A recently identified chemokine, fractalkine, is a member of the chemokine gene family, which consists principally of secreted, proinflammatory molecules. Fractalkine is distinguished structurally by the presence of a CX3C motif as well as transmembrane spanning and mucin-like domains and shows atypical constitutive expression in a number of nonhematopoietic tissues, including brain. We undertook an extensive characterization of this chemokine and its receptor CX3CR1 in the brain to gain insights into use of chemokine-dependent systems in the central nervous system. Expression of fractalkine in rat brain was found to be widespread and localized principally to neurons. Recombinant rat CX3CR1, as expressed in Chinese hamster ovary cells, specifically bound fractalkine and signaled in the presence of either membrane-anchored or soluble forms of fractalkine protein. Fractalkine stimulated chemotaxis and elevated intracellular calcium levels of microglia; these responses were blocked by anti-CX3CR1 antibodies. After facial motor nerve axotomy, dramatic changes in the levels of CX3CR1 and fractalkine in the facial nucleus were evident. These included increases in the number and perineuronal location of CX3CR1-expressing microglia, decreased levels of motor neuronexpressed fractalkine mRNA, and an alteration in the forms of fractalkine protein expressed. These data describe mechanisms of cellular communication between neurons and microglia, involving fractalkine and CX3CR1, which occur in both normal and pathological states of the central nervous system.Chemokines mediate the directed migration of a variety of leukocyte subsets and consist of at least four subfamilies based principally on the relative position of highly conserved cysteine residues in their amino acid sequences (1, 2). Most chemokine peptides are characterized as secreted proteins of Ϸ7-10 kDa. The recent discovery of a chemokine termed fractalkine has revealed additional distinctive structural features in this gene family. These features include a CX3C motif and a mucin-like stalk that tethers the chemokine domain to transmembrane (TM) spanning and short intracellular domains (3, 4). Evidence from transfected cell systems indicates that fractalkine can exist as membrane-anchored, pro-adhesive, and secreted, chemotactic forms. Furthermore, unlike most chemokine peptides, fractalkine expression is demonstrable in nonhematopoietic tissues including brain, kidney, lung, and heart. In particular, the relatively high levels of fractalkine in the brain raises questions related to the function of chemokines in the central nervous system (CNS).G-protein coupled receptors for chemokine peptides have been characterized extensively in transfected cells and peripheral leukocytes (2). However, very little is known regarding chemokine receptor expression and function in the CNS. Some chemokine receptors, including CCR5, CCR3 (5-7), CXCR4 (7-10), CXCR1, and DARC (11) have been demonstrated to be expressed in either normal brain tissue or cells derived from the...

We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.

We describe a general method for the preparation of cyclic peptides by intramolecular thiazolidine formation from linear, unprotected peptide precursors. The precursors contain a protected 1,2-aminothiol from an N-terminal cysteine and a 1,2-amino alcohol or 1,2-diol as a masked aldehyde. Thiazolidine formation was effected by oxidation of the 1,2-amino alcohol or 1,2-diol by sodium periodate to give an aldehyde, followed by deprotection of the masked 1,2-aminothiol. The cyclization could be effected at concentrations as high as 20 mM and was free from any polymerized side products. Such high efficiency of macrocyclization may be attributed to the ring-chain" tautomerism of the open chain amino-aldehyde precursor that favors a macrocyclic thiazolidine ring. Thiazolidine formation was further exploited as a capture device to position the N and C termini covalently close together and then to allow a proximity-driven O to N acyl transfer through a novel tricyclic ring contraction to yield an all amide, end-to-end cyclic lactam. These macrocyclization methods have been applied to the synthesis of cyclic peptides containing 5 to 26 amino acids.

In this paper we describe “solid phase chemical ligation” (SPCL), the application of the principles of polymer-supported organic synthesis to the construction of large polypeptide chains for the total chemical synthesis of proteins. In this method, each building block used is an unprotected peptide segment of 20 or more amino acids. These are consecutively reacted by chemical ligation, the chemoselective reaction of the unprotected peptide segments from aqueous solution, to make the polymer-supported target polypeptide. In a final step, the assembled full-length target polypeptide is released from the aqueous-compatible polymer support. Here we report chemistries for the attachment of the first segment to a polymer support, and for the assembly of the target polypeptide chain starting from the polymer-bound peptide segment. In this solid phase protein synthesis method, large target polypeptide chains can be built efficiently and rapidly by SPCL and, after release from the polymer support, folded to give functional protein molecules. Several examples of the application of SPCL are given:  model peptides consisting of 27 and 68 amino acids, and polypeptides corresponding to the proteins C5a (74 amino acids) and MIF (115 amino acids), were each made in good yield and purity from the consecutive solid phase ligation of peptide segments. In addition, we report the total synthesis by SPCL of the enzyme “human group V secretory phospholipase A2” (GV-PLA2), which comprises a polypeptide of 118 amino acids containing 6 disulfide bonds. As demonstrated by these examples, SPCL is an important extension of our capabilities for total chemical protein synthesis.