Chemokine N-terminus

This is a general protocol for purification of chemokines with expression sequences that include an N-terminal Hisg tag and a SMT3 cleavage site. Removal of the His6-SMT3 from the chemokine with ubiquitin fike protease-1 (ULPl or SUMO-protease-1) results in a native, mature chemokine N-terminus that is suitable for all experimental appfications (Lu et al., 2009). 

The length and composition of the chemokine N-terminus play a crucial role in this process and can determine whether the ligand-receptor... 

Zoffmann S, Chollet A, Galzi JL. Identification of the extracellular loop 2 as the point of interaction between the N terminus of the chemokine MIP-lalpha and its CCR1 receptor. Mol Pharmacol 2002 62(3) 729-736. 

Understanding the nature of the domains or elements on the chemokine receptor coreceptors is also of significant importance, especially with regard to the development of coreceptor-blocking therapeutics. HIV-1 gpl20 interacts with multiple extracellular domains of the chemokine receptors to mediate binding, and the N-terminus and extracellular loop (ECL) number 2 of CCR5... 

In addition, HIV-1 infection also induces the secretion of certain metallo-proteinases (MMP-2, MMP-9) that are capable of cleaving the N-terminus of SDF-1 but not that of the P-chemokines, and this decreases SDF-1 affinity for CXCR4 binding (227-229). Proteolytic modification of SDF-1 by a serum factor(s) that is different from CD26 and MMPs, which results in reduced anti-HIV activity, has been described as well (230). How SDF-1 proteolytic... 

Also termed 7-chemokines, C Chemokines are composed of only two cysteines one on the N-terminus and the other a downstream cysteine. There are two chemokines in this group, lymphotactin-a (XCL1) and lymphotactin-(3 (XCL2). Their function is the attraction of T-cell precursors to the thymus. 

Fig 2. Expression of chemokines with and without N-terminal tags. Murine RANTES and human herpesvirus 8 vMIP-lB were expressed either as mature proteins (native) or with the addition of the residues MKKKWPR- to the N-terminus (K3) using the T7 based expression vector pET24d in either BL21(DE3) or BL21(DE3)pLysS cells. Each lane of this Coomassie stained SDS-polyacrylamide gel contains 0.04 OD600 units of the indicated strain following a 3-h induction. 

N-terminus For chemokines it is well-known that the N-terminal residues are critical for function (2-6,9). Sometimes the N-terminus of the mature protein is not known. Synthesis allows generation of multiple N-terminal forms in a single synthesis, thus allowing determination of the correct form. 

Fig. 4. Schematic illustration of chemokine-receptor interactions. The transmembrane helices of the receptor, shown as blue tubes, were derived from the structure of rhodopsin (PDB code 1L9H). The chemokine (pink) and the N-terminal extracellular domain of the receptor were derived from the IL8-CXCR1 peptide complex shown in Fig. 5 (PDB code lILP). Except for the N-terminus, the loops of the receptor are not displayed. The figure illustrates the relative size of the receptor and ligand, and the interaction of the receptor N-terminus along one face of the chemokine. The interaction orients the N-terminal signal domain towards the receptor as displayed here it is oriented towards the helical bundle, which may or may not be correct for some chemokines. (See Color Insert.)...

Ho, H. H., Du, D., and Gershengom, M. C. (1999). The N terminus of Kaposi s sarcoma-associated herpesvirus G protein-coupled receptor is necessary for high affinity chemokine binding but not for constitutive activity. J. Biol. Chem. 274, 31327-31332. 

---