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Abstract
N-oligosaccharides of Saccharomyces cerevisiae glycoproteins are classified as core and mannan types. The former contain 13-14 mannoses whereas mannan-type structures consist of an inner core extended with an outer chain of up to 200-300 mannoses, a process known as hyperglycosylation. The selection of substrates for hyperglycosylation poses a theoretical and practical question. To identify hyperglycosylation determinants, we have analyzed the influence of the second amino acid (Xaa) of the sequon in this process using the major exoglucanase as a model. Our results indicate that negatively charged amino acids inhibit hyperglycosylation, whereas positively charged counterparts promote it. On the basis of the tridimensional structure of Exg1, we propose that Xaa influences the orientation of the inner core making it accessible to mannan polymerase I in the appropriate position for the addition of alpha-1,6-mannoses. The presence of Glu in the Xaa of the second sequon of the native exoglucanase suggests that negative selection may drive evolution of these sites. However, a comparison of invertases secreted by S. cerevisiae and Pichia anomala suggests that hyperglycosylation signals are also subjected to positive selection.
Highlights
Protein glycosylation in eukaryotic cells is thought to play an essential role in many processes such as protein folding and transport, maintenance of protein and cell structure, and cell recognition and adhesion, as well as other functions
Additional studies in vitro again using the rabies virus glycoprotein as a model have indicated that introduction of specific amino acids, such as Trp, Asp, Glu, or Leu, in the X position convert the sequon to a poor oligosaccharide acceptor [18, 19]
Exg1b, contains 12% carbohydrate distributed into two short oligosaccharides, each consisting of a regular inner core whose outer chain is reduced to two or three residues of mannose, indicating that the ␣-1,6-mannose added by Och1 is capped by a stop-signal ␣-1,2-mannose, which may be elongated with a terminal ␣-1,3-mannose [23]
Summary
Exg1b, contains 12% carbohydrate distributed into two short oligosaccharides, each consisting of a regular inner core whose outer chain is reduced to two or three residues of mannose, indicating that the ␣-1,6-mannose added by Och is capped by a stop-signal ␣-1,2-mannose, which may be elongated with a terminal ␣-1,3-mannose [23] These oligosaccharides are attached to both potential glycosylation sites (Asn165-Asn166-Ser167 and Asn325-Glu326-Ser327) present in the polypeptide [17, 24]. In this article we describe the effect of sequon composition, in particular the influence of the second amino acid of the tripeptide sequence (X) in the hyperglycosylation of Exg, and we provide a structure-based hypothesis to explain our results. We have constructed mutated versions of the EXG1 gene in which the two sequons of the protein have been systematically mutated
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