N-glycosylation triggered a dual evolutionary pressure in eukaryotic secretory pathway proteins

LOPEZ MEDUS M.; COUTO, PM; GÓMEZ G.E; CORTI BIELSA R; ZACCHI L; CARAMELO, J.J.

Vermont

Congreso; FASEB Summer Research Conference "Protein Folding in the Cell"; 2016

FASEB

Nearly one third of the eukaryotic proteins belong to the secretory pathway. These proteins fold in the endoplasmic reticulum before continuing their journey. In addition, in the ER lumen proteins are N-glycosylated. This is one of the most abundant and drastic protein modifications in which a high mannose oligosaccharide is attached to the lateral chain of Asn residues within the context Asn-XX-Ser/Thr (XX cannot be Pro), a motif known as N-glycosylation ?sequon?. The transfer of N-glycan takes place either co- and post-translocationaly, and in most cases it happens before the folding process is completed. The presence of a sequon is a condition necessary but no sufficient for the N-glycosylation. It is estimated that about 20 % of all potential sequons remain vacant. In addition, in non-secretory pathway proteins protein near 18 % of Asn displayed in such context are placed in structurally buried positions. Consequently, there are two possible scenarios during the evolution of secretory pathway proteins: (1) the proportion of sequons in buried positions is similar to that of the non-secretory pathway protein, is this situation these sequons should remain vacant, or (2) buried sequons were deleted from secretory pathway proteins. To address this issue we classified the PDB according to the cellular location and we performed a residue surface exposition analysis. Results show that the second scenario was by far the most favored, being the absence of buried sequons in secretory pathway proteins their most distinctive feature at the level of residue exposition. In addition, sequons were also deleted from most protein-protein interfaces. As a proof of concept, we studied the effect of introducing sequons in BiP, the HSP70 ER homologue. While all cytosolic and mitochondrial HSP70s display several sequons, those motifs are completely absent in BiP. A complementation assay in yeast reveals that BiPs displaying sequons that are conserved in its cytosolic counterpart are non-functional. In summary, eukaryotic secretory pathway proteins suffered a dual evolutionary pressure with respect of N-glycosylation. On one hand there was a pressure that increased the sequons density on exposed positions while in parallel they were deleted from structurally buried sites.