Effect of sialic acid content on glycoprotein pI analyzed by two‐dimensional electrophoresis

Abstract

2-DE is broadly used for quantitative analysis of differential protein expression in complex mixtures such as serum samples or cell lysates. PTMs directly influence the 2-DE pattern, and knowledge of the rules of protein separation is required in order to understand the protein distribution in a 2-DE gel. Glycosylation is the most common PTM and can modify both the molecular weight and the pI of a protein. In particular, the effect of charged monosaccharides (mainly sialic acids, SAs) on the 2-DE pattern of a protein is of major interest since changes in sialylation are regularly observed in comparative studies. Little is known about the pI shift of a glycoprotein induced by the presence of SAs, or whether this shift is the same for all glycoproteins. To address this issue, this study examined the influence of SA on the 2-DE pattern of three serum glycoproteins (haptoglobin, α1-antitrypsin and ribonuclease 1), which N-glycan chains had been previously characterised, and reviewed existing bibliographic data. The SA content of the different glycoforms of a glycoprotein showed a negative linear correlation with the pI, although the slope varied among the studied glycoproteins. We also described a positive correlation between the protein pI and the pI decrease per SA molecule.

Characterization of transferrin glycopeptide structures in human cerebrospinal fluid

“…Furthermore, Tf glycovariants in Spots 6 and 7 contained high levels of N-acetylhexosamine (HexNAc) residues (up to 7 residues) compared to Tf glycovariants in Spots 1 to 5. This is in agreement with a previous study showing that the number of terminal sialic acid residues contributes to the negative shift in the pI of a glycoprotein in 2-DE analysis [18]. Table 2 summarizes the glycoforms detected in each spot, the approximate protein abundance in each spot and the relative mass spectral intensity ratios when a glycopeptide mixture was detected in a spot.…”

Lack of phosphomannomutase 2 affects Xenopus laevis morphogenesis and the non‐canonical Wnt5a/Ror2 signalling

“…To find out if the Wnt5a/Ror2 signalling (Schambony and Wedlich 2007) as a branch of the β-catenin independent Wnt pathways is affected by the Pmm2 knockdown, we first analysed the glycosylation status of Wnt5a of the Pmm2 morphants and found it to be significantly altered. As Yamamoto and co-workers described recently for MDCK II cells (Yamamoto et al 2015), Wnt5a is glycosylated with two mannose-rich type N-glycans and one hybrid-type oligosaccharide carrying one sialic acid residue, we associate the noticed shifting of Wnt5a-flag proteins towards a more basic isoelectric point (Barrabés et al 2010) in 2D-electrophoresis with the loss of the negatively charged hybrid-type N-glycan provoked by the Pmm2 deficiency. This is further supported by the detected loss of signal strength in the lectin profiling.…”

ADAMTS‐13 glycans and conformation‐dependent activity

“…In the case of the FRETS-VWF73 assay, Muia when used to digest FRETS-VWF73 at pH 7.4, PNG-AD13 had reduced activity as compared with Neu-AD13; however, this was not observed under shear stress, with both Neu-AD13 and PNG-AD13 being equally less active, suggesting that loss of sialic acid was responsible for the reduction in activity. The pH sensitivity is consistent with studies showing that loss of sialic acid can significantly alter the protein pI [31]. Some of these observations are consistent with the study of Zhou et al, in which PNG-AD13 showed normal activity against FL-VWF in the presence of guanidine chloride [22].…”