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Glycobiology and Extracellular Matrices: Identification of Neutrophil Granule Glycoproteins as Lewis x-containing Ligands Cleared by the Scavenger Receptor C-type Lectin Sarah A. Graham, Aristotelis Antonopoulos, Paul G. Hitchen, Stuart M. Haslam, Anne Dell, Kurt Drickamer and Maureen E. Taylor Downloaded from J. Biol. Chem. 2011, 286:24336-24349. doi: 10.1074/jbc.M111.244772 originally published online May 11, 2011 http://www.jbc.org/ Access the most updated version of this article at doi: 10.1074/jbc.M111.244772 Find articles, minireviews, Reflections and Classics on similar topics on the JBC Affinity Sites. at Imperial College London on October 7, 2014 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts Supplemental material: http://www.jbc.org/content/suppl/2011/05/11/M111.244772.DC1.html This article cites 56 references, 21 of which can be accessed free at http://www.jbc.org/content/286/27/24336.full.html#ref-list-1 THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 27, pp. 24336–24349, July 8, 2011 Author’s Choice © 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Identification of Neutrophil Granule Glycoproteins as Lewisx-containing Ligands Cleared by the Scavenger Receptor C-type Lectin*□S Received for publication, March 28, 2011, and in revised form, April 26, 2011 Published, JBC Papers in Press, May 11, 2011, DOI 10.1074/jbc.M111.244772 Sarah A. Graham, Aristotelis Antonopoulos, Paul G. Hitchen, Stuart M. Haslam, Anne Dell, Kurt Drickamer, and Maureen E. Taylor1 From the Division of Molecular Biosciences, Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom The scavenger receptor C-type lectin (SRCL) is a glycan- mediate endocytosis of several classes of ligands (1–3). The binding receptor that has the capacity to mediate endo- extracellular portion of the receptor consists of three distinct cytosis of glycoproteins carrying terminal Lewisx groups regions as follows: a coiled coil of ␣-helices ϳ450 Å in length; a (Gal␤1–4(Fuc␣1–3)GlcNAc). A screen for glycoprotein ligands collagen-like region of a similar length; and a globular C-termi- Downloaded from for SRCL using affinity chromatography on immobilized SRCL nal C-type carbohydrate-recognition domain (CRD). These followed by mass spectrometry-based proteomic analysis regions are predicted to be linked by flexible joints (4). revealed that soluble glycoproteins from secondary granules of Although the first two types of domains are found in other class neutrophils, including lactoferrin and matrix metalloprote- A scavenger receptors, SRCL is the only example of a scaven- inases 8 and 9, are major ligands. Binding competition and sur- ger-type receptor in this class that contains a C-type CRD (5). http://www.jbc.org/ face plasmon resonance analysis showed affinities in the low In functional terms, the ␣-helical region in SRCL probably micromolar range. Comparison of SRCL binding to neutrophil forms a stalk projecting from the cell surface, and studies with and milk lactoferrin indicates that the binding is dependent on truncated forms of the receptor indicate that, as with other cell-specific glycosylation in the neutrophils, as the milk form of scavenger receptors, yeast and other microbes bind to the col- the glycoprotein is a much poorer ligand. Binding to neutrophil lagen-like domains, probably through regions containing clus- glycoproteins is fucose-dependent, and mass spectrometry- ters of positively charged amino acid residues (1). The CRD at Imperial College London on October 7, 2014 based glycomic analysis of neutrophil and milk lactoferrin was shows high selectivity for glycans terminating with the Lewisx used to establish a correlation between high affinity binding to trisaccharide (Gal␤1–4(Fuc␣1–3)GlcNAc and recognizes x SRCL and the presence of multiple clustered terminal Lewis Lewisx-containing glycoconjugates (6), but no physiological groups on a heterogeneous mixture of branched glycans, some ligands for the receptor have been reported. The endothelial with poly N-acetyllactosamine extensions. The ability of SRCL expression of SRCL suggests that the receptor could be to mediate uptake of neutrophil lactoferrin was confirmed using involved in mediating adhesion of circulating cells to the endo- fibroblasts transfected with SRCL. The common presence of thelium during leukocyte extravasation, a function analogous x Lewis groups in granule protein glycans can thus target granule to that of the selectin family of C-type lectins (7). Consistent proteins for clearance by SRCL. PCR and immunohistochemical with a role for SRCL in cell adhesion, the Lewisx structure is analysis confirm that SRCL is widely expressed on endothelial found on the surfaces