N-Glycosylation of Asparagine Residues in Subtilisin, Lysozyme, and Synthetic Peptides by Microsomal Transferases

Abstract

The chapter focuses on the structural studies on glycoprotein's containing N -glycosically linked oligosaccharides. A search was initiated to find tripeptide sequence in nonglycosylated form to examine the specificity of the enzyme system involved in the N -glycosylation of proteins. It has been reported that RNase A acts as an acceptor for the transfer of GlcNAc from UDP-GlcNAc in the presence of rabbit liver extracts or human serum. Evidence for the participation of mannose-containing dolichol intermediates in the glycosylation of exogenous proteins has been reported. The optimal conditions for the transfer of GlcNAc were determined by use of RNAase A as the acceptor. It is deduced that both RNAase and denatured subtilisin acted as acceptors for the transfer of GlcNAc to protein, when either NAD+ or GDP-mannose was added to the reaction mixture. These nucleotides partially protect the substrate from enzymatic degradation during long periods of incubation. Most of the activity is associated with a GlcNAc : glycosyltransferase present in the microsomes. A number of different amino acids are found in the variable position of the tripeptide sequence. The chapter illustrates the extent of incorporation of GlcNAc into these peptides in the standard assay system. The present studies using low-molecular-weight peptides strongly suggest that the presence of this tripeptide sequence is sufficient for N -glycosylation and the initiation of oligosaccharide synthesis.