Chemokines and Glycosaminoglycans

Although it was not known at the time, the chemokine field started with the identification of a protein byHeparin Sepharose affinity chromatography, platelet factor 4 (PF4) (1) now called CXCL4 in the systemic nomenclature introduced in 2000. As with many pathways in scientific research, our entry into the field of chemokines and glycosaminoglycans (GAGs) was a series of fortuitous coincidences. Christine Power, working on the chemokine project in the Glaxo Institute of Molecular Biology (GIMB) under the leadership of Tim Wells, had hired a postdoctoral scientist, Arlene Hoogewerf to clonemurine CCR4, and to generate the KOmouse. Arlene had previously studied the role of proteoglycans in regulating the function of othermolecules and had also previously published a paper on the enzymatic activity of CTAP-III, and NAP-2, truncated forms of platelet basic protein/CXCL7, to degrade heparin. Since our group was currently working on these chemokines derived from the β-thromboglobulin precursor, and moreover we were all biochemists, our interest was piqued by the relationship between chemokines and GAGs. This interest was of course inspired by the paper recently published by Antal Rot demonstrating the evidence for haptotaxis as opposed to chemotaxis (2), earning him the title of Godfather of Chemokine–GAG biology. Arlene then followed a dual Post-doc pathway, partially hijacked by Tim to collaborate with him and his Ph.D. student, Gaby Kuschert (now Gaby Campanella) to lay much of the biochemical groundwork of chemokine–GAG interactions, and also fulfilling her goal of creating the CCR4−/− mouse. They were able to show that chemokines demonstrated selectivity in their interaction with GAGs, beyond the obvious electrostatic interactions between basic and acidic molecules, and importantly made the observation that this interaction could trigger oligomerization of chemokines (3, 4). In addition, theywere able to define the pharmacophore responsible forGAGbinding of the chemokine IL-8/CXCL8 (5). My lab became more directly involved in this research direction through the serendipitous encounter of a Ph.D. student, Sarah Fritchley, working in Simi Ali’s lab in Newcastle, UK, who was interested in expressing the putative GAG binding mutant of RANTES/CCL5 in E. coli, but who did not have a viable expression system. Sarah spent a couple of months in the lab under the tuition of our expert chemokine protein chemist, Fred Borlat, successfully producing the 40’s mutant, as we colloquially called it. We published it in JBC with its correct biochemical nomenclature, 44AANA47RANTES (6). Perhaps an omen as to the importance of thismutant for us was that it was an exception among most papers we had submitted as it was accepted overnight! We were heavily involved in screening for chemokine receptor antagonists at this time, and Marie Kosco-Vilbois had set up a simple cell recruitment assay to test putative inhibitors in vivo – chemokine-induced peritoneal recruitment in mice. Therefore, to investigate the effect of the abrogation of GAG binding in vivo, we asked her technician, Suzanne Herren, to test it for us. Being very rigorous, and accustomed to testing compounds for their ability to inhibit chemokine induced recruitment, in this instance RANTES, Suzanne tested it both for its agonist and antagonist activity. I will never forget my amazement in seeing that the mutant was not only unable to recruit cells but actually inhibited the recruitment induced by RANTES. Again serendipity stepped in. I gave a talk at a BALR meeting in the UK, and after dinner at the speaker’s table, joined the youngsters at the adjacent table for a post-prandial “relaxation”. . . (which