A Fluorescent Interleukin‐8 Receptor Probe Produced by Targetted Labelling at the Amino Terminus
- 1 January 1995
- journal article
- Published by Wiley in European Journal of Biochemistry
- Vol. 227 (1-2) , 328-334
- https://doi.org/10.1111/j.1432-1033.1995.tb20393.x
Abstract
Interleukin-8 is the most extensively characterised member of the structurally related chemotactic and pro-inflammatory proteins collectively called chemokines. It binds to two closely related members of the seven transmembrane chemokine receptor family found on a variety of leukocyte cell types. In order to study the interaction of interleukin-8 with its receptors, and their distribution, we have produced a fluorescently labelled protein as an alternative to the radioactive 125I-interleukin-8 ligand. Interleukin-8 is naturally produced as two forms, a 72-residue polypeptide by monocytes and a 77-residue form produced by endothelial cells which has an extension of five amino acids at the amino terminal. Both forms are active at nanomolar concentrations, implying that chemical modification to the amino terminus of the 72-residue form will not destroy activity. The 72-residue interleukin-8 sequence starts with a serine residue, which can be oxidised under mild conditions to give a reactive glyoxylyl function which is then reacted with a nucleophilic fluorescein derivative. The site-specifically labelled protein was easily isolated by reverse-phase HPLC. The dissociation constant of the fluorescently labelled interleukin-8 from its receptors on neutrophils was measured by displacement of 125I-interleukin-8 and found to be 10 nM compared to 1 nM for the unmodified protein. The modified protein is highly active in in vitro bioassays using human neutrophils, giving an EC50 of 7 nM in chemotaxis and an EC50 of 0.62 nM for shape change. The binding of the fluorescent protein to neutrophils can also be measured by fluorescent automatic cell sorter (FACS) analysis, and can be competed by unlabelled interleukin-8. The amino-terminal modification of interleukin-8 has produced a reagent which is useful for the quantification of interleukin-8 receptor expression, and will also be useful in monitoring the fate of the ligand after receptor binding.Keywords
This publication has 25 references indexed in Scilit:
- The interleukin-8-receptor family: from chemokines to malariaImmunology Today, 1994
- Protein conjugates of defined structure: Synthesis and use of a new carrier moleculeBioconjugate Chemistry, 1993
- Cloning of Complementary DNA Encoding a Functional Human Interleukin-8 ReceptorScience, 1991
- Structure and Functional Expression of a Human Interleukin-8 ReceptorScience, 1991
- A novel derivative of the chelon desferrioxamine for site‐specific conjugation to antibodiesInternational Journal of Cancer, 1989
- The Neutrophil-Activating Protein (NAP-1) Is Also Chemotactic for T LymphocytesScience, 1989
- Identification and characterization of specific receptors for monocyte-derived neutrophil chemotactic factor (MDNCF) on human neutrophils.The Journal of Experimental Medicine, 1989
- Molecular cloning of a human monocyte-derived neutrophil chemotactic factor (MDNCF) and the induction of MDNCF mRNA by interleukin 1 and tumor necrosis factor.The Journal of Experimental Medicine, 1988
- N-terminal modification of proteins—a reviewProtein Journal, 1984
- Size and refractive index dependence of simple forward angle scattering measurements in a flow system using sharply-focused illumination.Journal of Histochemistry & Cytochemistry, 1977