Identification and Characterization of a Glycosaminoglycan Recognition Element of the C Chemokine Lymphotactin

Abstract

Chemokine-mediated recruitment of leukocytes in vivo depends on interactions with cell surface glycosaminoglycans. Lymphotactin, the unique member of the "C" chemokine subclass, is a highly basic protein that binds heparin, a glycosaminoglycan, with high affinity (ϳ10 nM). We detected lymphotactin-heparin binding by NMR and mapped this interaction to a narrow surface that wraps around the protein. Substitutions in and around this binding site and surface plasmon resonance analysis of heparin binding affinity identified two arginine residues of lymphotactin as critical for glycosaminoglycan binding. Both arginine mutant proteins and the combined double mutant had dramatically diminished in vivo activity in a leukocyte recruitment assay, suggesting that the lymphotactin-glycosaminoglycan interactions detected in vitro are important for the function of this chemokine. Our results demonstrate that like other chemokines, lymphotactin utilizes highly specific glycosaminoglycan-binding sites that represent potential targets for drug development.A family of small secreted proteins, chemokines recruit leukocytes from the circulatory system to mediate inflammatory responses in host defense and wound healing (1), control angiogenesis, and regulate lymphoid development (2, 3). Chemokine sequences are divided into four subfamilies, based on the number and spacing of conserved cysteine residues. Many chemokine structures are known, and members of all four subfamilies adopt a canonical tertiary fold, typically stabilized by a pair of disulfide bonds (4); some also form homodimeric structures (5). Chemokines stimulate leukocyte migration by activating specific G protein-coupled receptors expressed in their target cell populations (3, 6). This signaling network of roughly 50 chemokines and 20 receptors is both selective and redundant: specific leukocyte types express a particular receptor and are recruited only in response to their cognate chemokines, however, many receptors can be activated by more than one chemokine ligand (7).Human lymphotactin (hLtn/XCL1) 1 recruits T lymphocytes and natural killer cells through its specific receptor XCR1 in normal immune function and chronic inflammatory conditions (8, 9). For example, an inappropriate or uncontrolled T cell infiltration driven by hLtn expression is a factor in both Crohn's disease (10) and rheumatoid arthritis (11). Interestingly, the unique ability of hLtn to specifically chemoattract lymphocytes has been exploited therapeutically in combination with the T cell-activating cytokine interleukin-2 (IL-2). T cells recruited by hLtn and activated by IL-2 generated potent antitumor immunity in human clinical trials for treatment of neuroblastoma, where studies with either hLtn or IL-2 alone failed to generate a significant response (12). Hence, hLtn and its analogs with activity toward XCR1 will be extremely valuable as agents for T cell recruitment in cancer vaccines (6), whereas specific antagonists of hLtn activity may be useful for treating certain autoimmune or inflammator...

Interconversion between two unrelated protein folds in the lymphotactin native state

“…Like other chemokines (16), Ltn binds heparin with high affinity, and its in vivo activity depends on GAG interactions (17). Wild-type Ltn elutes from a heparin-Sepharose column in two distinct fractions (Fig.…”

The Lymphotactin Receptor Is Expressed in Epithelial Ovarian Carcinoma and Contributes to Cell Migration and Proliferation

“…These data could indicate that very little soluble XCL1 is released into the ascitic fluid. Thus, likely sources of XCL1 in the peritoneal cavity include areas in the vicinity of the XCL1-producing cells of the immune system, ovary, and other peritoneal organs as well as cell surface-attached GAGs harboring XCL1 (13,30,31). To further investigate the possible source of XCL1 in EOC, we tested the EOC cell lines IGROV-1, SKOV-3, and A2780 and found that XCL1, but not XCL2, RNA is expressed by these EOC cell lines (Fig.…”

Dromedary glycosaminoglycans: Molecular characterization of camel lung and liver heparan sulfate

“…Heparin and HS are closely related, overlapping structures that are widely distributed in invertebrates (Medeiros et al, 2000;Nobuo, 2003;Cesaretti et al, 2004), as well as in vertebrates (Gomes and Dietrich, 1982). Moreover, HS GAGs modulate many biological activities including signal transduction (Ibrahimi et al, 2005), inflammation (Peterson et al, 2004), complement activation (Yu et al, 2005) and cAMP-dependent substrate phosphorylation catalyzed by protein kinase (Dittmann et al, 1998). Some of the biological roles of GAGs have been exploited for the design and preparation of therapeutic drugs.…”