title: The role of the glycan moiety on the structure-function relationships of PD-L1, type 1 ribosome-inactivating protein from P. dioica leaves.
abstract: N-glycosylation is one of the major naturally occurring covalent co-translational modifications of proteins in plants, being involved in proteins structure, folding, stability and biological activity. In the present work the influence of carbohydrate moieties on the structure-function relationships of type 1 ribosome-inactivating proteins (RIPs) was investigated. To this aim, PD-Ls, RIPs isolated from Phytolacca dioica L. leaves, differing for their glycosylation degree, were used as an experimental system. In particular, comparative structural and biological analyses were performed using native and unglycosylated recombinant PD-L1, the most glycosylated P. dioica RIP isoform. The glycans influence on protein synthesis inhibition and adenine polynucleotide glycosidase activity was investigated. The interaction with adenine, the product of the de-adenylation reaction, was also investigated for native and recombinant PD-L1 by fluorescence spectroscopy. Furthermore, the crystal structure of PD-L1 in complex with adenine was determined. Our data confirm that the absence of glycan moieties did not affect the biological activity in terms of protein synthesis inhibition. However, the removal of carbohydrate chains significantly increased the deadenylation capability, likely as a consequence of the increased accessibility of substrates to the active site pocket. Furthermore, preliminary data on cellular uptake showed that all PD-L isoforms were internalized and, for the first time, that the vesicular distribution within cells could be influenced by the protein glycosylation degree.
authors.affiliation: Dipartimento di Scienze della Vita, Seconda Università di Napoli, Via Vivaldi 43, I-81100 Caserta, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: Severino
authors.forename: Angela
authors.initials: A
authors.lastname: Chambery
authors.forename: Antimo
authors.initials: A
authors.lastname: Di Maro
authors.forename: Daniela
authors.initials: D
authors.lastname: Marasco
authors.forename: Alessia
authors.initials: A
authors.lastname: Ruggiero
authors.forename: Rita
authors.initials: R
authors.lastname: Berisio
authors.forename: Francesco
authors.initials: F
authors.lastname: Giansanti
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ippoliti
authors.forename: Augusto
authors.initials: A
authors.lastname: Parente
mesh_terms: D000225:Adenine / Q000378:metabolism; D000818:Animals; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D005813:Genes, Synthetic; D006031:Glycosylation; D013058:Mass Spectrometry; D051379:Mice; D008958:Models, Molecular; D041681:NIH 3T3 Cells; D029603:Phytolacca / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry / Q000378:metabolism*; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013696:Temperature
chemical_list: D010940:Plant Proteins; D011134:Polysaccharides; D020033:Protein Isoforms; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D000225:Adenine