The addition of polysialic acid to N- and/or O-linked glycans, referred to as polysialylation, is a rare posttranslational modification mainly known to control developmental plasticity of the nervous system. Here we show that CCR7, the central chemokine receptor controlling immune cell trafficking to secondary lymphatic organs, carries polysialic acid. This modification is essential for recognition of the CCR7 ligand CCL21. As a consequence, dendritic cell trafficking is abrogated in polysialyltransferase deficient mice, manifesting in disturbed lymph node homeostasis and unresponsiveness to inflammatory stimuli. Structure-function analysis of chemokine-receptor interactions reveals that CCL21 adopts an autoinhibited conformation, which is released upon interaction with polysialic acid. Thus, we describe a glycosylation-mediated immune cell trafficking disorder and its mechanistic basis.
CCL19 and CCL21 are chemokines involved in the trafficking of immune cells, particularly within the lymphatic system, through activation of CCR7. Concurrent expression of PSGL-1 and CCR7 in naive T-cells enhances recruitment of these cells to secondary lymphoid organs by CCL19 and CCL21. Here the solution structure of CCL19 is reported. It contains a canonical chemokine domain. Chemical shift mapping shows the N-termini of PSGL-1 and CCR7 have overlapping binding sites for CCL19 and binding is competitive. Implications for the mechanism of PSGL-1’s enhancement of resting T-cell recruitment are discussed.
In Neurospora the initial enzymes of pyrimidine synthesis, pyrimidine-specific carbamyl phosphate synthetase and aspartate transcarbamylase, are specified by the same locus, pyr-3. A purification technique devised for the most labile of these two enzymes, the synthetase, resulted in the copurification of aspartate transcarbamylase. The two activities remained associated throughout (NH&S04 precipitation, calcium phosphate gel adsorption, gel filtration chromatography, DEAE-cellulose chromatography, and sucrose density gradient centrifugation. An enzyme preparation estimated to be 50 pure was obtained containing pyrimidinespecific carbamyl phosphate synthetase and aspartate transcarbamylase at specific activities 50 and 36 times that found in the original derepressed extract (250 and 180 times that found in wild type crude extracts). The molecular weight of the enzyme complex was estimated to be 650,000 g per mole on the basis of its elution pattern from agaiose gel N eurospora has been shown to possess two enzymes which form carbamyl phosphate, an intermediate of both arginine and pyrimidine synthesis (Williams and Davis, 1970;. The two carbamyl phosphate synthetases are under control of separate genetic loci and can be clearly distinguished on the basis of molecular weight, feedback inhibitors, derepression conditions, and stabilization factors. Irt vivo one enzyme produces carbamyl phosphate specifically for use in the arginine pathway while the second synthesizes carbamyl phosphate specifically for utilization via the pyrimidine pathway (Davis, 1967). The syntheses of CPSp,,l and ATC (carbamoy1phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2.) are coderepressed by pyrimidine starvation. The CPSp,, activity can be completely inhibited by 5-10 X lou4 M UTP (uridine triphosphate). ATC activity is not inhibited by UTP. CPSp,, is cold labile and has much greater affinity for L-glutamine than for ammonia as a nitrogen source (Williams and Davis, 1970). . To whom reprint requests should be directed.Abbreviations used are: CPSpyr, pyrimidine-specific carbamyl phosphate synthetase; ATC, aspartate transcarbamylase.GM 19703-02). filtration columns and its sedimentation properties in sucrose density gradients. The synthetase activity of the purified complex is completely inhibited by the end product, UTP (uridine triphosphate), at 1 x 10-3 M. On sucrose density gradients the complex sediments at a value of 21 S in the absence of UTP and at 15 S in the presence of UTP. This suggests the dissociation of enzyme subunits in the presence of the feedback inhibitor; however no molecular weight change is observed, in the presence of UTP, in agarose gel filtration. Earlier studies from this laboratory have shown that under normal in viuo conditions carbamyl phosphate produced by the pyrimidine-enzyme complex is unavailable as a substrate for ornithine transcarbamylase in the arginine synthetic pathway. The observed confinement of carbamyl phosphate in pyrimidine synthesis could be explained if carbamyl phosphate existed as an...
RNA interference technology, silencing targeted genes in mammalian cells, has become a powerful tool for studying gene function. For the first time in cancer research, we show that direct injection of a pool of naked small interfering RNA (siRNA) duplexes can prevent tumorigenesis in an animal model, suggesting a novel preventive and therapeutic strategy for cancer management. As a model system, we used siRNA duplexes of CXCR4 to block breast cancer metastasis. Here, we show that blocking CXCR4 expression at the mRNA level by a combination of two siRNAs impairs invasion of breast cancer cells in Matrigel invasion assay and inhibits breast cancer metastasis in an animal model. Targeting more than one site of the target gene may be important to overcome the functional redundancy of other variants of a single gene, especially in in vivo experiments. Moreover, our studies confirm the necessity of CXCR4 in breast cancer metastasis.
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