Generation and evaluation of glycosylation and RNase mutants of recombinant omega-1. (A) The amino acid sequence of omega-1 (NCBI Protein database accession no. ABB73003.1) is shown in which the mutation sites are depicted. The two conserved amino acid sequence (CAS) domains essential for catalytic activity are marked in gray, and the two N-linked glycosylation sites are boxed. (B) RNA from PBMCs was incubated for 1 h with the different omega-1 variants (500 and 100ng/ml) and analyzed on a 2% agarose gel for breakdown. RNase A was used as a positive control. One of two experiments is shown. (C) The omega-1 mutants were run under nonreducing conditions by SDS-PAGE and silver stained. A Western blot by staining with a specific anti–omega-1 monoclonal antibody was in line with the absence of glycosylation only on the omega-1 glycosylation mutant. (D) MALDI-TOF mass spectrum of glycopeptides from a tryptic digest of recombinant WT omega-1, covering the glycosylation site N176. Recombinant omega-1 was subjected to SDS-PAGE under reducing conditions and stained with Colloidal blue. Stained bands were excised, subjected to reduction and alkylation, and digested with trypsin. The MALDI-TOF-MS spectrum derived from the upper band in the SDS-PAGE pattern is depicted. Signals ([M⁺H⁺]) are labeled with monoisotopic masses. Composition of the glycan moieties are given in terms of hexose (H), N-acetylhexosamine (N), and fucose (F). Differences in fucose content are indicated by double-headed arrows. Signals that cannot be assigned to glycopeptides are marked with asterisks.