of leukocytes and has been implicated in cells and thus represents a distributed system that could scav- neutrophil adhesion (8). It has also been proposed that SRCL enge released neutrophil glycoproteins both locally at sites of mediates adhesion of Lewisx-expressing cancer cells to endo- inflammation or systemically when they are released in the thelium (9). However, a role of SRCL in cell adhesion remains to circulation. be established definitively. The endocytic activity of SRCL supports the alternative sug- gestion that this receptor may be involved in uptake and degra- The scavenger receptor C-type lectin (SRCL,2 also desig- dation of glycoproteins (6). Several other receptors that contain nated collectin P1) is an endothelial cell surface receptor able to C-type CRDs as well as other types of sugar-binding domains have been implicated in glycoprotein turnover (10). The roles of * This work was supported by Grant 075565 from the Wellcome Trust (to M. E. T. and K. D.) and Grants B19088, SF19107, BBF0083091, and the mannose receptor on macrophages in the clearance of gly- BBC5196701 from the Biotechnology and Biological Sciences Research coprotein hormones tagged with GalNAc-4-SO4 and released Council (to A. D.). □ lysosomal enzymes bearing high mannose oligosaccharides S The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1–S11 and Figs. S1–S10. have been particularly well characterized (11, 12), and the Author’s Choice—Final version full access. importance of the hepatic asialoglycoprotein receptor in clear- 1 To whom correspondence should be addressed: Division of Molecular Bio- ance of specific subclasses of serum glycoproteins has been sciences, Biochemistry Bldg., Imperial College, London SW7 2AZ, United Kingdom. Tel.: 44-20-7594-5281; Fax: 44-20-7594-3057; E-mail: maureen. extensively studied (13, 14). [email protected]. In the studies reported here, the function of SRCL was 2 The abbreviations used are: SRCL, scavenger receptor C-type lectin; probed by identifying specific glycoprotein ligands associated CRD, carbohydrate-recognition domain; LNFP, lacto-N-fucopentaose; CEACAM, carcinoembryonic antigen cell adhesion molecule; Bicine, with cells in the blood. The predominant SRCL ligands in neu- N,N-bis(2-hydroxyethyl)glycine. trophils were found to be soluble granule proteins rather than 24336 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286•NUMBER 27•JULY 8, 2011 Clearance of Neutrophil Granule Glycoproteins by SRCL cell surface proteins, suggesting that a major role for SRCL is phosphate/nitro blue tetrazolium substrate (Calbiochem). likely to be in the clearance of glycoproteins released at sites of Immunoblotting was performed using rabbit polyclonal anti- inflammation, which may be tagged with Lewisx epitopes to bodies to pan-CEACAM (Abcam) at a 1:500 dilution fol- facilitate their uptake after release. lowed by visualization using alkaline phosphatase-conju- gated protein A. EXPERIMENTAL PROCEDURES Purification of Ligands from Human Granulocytes by Affinity Generation of Biotin-tagged CRD of SRCL—The coding Chromatography—An SRCL affinity column was created by sequence for the biotinylation tag Gly-Leu-Asn-Asp-Ile-Phe- passing ϳ5 mg of biotinylated CRD of SRCL in eluting buffer Glu-Ala-Gln-Lys-Ile-Glu-Trp-His-Glu, in which the lysine res- over a 2-ml column of avidin-agarose (Pierce) pre-rinsed with idue is a target for biotinylation (15), was added to the C termi- the same buffer. The column was washed extensively with elut- nus of the CRD in the expression vector pT5T (16). Escherichia ing buffer followed by loading buffer. A sample of the washed coli strain BL21/DE3 cells containing this expression plasmid as resin was analyzed by SDS-PAGE to confirm binding of the well as a plasmid containing the chloramphenicol resistance CRD to the resin. gene and the birA biotin ligase gene (17) were grown in 1-liter Leukocytes were isolated from ϳ50 ml of fresh human blood batches, and inclusion bodies were isolated and dissolved in containing 1.5 mg/ml EDTA as an anticoagulant. Granulocytes guanidine as described previously for preparation of other bio- were isolated by centrifugation through Ficoll-Paque PLUS 3 tin-tagged CRDs (18). Following renaturation by dilution into 4 (d ϭ 1.077 g/cm ; Amersham Biosciences) followed by lysis of Downloaded from volumes of loading buffer (0.5 M NaCl, 25 mM Tris-Cl, pH 7.8, erythrocytes in ammonium bicarbonate (20). Granulocytes 25 mM CaCl2) and dialysis against three changes of the same were lysed by sonication in low salt loading buffer containing buffer, the tagged CRD was isolated following the procedure 1% Triton X-100 and a protease inhibitor mixture (Calbiochem

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