Naslov
THE DISULPHIDE 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, znanstveni

Izvornik
Book of Abstracts of the 3rd Congress of Croatian Geneticists with international participation / Jasna Franekić, Verica Garaj-Vrhovac - , 2012

Skup
3rd Congress of Croatian Geneticists with international participation

Mjesto i datum
Krk, Hrvatska, 13.05.2012. - 16.05.2012

Vrsta sudjelovanja
Predavanje

Vrsta recenzije
Nije recenziran

Ključne riječi
adenovirus retargeting; disulphide bonds; RGD4C
(adenovirus; retargeting; disulphide bonds; RGD4C)

Sažetak
The αv-integrin binding motif RGD4C (CDCRGDCFC) has been used extensively to circumvent inefficient adenovirus type 5 (Ad5) transduction of cells expressing low levels of the coxsackie B and adenovirus receptor (CAR), its primary receptor. However, 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. Finally, to determine the number and position of disulphide 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 disulphide bond in the C547DC549RGDC553FC555 retargeting motif, that is crucial for Ad5RGD4C retargeting to αv-integrins.

Izvorni jezik
Engleski

Znanstvena područja
Biologija

POVEZANOST RADA