title: Identification of neutrophil granule glycoproteins as Lewis(x)-containing ligands cleared by the scavenger receptor C-type lectin.
abstract: The scavenger receptor C-type lectin (SRCL) is a glycan-binding receptor that has the capacity to mediate endocytosis of glycoproteins carrying terminal Lewis(x) groups (Galβ1-4(Fucα1-3)GlcNAc). A screen for glycoprotein ligands for SRCL using affinity chromatography on immobilized SRCL followed by mass spectrometry-based proteomic analysis revealed that soluble glycoproteins from secondary granules of neutrophils, including lactoferrin and matrix metalloproteinases 8 and 9, are major ligands. Binding competition and surface plasmon resonance analysis showed affinities in the low micromolar range. Comparison of SRCL binding to neutrophil and milk lactoferrin indicates that the binding is dependent on cell-specific glycosylation in the neutrophils, as the milk form of the glycoprotein is a much poorer ligand. Binding to neutrophil glycoproteins is fucose-dependent, and mass spectrometry-based glycomic analysis of neutrophil and milk lactoferrin was used to establish a correlation between high affinity binding to SRCL and the presence of multiple clustered terminal Lewis(x) groups on a heterogeneous mixture of branched glycans, some with poly N-acetyllactosamine extensions. The ability of SRCL to mediate uptake of neutrophil lactoferrin was confirmed using fibroblasts transfected with SRCL. The common presence of Lewis(x) groups in granule protein glycans can thus target granule proteins for clearance by SRCL. PCR and immunohistochemical analysis confirm that SRCL is widely expressed on endothelial cells and thus represents a distributed system that could scavenge released neutrophil glycoproteins both locally at sites of inflammation or systemically when they are released in the circulation.
authors.affiliation: Division of Molecular Biosciences, Department of Life Sciences, Imperial College, London SW7 2AZ, United Kingdom.
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Graham
authors.forename: Aristotelis
authors.initials: A
authors.lastname: Antonopoulos
authors.forename: Paul G
authors.initials: PG
authors.lastname: Hitchen
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Haslam
authors.forename: Anne
authors.initials: A
authors.lastname: Dell
authors.forename: Kurt
authors.initials: K
authors.lastname: Drickamer
authors.forename: Maureen E
authors.initials: ME
authors.lastname: Taylor
mesh_terms: D037222:Collectins / Q000235:genetics / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005643:Fucose / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016256:Lewis X Antigen / Q000031:analogs & derivatives; D008024:Ligands; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D009928:Organ Specificity / Q000502:physiology; D011485:Protein Binding; D051116:Receptors, Scavenger / Q000235:genetics / Q000378:metabolism*; D022142:Secretory Vesicles / Q000235:genetics / Q000378:metabolism*; D014312:Trisaccharides / Q000235:genetics / Q000378:metabolism*
chemical_list: C422442:COLEC12 protein, human; D037222:Collectins; D006023:Glycoproteins; D016256:Lewis X Antigen; D008024:Ligands; D051116:Receptors, Scavenger; D014312:Trisaccharides; C110530:galactosyl-(1,4)-fucopyranosyl-(1,3)-N-acetylglucosamine; D005643:Fucose