Abstract
To gain an understanding of why the polymannose-type oligosaccharide chain of bovine pancreatic ribonuclease B is not processed to a complex-type chain in vivo, the processing of this glycoprotein was studied in two cell-free systems. Addition of native 125I-ribonuclease B to rat liver Golgi membranes in the presence of UDP-GlcNAc resulted in the conversion of the high mannose chain to a complex type as evidenced by the acquisition of resistance to digestion with endoglucosaminidase H. Processing was linearly dependent on time and on the amount of Golgi membranes. Omission of UDP-GlcNAc from the reaction mixtures completely abolished processing of the glycoprotein. Product identification studies confirmed that the formation of ribonuclease that was resistant to digestion with endoglucosaminidase H was accompanied by the appearance of a complex-type oligosaccharide that contained one or more terminal beta-GlcNAc residues. In vitro processing of 125I-ribonuclease B that had been denatured by reduction and alkylation revealed that the rate of complex chain formation was only slightly greater than that observed with the native enzyme. In contrast to the results obtained with the heterologous rat liver system, Golgi membranes from bovine pancreas failed to process native ribonuclease B to the complex form. However, bovine pancreas Golgi membranes did readily process the denatured form of the enzyme. The presence of a factor in bovine pancreas that binds only to native ribonuclease B and thereby prevents its oligosaccharide chain from being processed was considered to be unlikely on the basis of gel filtration studies and mixing experiments. These findings indicate that some aspect of the conformation of native ribonuclease B prevents one or more of the processing enzymes of bovine pancreas from acting on the oligosaccharide chain. In addition, the substrate specificity of this processing enzyme(s) differs markedly from its counterpart in rat liver. These two factors, conformation of the substrate and specificity of the processing enzymes, apparently combine to produce the high mannose oligosaccharide chain of ribonuclease B observed in vivo.
Highlights
To gain an understanding of why the polymannose- duce the high mannose oligosaccharidechain of ribontype oligosaccharide chain of bovine pancreatic ribon- uclease B observed in vivo
Many glycoproteinspossessoligosaccharideswhich are elease B to rat liver Golgi membranes in the presence linked to asparagine residues by an N-glycosidic bond.These ipf UDP-GlcNAc resulted in theconversion of the high oligosaccharides have been broadly classifiedinto two groups mannose chain to a complex type as evidenced by the designated as high mannose and complex onthe basis of their acquisition of resistance todigestion with endoglucos- component sugars
Product iden- a series of processing reactions lead to theformation of either tification studies confirmed that the formation of ri- high mannose or complex chains
Summary
[10] used endo H’ as a probe to determine the ings indicate that some aspect of the conformation of relative accessibility of the oligosaccharides of Sindbis virus native ribonuclease B prevents one or more of the E, and Ez glycoproteins They showed that oligosaccharides processing enzymes of bovine pancreas from acting on which were destined to become complex were accessible to the oligosaccharide chain. The substrate the probe whilethose destined to remain in the high mannose specificity of this processing enzyme(s) differs mark- form were relatively resistant to digestion They concluded edly from its counterpart in rat liver.
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