| The life expectancy of humans is continuously rising and consequently ageing-related disorders increasingly challenge the quality of life of the elderly. Ageing is tightly associated with a negative Ca2+ balance leading to among others osteoporosis, arteriosclerosis and ectopic calcification. Recently, klotho (named after the Greek goddess who spins the thread of life) was identified as new ?anti-ageing? hormone being instrumental in the process of age-related adaptations in the Ca2+ balance (1, 2). Klotho is a type I membrane glycoprotein which shares homology to ?-glucosidase enzymes. Interestingly, klotho gene ablation resulted in a syndrome closely resembling human ageing, including short life span, bone aberrations, skin atrophy and a disturbed Ca2+ balance together with high serum vitamin D levels. In addition, polymorphisms in the klotho gene have been linked to reduced bone mineral density in humans. However, the molecular function of klotho and the down-stream targets of this anti-ageing hormone remain to be identified. Recently, we showed that klotho is predominantly expressed in kidney, where it is secreted in the pro-urine (3). Furthermore, we demonstrated that the epithelial Ca2+ channel (TRPV5), which is the gatekeeper in the process of Ca2+ active reabsorption, is stimulated by klotho via a novel extracellular activation mechanism which we recently published in Science (3). We hypothesize that klotho hydrolyses via its ?-glucuronidase activity the extracellular TRPV5 sugar residues, entrapping the channel in the plasma membrane to maintain durable Ca2+ transport activity in kidney. TRPV5 is glycosylated at one aspagarine residue in the first extracellular part of the protein. These results indicate that glycosylation of TRPV5 and possibly other membrane transporters provides an important feature to regulate ion transport across the plasma membrane of cells. Key objectives and experimental approach: The general aim of this project is to delineate the function of klotho in the regulation TRPV5 and the consequences for the Ca2+ balance providing new insights into the molecular mechanism of ageing. To this end the following key objectives will be addressed: i) Mechanism of klotho-mediated hydrolysis of the TRPV5 N-glycosylation. We will characterize the complex structure of the glycosylated sugar residues of TRPV5 and investigate the modification by klotho using Mass Spectrometry (MS), electron capture dissociation (ECD) and infrared multiphoton dissociation (IRMPD) after treatment with specific glucosidases. Using this approach the N-glycan of TRPV5 will be delineated. ii) TRPV5 channel activation by klotho. This key objective will address the specificity of the klotho effect on TRPV5 by using a comprehensive set of glucosidases. In addition, the molecular identity of the klotho-mediated signaling cascade and the effect of klotho on the plasma membrane recycling process of TRPV5 will be studied using a combined biochemical, electrophysiological and cell biological approach including Function Recovery After Chemo¬bleaching (FRAC). In addition, this key objective aims to elucidate the identity of plasma membrane retention proteins that entrap TRPV5 in the membrane upon klotho treatment. iii) Secretion mechanism and hormonal regulation of klotho in the urine. This key objective will address the molecular mechanism of klotho release into the extracellular body fluids. To this end, the cellular release location in the kidney and the protein cleavage site in klotho will be determined. These results will be instrumental to identity the protease involved in klotho secretion. In addition, the effect of calciotropic hormones will be studied on this secretion process. iv) The ?in vivo? function of klotho on renal Ca2+ handling. The mechanism of TRPV5 activation into the pro-urine will be studied in isolated renal tubules and animal models using chemical oligosaccharide engineering. Subsequently, the action of klotho on TRPV5, Ca2+ reabsorption and ultimately the Ca2+ balance will be investigated using biosynthetic intro duction of unnatural sugars into the glycosylation of TRPV5. v) Role of glucosidases in the activation of renal transporters. Here will be addressed the glycosylation of other renal ion transporters and channels, (i.e. Na,Cl-cotrans¬porter (NCC), Na,Cl,K-cotransporter (NKCC2), Mg2+ channels (TRPM6 & TRPM7) and osmosensor TRPV4). Modification of N-glycosylation present on these ion trans¬porters will be studied to invest whether the ion permeability in kidney cells can be modulated. These results will indicate whether this novel concept of regulation is a general activation mechanism or specifically restricted to TRPV5 to maintain the Ca2+ balance by klotho. |
