Naslov
The disulfide bond of an RGD4C motif inserted within the HI loop of adenovirus type 5 fiber protein is critical for retargeting to αv-integrins

Autori
Majhen, Dragomira ; Richardson, Jennifer ; Vukelić, Bojana ; Dodig, Ivana ; Cindrić, Mario ; Benihoud, Karim ; Ambriović-Ristov, Andreja

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, ostalo

Izvornik
5th Croatian Congress of Microbiology / Černi, Silvija ; Šeruga Musić, Martina ; Škorić, Dijana - Zagreb : Hrvatsko mikrobiološko društvo, 2012, 35-35

ISBN
978-953-778-05-7

Skup
5th Croatian Congress of Microbiology with International Participation

Mjesto i datum
Primošten, Hrvatska, 26.09.2012. - 30.09.2012

Vrsta sudjelovanja
Poster

Vrsta recenzije
Nije recenziran

Ključne riječi
αv integrins; coxsackie and adenovirus receptor; adenovirus type 5; disulfide bond; RGD4C targeting motif

Sažetak
Human adenovirus type 5 (Ad5) is an attractive gene delivery vector for cancer gene therapy. Ad5 binds to coxsackievirus B and adenovirus receptor (CAR) and internalizes upon interaction between the conserved arginine-glycine-aspartate (RGD) sequence of the penton base proteins and αv-integrins. To overcome low or lack of expression of CAR in tumor tissues and cell lines, a strategy has been implemented based on tropism modification by genetic incorporation of a targeting ligand into the HI loop of Ad5 fiber protein. While several retargeting motifs have been evaluated as insertions in the HI loop, peptide RGD4C (CDCRGDCFC) originally isolated from a phage-displayed peptide library has been used most extensively. The reason for using cysteines in RGD4C is that the cysteines located in both flanks are expected to form disulfide bonds and create a stem-like structure with the targeting peptide in a center. Up until now it has not been clear whether cysteines in the RGD4C motif influence the retargeting potential of RGD4C-retargeted Ad5. In a panel of cancer cell lines we measured transduction efficacy of replication deficient Ad5 bearing wild type fiber (Ad5wt) or fibers bearing RGD4C or RGD4G inserts within the HI loop (Ad5RGD4C or Ad5RGD4G). The increment in transduction efficacy with Ad5RGD4C was highest in the CAR-negative cell lines while the transduction efficacy of Ad5RGD4G was only slightly increased in comparison to Ad5wt. The increased Ad5RGD4C transduction efficiency was abolished when transduction was performed in the presence of function-blocking anti-integrin αvβ3 or αvβ5 antibodies. Treatment with reducing agent DTT prior to measuring cell transduction, had no effect on cell transduction by wild type Ad5wt and Ad5RGD4G. By contrast, DTT treatment abolished the increased transduction efficacy of Ad5RGD4C in all analyzed cell lines in a dose-dependent manner. To determine the number and position of disulfide bond(s) within the RGD4C sequence inserted in the HI-loop of Ad5 fiber protein, we performed mass spectrometry analysis of fiber protein isolated from purified Ad5RGD4C. We showed that, within the HI-loop of the Ad5 fiber protein, cysteines C547 and C549 form a single disulfide bond in the C547DC549RGDC553FC555 retargeting motif, that is crucial for Ad5RGD4C retargeting to αv-integrins. This is an unexpected finding proving that there is just one disulfide bond within the RGD4C and that it does not link cysteine residues in opposing flanks.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

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