concentrations of free carboxylated or carboxylate-neutralized glycans. Each point is the mean two determinations. C. Time course of complexation between S100A12 and carboxylated glycans (0.1M, moderately charged, 0.3M highly charged) in the presence or absence of calcium was measured by light scattering at 420 nm.
Figure 3
Figure 3. Glycosylation of sRAGEs expressed in different cells
A. sRAGEs were expressed in different mammalian cells using retroviral vectors and in SF9 cells and purified as described under Methods. Purity was confirmed by SDS-PAGE and Coomassie brilliant blue staining. Western blots show that sRAGEs from different sources are all glycosylated. Lane 1: undigested. Lane 2: Endo H digested Lane 3: PNGase F digested. B. mAbGB3.1 reactivity of the different preparations as determined by ELISA. C. There is direct correlation between mAbGB3.1 reactivity of the human sRAGEs and bovine RAGE, and their maximum binding sites for human S100A12.
Figure 4
Figure 4. RAGE forms higher-order oligomers
A. Field of view of negatively stained sRAGE and in inset mAbGB3.1-enriched fraction as captured by EM imaging. Protein densities are white on a dark background. For display purposes, some of the oligomers in this image have been marked using white boxes for small oligomers, possibly dimers, and white circles for slightly larger oligomers which are enriched in the mAbGB31. fraction. Yellow circles mark larger, multi size oligomers. Scale bar is 100 nm. The mAbGB3.1-enriched fraction (inset) seems to be more homogeneous with molecular distribution corresponding to the white circles B. Results from reference-free, K-means clustering. Class averages of five slef consistent classes with the percentage of classified images. The image marked with sym represents the symmetrized average (2-fold) of the most self-consistent, 70% of the data. All images display similar morphology with a central mass and two equivalent lobes attached. C. Comparison of the symmetrized average with the surface representation of four homology models of sRAGE molecules suggests that this enriched-fraction consists of four or more sRAGE oligomerized receptor.
Figure 5
Figure 5. Analysis of RAGE-S100A12 complex formation
Purified bovine RAGE, mAbGB3.1 enriched bovine RAGE, deglycosylated mAbGB3.1-enriched bovine RAGE, human sRAGE expressed in HeLa cells and and mAbGB3.1-enriched sRAGEHeLa, and human sRAGE expressed in E.coli were analyzed as such or mixed with radioiodinated human S100A12 and the complexes formed were analyzed on precalibrated Superdex 200 gel filtration column (fractionation range 10,000–600,000). Fractions were collected and RAGE quantitated by ELISA. S100A12 was measured by quantitating radioactivity (not shown). Molecular weights of the complexes were determined from a calibration curve using marker protein standards (shown by arrows on the first panel for bovine RAGE) 1: bovine thyroglobulin (670 kDa) 2: ferritin (440 kDa) 3: IgG (150 kDa) 4: bovine albumin (66 kDa), and 5: ribonuclease (13.7 Da). RAGE multimerization is evident in solution. Unfractionated RAGE forms complexes with S100A12 corresponding to ratios of at least RAGE3: S100A126 (MW 220 kDa), while mAbGB3.1 enriched populations form higher order complexes of hexamer:dodecamer and above. Deglycosylation significantly reduces the ability to form complexes.
Figure 6
Figure 6. Activation of NF-κB by S100A12 ligation of RAGE is inhibited by mAbGB3.1 and sRAGE
Stable transfectants of HeLa cells expressing full length and cytoplasmic tail deleted (signaling deficient) RAGE were generated as described. Expression of RAGE in cells was confirmed by A. Western blotting using anti-RAGE, Lane 1, untransfected HeLa cells; Lane 2, Full length RAGE transfectants and Lane 3, Tail deleted RAGE transfectants and by B. FACS analysis. Unstained cells are represented in the background. Low-level endogenous expression is seen in untransfected cells. C. Activation of NF-κB by S100A12 ligation of RAGE is inhibited by mAbGB3.1 and sRAGE: HeLa cells expressing full length or tail deleted RAGE were transiently transfected with an NF-κB-responsive cis-reporter gene and a pSV-β-Galactosidase control vector. S100A12 activation in the presence and absence of inhibitors and analysis of enzymes in lysates was done as described. The ratio of Luc/Gal served to normalize for Luc activity. Luc/Gal ratio in transfected unactivated control, which accounted for endogenous activity, was considered 100%. Activity in tail deleted RAGE accounted for RAGE-unrelated background. All values represent the mean ± S.D. (n = 2).
Figure 7
Figure 7. Schematic representation of RAGE receptor clustering and formation of signaling assembly following carboxylated glycan-mediated S100A12 binding
A Lateral view: Our EM studies as well as studies from other labs suggest the presence of a pre-assembled RAGE multimer on cell surfaces. The V-domains are modified by hybrid, high mannose and complex glycan chains, with a subpopulation modified by carboxylated glycans (marked by a star). Ca (and Zn) promotes hexamerization of extracellular S100A12 released from cells in response to inflammatory and other stimuli. Carboxylated glycans could provide initial high affinity binding sites on RAGE for S100A12, and promote increased initial rate of complexation, leading to subsequent domain interactions and formation of a signaling assembly. Multiple binding sites could be provided on a single RAGE domain by the display of multi-antennary glycan chains modified by one or more carboxylated-glycan epitopes (only one epitope is shown for sake of space and clarity). Whether this ligand-receptor assembly occurs on organized membrane microdomains remains to be determined B. A view from the top of the cell membrane showing preassembled receptor and ligand-induced formation of signaling complexes. By providing multi-valency or multiple binding sites, caboxylated-glycans could promote higher order complexes (as seen in Fig 5) with mAbGB3.1-enriched RAGE.
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References

    1. Bierhaus A, Humpert PM, Morcos M, Wendt T, Chavakis T, Arnold B, Stern DM, Nawroth PP. Understanding RAGE, the receptor for advanced glycation end products. J Mol Med. 2005;83:876–86. - PubMed
    1. Bierhaus A, Nawroth PP. Multiple levels of regulation determine the role of the receptor for AGE (RAGE) as common soil in inflammation, immune responses and diabetes mellitus and its complications. Diabetologia. 2009;52:2251–63. - PubMed
    1. Bierhaus A, Schiekofer S, Schwaninger M, Andrassy M, Humpert PM, Chen J, Hong M, Luther T, Henle T, Kloting I, Morcos M, Hofmann M, Tritschler H, Weigle B, Kasper M, Smith M, Perry G, Schmidt AM, Stern DM, Haring HU, Schleicher E, Nawroth PP. Diabetes-associated sustained activation of the transcription factor nuclear factor-kappaB. Diabetes. 2001;50:2792–808. - PubMed
    1. Brewer CF, Miceli MC, Baum LG. Clusters, bundles, arrays and lattices: novel mechanisms for lectin-saccharide-mediated cellular interactions. Curr Opin Struct Biol. 2002;12:616–23. - PubMed
    1. Cairo CW, Gestwicki JE, Kanai M, Kiessling LL. Control of multivalent interactions by binding epitope density. J Am Chem Soc. 2002;124:1615–9. - PubMed

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