Abstract
Although posttranslational protein modifications are generally thought to perform important cellular functions, recent studies showed that a large fraction of phosphorylation sites are not evolutionarily conserved. Whether the same is true for other protein modifications, such as N-glycosylation is an open question. N-glycosylation is a form of cotranslational and posttranslational modification that occurs by enzymatic addition of a polysaccharide, or glycan, to an asparagine (N) residue of a protein. Examining a large set of experimentally determined mouse N-glycosylation sites, we find that the evolutionary rate of glycosylated asparagines is significantly lower than that of nonglycosylated asparagines of the same proteins. We further confirm that the conservation of glycosylated asparagines is accompanied by the conservation of the canonical motif sequence for glycosylation, suggesting that the above substitution rate difference is related to glycosylation. Interestingly, when solvent accessibility is considered, the substitution rate disparity between glycosylated and nonglycosylated asparagines is highly significant at solvent accessible sites but not at solvent inaccessible sites. Thus, although the solvent inaccessible glycosylation sites were experimentally identified, they are unlikely to be genuine or physiologically important. For solvent accessible asparagines, our analysis reveals a widespread and strong functional constraint on glycosylation, unlike what has been observed for phosphorylation sites in most studies, including our own analysis. Because the majority of N-glycosylation occurs at solvent accessible sites, our results show an overall functional importance for N-glycosylation.