ABSTRACT
Four cyclic pentapeptides and two cyclic heptapeptides modelled on the 3(10) helical Pro138-Gly144 segment of the water channel aquaporin-4 (AQP4) postulated to mediate adhesive interactions between AQP4 tetramers were synthesised by olefin metathesis. Three related acyclic pentapeptides Boc-Ser(All)-Xaa1-Val-Ser(All)-Gly-OMe (Xaa1 = Val, Aib; Boc = tert-butoxycarbonyl; All = allyl) and Boc-Ser(Bn)-Val-Val-Gly-Gly-OMe (Bn = benzyl) and two acyclic heptapeptides Boc-Pro-Pro-Ser(All)-Val-Val-Ser(All)-Gly-OMe and Boc-Pro-Pro-Ser(Bn)-Val-Val-Gly-Gly-OMe were also prepared. NMR, CD and IR data provided evidence that the peptides can access a 3(10) helical structure in apolar solvents and pointed to a significant stabilising effect of the olefinic bridge on helicity in an aqueous environment. Thus we could demonstrate the viability of using ring closing olefin metathesis to stabilise short protein segments in the helical conformation that they adopt in their native protein environment. Our approach provides access to a set of peptides with potential binding affinity for AQP4.
Subject(s)
Alkenes/chemical synthesis , Aquaporin 4/chemical synthesis , Glycine/chemical synthesis , Peptides, Cyclic/chemical synthesis , Proline/chemical synthesis , Alkenes/chemistry , Aquaporin 4/chemistry , Circular Dichroism , Glycine/chemistry , Humans , Nuclear Magnetic Resonance, Biomolecular , Peptides, Cyclic/chemistry , Proline/chemistry , Protein Stability , Protein Structure, Secondary , Spectrophotometry, InfraredABSTRACT
Aquaporin-4 (AQP4) is a brain aquaporin implicated in the pathophysiology of numerous clinical conditions including brain edema. Here we show that rat AQP4 has six cDNA isoforms, formed by alternative splicing. These are named AQP4a-f, where AQP4a and AQP4c correspond to the two classical M1 and M23 isoforms, respectively. The various isoforms are differentially expressed in kidney and brain, and their prevalence does not correspond to the level of the respective mRNAs, pointing to posttranscriptional regulation. The three isoforms lacking exon 2, AQP4b, AQP4d, and AQP4f, have an intracellular localization when expressed in cell lines and do not transport water when expressed in Xenopus oocytes. In contrast, the largest of the new isoforms, AQP4e, which contains a novel N-terminal domain, is localized at the plasma membrane in cell lines and functions as a water transporter in Xenopus oocytes.