title: Glycosylation and specific deamidation of ribonuclease B affect the formation of three-dimensional domain-swapped oligomers.
abstract: RNase A oligomerizes via the three-dimensional domain-swapping mechanism to form a variety of oligomers, including two dimers. One, called the N-dimer, forms by swapping of the N termini of the protein; the other, called the C-dimer, forms by swapping of the C termini. RNase B is identical in protein sequence and conformation to RNase A, but its Asn34 bears an oligosaccharide chain that might affect oligomerization. The ability of RNase B to oligomerize under two sets of conditions has been examined. The amount of oligomers formed via lyophilization was somewhat lower for RNase B than RNase A, and RNase B oligomerized more rapidly in 40% ethanol solution at high temperature than RNase A. The ratio of the N-dimer to C-dimer formed increased with the size of the carbohydrate chain under both sets of conditions. These results suggest that the oligosaccharide chain either favors productive collisions or stabilizes the oligomers, especially the N-dimer. Endoglycosidase H treatment of RNase B partially restored RNase A-like oligomerization. Derivatives of RNase A conjugated at the amine groups to polyethylene glycol chains showed a greatly reduced capacity for oligomerization, suggesting that oligomerization can be impeded sterically. Commercial preparations of RNase B eluted as two main peaks by cation exchange chromatography. Using chromatography, mass spectroscopy, and two-dimensional NMR, the major peak was identified as RNase B selectively deamidated at Asn67. This deamidated protein showed a >4 degrees C drop in thermal stability, disruption of the native structure of residues 67-69, and a decreased ability to oligomerize compared with unmodified RNase B.
authors.affiliation: Dipartimento di Scienze Neurologiche e della Visione, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università di Verona, Strada Le Grazie 8, I-37134 Verona, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Gotte
authors.forename: Massimo
authors.initials: M
authors.lastname: Libonati
authors.forename: Douglas V
authors.initials: DV
authors.lastname: Laurents
mesh_terms: D000577:Amides; D002852:Chromatography, Ion Exchange; D019281:Dimerization; D005612:Freeze Drying; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000494:pharmacology; D006031:Glycosylation; D017434:Protein Structure, Tertiary; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000577:Amides; D005656:Fungal Proteins; D012260:Ribonucleases; C029600:ribonuclease B; D012259:Ribonuclease, Pancreatic; D006026:Glycoside Hydrolases