Lianglu Wan

Date of Award


Degree Type


Degree Name

Doctor of Philosophy


The cationic (C.PRX) and the anionic (A.PRX) peroxidase isozymes secreted into the medium of cell cultures are both 20% glycosylated proteins. They are stable and occur in high amounts (C.PRX, 5 mg and A.PRX 0.5 mg per litre of spent culture medium), and so provide an ideal system for the study of the effect of glycosylation on the structure and function of glycoproteins.;A novel method to detect glycoproteins and glycopeptides is proposed using periodic-acid Schiff's reagent dot-blotting assay on nitrocellulose membrane. At least 0.1 ug of glycan can be detected within 40 minutes.;The amino acid sequence of C.PRX is confirmed to be the same as the base sequence of cDNA clone prxPNC1 by the amino acid sequencing of the smaller formic acid cleaved peptide fragment. Out of five potential N-glycosylation sites only three, Asn{dollar}\sb{lcub}60{rcub}{dollar}, Asn{dollar}\sb{lcub}144{rcub}{dollar} and Asn{dollar}\sb{lcub}185{rcub}{dollar} are used. Both Asn{dollar}\sb{lcub}209{rcub}{dollar} and Asn{dollar}\sb{lcub}275{rcub}{dollar} have a proline residue at the C-terminal of the consensus sequence and are not glycosylated. Computer modelling showed that Asn{dollar}\sb{lcub}60{rcub}{dollar} and Asn{dollar}\sb{lcub}185{rcub}{dollar} are located in hydrophilic {dollar}\beta{dollar}-turns, while Asn{dollar}\sb{lcub}144{rcub}{dollar} in a hydrophobic {dollar}\beta{dollar}-sheet. The glycosylated asparagines present a preference for {dollar}\beta{dollar}-turns.;On the basis of Concanavalin A binding, C.PRX is fractionated into two forms, either binding, (CP+), or not binding, (CP{dollar}-{dollar}), to Concanavalin A column. Further investigation showed that CP{dollar}-{dollar} and CP+ have the same peptide chain and differ in carbohydrate moiety. All the three glycans of CP{dollar}-{dollar} and CP+ probably have the same core structure but different lengths and terminal sugars. This microheterogeneity of glycans is caused by a co-secreted {dollar}\beta{dollar}-galactosidase, which has two isozymes with mass 60 kd, pl 7.3 and mass 66 kd, pl 7.6 individually.;The antisera against TPCK-tryptic glycopeptides of C.PRX and A.PRX were raised and the antibodies directed to glycans were purified. The epitope mapping showed that the immunogenicity of the three glycans of C.PRX is similar and is mainly directed towards the core structure (Xyl) (Man){dollar}\sb3{dollar}(Fuc) (GlcNAc){dollar}\sb2{dollar}. The carbohydrate moiety of many plant proteins may be the basis of often observed antigenic cross reaction among different glycoproteins that are distinct in both function and structure of peptide moiety.;PNGase eliminates C.PRX enzyme activity and this reaction is dose- and time-dependent. Antibodies directed towards glycans of C.PRX or A.PRX do not block enzyme activity, but the antibodies against whole C.PRX or A.PRX inhibit enzyme activity by 80% and 30% respectively. Glycans are probably not directly involved in the enzyme active centre but play a role in maintaining the conformation of the peptide. Removal of glycans may cause a change of the 3-D structure of the peptide and the loss of calcium and heme.



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