A not-glycosylated isoform of γ-conglutin, a hexameric glycoprotein of Lupinus albus seed, participates in the oligomerization equilibrium.

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have 'escaped' the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.

Direct Assessment of Oligomerization of Chemically Modified Peptides and Proteins in Formulations using DLS and DOSY-NMR.

PURPOSE: Protein higher order structure (HOS) including the oligomer distribution can be critical for efficacy, safety and stability of drug products (DP). Oligomerization is particularly relevant to chemically modified protein therapeutics that have an extended pharmacokinetics profile. Therefore, the direct assessment of protein oligomerization in drug formulation is desired for quality assurance and control. METHODS: Here, two non-invasive methods, dynamic light scattering (DLS) and diffusion ordered spectroscopy (DOSY) NMR, were applied to measure translational diffusion coefficients (Ddls and Dnmr) of proteins in formulated drug products. The hydrodynamic molecular weights (MWhd), similar to hydrodynamic size, of protein therapeutics were derived based on a log(Ddls) vs log(MWhd) correlation model established using protein standards. RESULTS: An exponent value of -0.40 ± 0.01 was established for DLS measured log(D) vs. log(MWhd) using protein standards and a theoretical exponent value of -0.6 was used for unstructured polyethylene glycol (PEG) chains. The analysis of DLS derived MWhd of the primary species showed the fatty acid linked glucagon-like peptide 1 (GLP-1) was in different oligomer states, but the fatty acid linked insulin and PEG linked proteins were in monomer states. Nevertheless, equilibrium and exchange between oligomers in formulations were universal and clearly evidenced from DOSY-NMR for all drugs except peginterferon alfa-2a. CONCLUSION: The correlation models of log(D) vs. log(MWhd) could be a quick and efficient way to predict MWhd of protein, which directly informs on the state of protein folding and oligomerization in formulation.