N-linked glycosylation of proteins is an essential and highly conserved co- and post-translational protein modification reaction that occurs in all eukaryotes. Oligosaccharyltransferase (OST), a multi-subunit membrane-associated enzyme complex, carries out this reaction. In the central reaction, a carbohydrate group is transferred to the side chain of a consensus asparagine residue in the newly synthesized protein. Genetic defects in humans cause a series of disorders known as congenital disorders of glycosylation (CDG) that include mental retardation, developmental delay, hypoglycemia etc. Complete loss of N-glycosylation is lethal in all organisms. In Saccharomyces cerevisiae, OST consists of nine non-identical protein subunits. Ost4p is the smallest subunit containing 36 residues. It bridges catalytic subunit Stt3p to Ost3p/Ost6p subunit. Mutation of Valine (V) at position 23 in Ost4p to Aspartate (D) causes defects in the N-glycosylation process. To understand the structure, function and role of Ost4p in N-glycosylation, characterization of Ost4p and its functionally important mutant/s are critical. We report the mutagenesis, heterologous overexpression, purification, reconstitution in DPC micelles and biophysical characterization of Ost4V23D and compare its secondary structure and conformation to that of Ost4p. CD and NMR data suggest that mutation of Val²³ to Asp impacts the secondary structure and conformation of Ost4p.

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Journal Article

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Chaudhary B, Mazumder S, Mohanty S

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