CCR5 tyrosine sulfation heterogeneity generates cell surface receptor subpopulations with different ligand binding properties.

BACKGROUND: Chemokine receptor tyrosine sulfation plays a key role in the binding of chemokines. It has been suggested that receptor sulfation is heterogeneous, but no experimental evidence has been provided so far. The potent anti-HIV chemokine analog 5P12-RANTES has been proposed to owe its inhibitory activity to a capacity to bind a larger pool of cell surface CCR5 receptors than native chemokines such as CCL5, but the molecular details underlying this phenomenon have not been elucidated. METHODS: We investigated the CCR5 sulfation heterogeneity and the sensitivity of CCR5 ligands to receptor sulfation by performing ELISA assays on synthetic N-terminal sulfopeptides and by performing binding assays on CCR5-expressing cells under conditions that modulate CCR5 sulfation levels. RESULTS: Two commonly used anti-CCR5 monoclonal antibodies with epitopes in the sulfated N-terminal domain of CCR5 show contrasting binding profiles on CCR5 sulfopeptides, incomplete competition with each other for cell surface CCR5, and opposing sensitivities to cellular treatments that affect CCR5 sulfation levels. 5P12-RANTES is less sensitive than native CCL5 to conditions that affect cellular CCR5 sulfation. CONCLUSIONS: CCR5 sulfation is heterogeneous and this affects the binding properties of both native chemokines and antibodies. Enhanced capacity to bind to CCR5 is a component of the inhibitory mechanism of 5P12-RANTES. GENERAL SIGNIFICANCE: We provide the first experimental evidence for sulfation heterogeneity of chemokine receptors and its impact on ligand binding, a phenomenon that is important both for the understanding of chemokine cell biology and for the development of drugs that target chemokine receptors.

Re-engineering the µ-conotoxin SIIIA scaffold.

Voltage-gated sodium (Nav) channels are responsible for generation and propagation of action potentials throughout the nervous system. Their malfunction causes several disorders and chronic conditions including neuropathic pain. Potent subtype specific ligands are essential for deciphering the molecular mechanisms of Nav channel function and development of effective therapeutics. µ-Conotoxin SIIIA is a potent mammalian Nav 1.2 channel blocker that exhibits analgesic activity in rodents. We undertook to reengineer loop 1 through a strategy involving charge alterations and truncations which led to the development of µ-SIIIA mimetics with novel selectivity profiles. A novel [N5K/D15A]SIIIA(3-20) mutant with enhanced net positive charge showed a dramatic increase in its Nav 1.2 potency (IC50 of 0.5 nM vs. 9.6 nM for native SIIIA) though further truncations led to loss of potency. Unexpectedly, it appears that SIIIA loop 1 significantly influences its Nav channel interactions despite loop 2 and 3 residues constituting the pharmacophore. This minimal functional conotoxin scaffold may allow further development of selective NaV blockers.

Emerging trends in genomic approaches for microbial bioprospecting.

Microorganisms constitute two out of the three domains of life on earth. They exhibit vast biodiversity and metabolic versatility. This enables the microorganisms to inhabit and thrive in even the most extreme environmental conditions, making them all pervading. The magnitude of biodiversity observed among microorganisms substantially supersedes that exhibited by the eukaryotes. These characteristics make the microbial world a very lucrative and inexhaustible resource for prospecting novel bioactive molecules. Despite their vast potential, over 99% of the microbial world still remains to be explored. The primary reason for this is that the culture-dependent methods used in the laboratories are grossly insufficient, as they support the growth of under 1% of the microorganisms found in nature. This limitation necessitated the development of techniques to circumvent culture dependency and gain access to the outstanding majority of the microorganisms. The development of culture-independent techniques has essentially reshaped the study of microbial diversity and community dynamics. Application of genomic and metagenomic approaches is contributing substantially towards characterization of the real microbial diversity. The amenability of these techniques to high throughput has opened the doors to explore the vast number of "uncultivable" microbial forms in substantially lesser time. The present article provides an update on the recent technological advances and emerging trends in exploring microbial community.