Deglycosylation analysis of LASV Z+GPC+NP VLP. Non-denatured LASV Z+GPC+NP VLP were subjected to deglycosylation with PNGase F (4A - D, lane 2), Endo H (4A - D, lane 3), neuraminidase (4A - D, lane 4), or were left untreated (4A - D, lane 1), followed by SDS-PAGE and western blot analyses. Blots were probed with α-GP1 (4A), α-GP2 (4B), α-6X-HIS (Z) (4D) mAbs, or α-NP PAb (4C). PNGase F completely deglycosylated both GP1 and GP2 (4A, 4B, lane 2, respectively), resulting in a mobility shift of both proteins corresponding to their unprocessed polypeptide backbone molecular weights, of 20 kDa and 23 kDa, respectively. Conversely, Endo H showed little affect of GP1 (4A, lane 3) but significantly deglycosylated GP2, generating a relatively uniform, partially glycosylated species of ~ 30 kDa (4B, lane 3). Following Endo H digestion, which cleaves high mannose and some hybrid oligosaccharides from the backbone of N-linked glycoproteins, ~ 7 kDa of the GP2 mass remains inaccessible to this enzyme. Similar results were obtained when pre-denatured VLP were used as input in the reaction. Neuraminidase had no affect on the glycosylation profile of GP1, GP2, or GPC (4A, 4B, lane 4). None of the deglycosidases affected the mobility of NP (4C, lanes 2 - 4) or Z (4D, lanes 2 - 4) proteins. Protein molecular weights in kDa are noted to the right of each blot.
Branco et al. Virology Journal 2010 7:279 doi:10.1186/1743-422X-7-279