How Human IgGs Against Myelin Basic Protein (MBP) Recognize Oligopeptides and MBP

text

May 4, 2017

Authors

Sergey Belov¹, Valentina N. Buneva^1,2, and Georgy A. Nevinsky^1,2

Organizations

Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia
Novosibirsk State University, Novosibirsk, Russia

Abstract

Myelin basic protein (MBP) is a major protein of myelin-proteolipid shell of axons, and it plays an important role in pathogenesis of multiple sclerosis. In the literature, there are no data on how antibodies recognize different protein antigens including MBP. A stepwise increase in ligand complexity was used to estimate the relative contributions of virtually every amino acid residue (AA) of a specific 12-mer LSRFSWGAEGQK oligopeptide corresponding to immunodominant sequence of MBP to the light chains and to intact anti-MBP IgGs from sera of patients with multiple sclerosis. It was shown that the minimal ligands of the light chains of IgGs are many different free AAs (Kd = 0.51–0.016 M), and each free AA interacts with the specific subsite of the light chain intended for recognition of this AA in specific LSRFSW oligopeptide. A gradual transition from Leu to LSRFSWGAEGQK leads to an increase in the affinity from 10−1 to 2.3 × 10−4 M because of additive interactions of the light chain with 6 AAs of this oligopeptide and then the affinity reaches plateau. The contributions of 6 various AAs to the affinity of the oligopeptide are different (Kd, M): 0.71 (S), 0.44 (R), 0.14 (F), 0.17 (S), and 0.62 (W). Affinity of nonspecific oligopeptides to the light chains of IgGs is significantly lower. Intact MBP interacts with both light and heavy chains of IgGs demonstrating 192-fold higher affinity than the specific oligopeptide. It is a first quantitative analysis of the mechanism of proteins recognition by antibodies. The thermodynamic model was constructed to describe the interactions of IgGs with MBP. The data obtained can be very useful for understanding how antibodies against many different proteins can recognize these proteins.

CovalX Technology Used (Click each option to learn more)

XL Kit

Outcomes

20 μL of reaction mixture that contained one of specific buffers 25 mM, 1 mg/mL IgGs, 50 μM 1 of 2 fluorescent OP analogs (X-OP5 and X-OP12) was cross-linked. The IgGs with OP analogs were cross-linked using 0,5 mM glutaric dialdehyde 25 mM HEPES-NaOH (pH 8). The Modification of Abs was performed using 0.27 mM CovalX reagent (ethylene glycol bis-succinimidylsuccinate, iodacetic acid N-hydroxysuccinimide ester, and octaneodic acid di-Nhydroxysuccinimide ester) HEPES-NaOh (pH 6.0). The mixtures were incubated at 37 °C for 4 hours. Some samples were incubated without or with 50 mM Dithiothreitol (DTT) at 100 °C. Light and heavy chains of the intact IgG interactions with OPs were performed using SDS-PAGE in 4% to 15% gradient gels under non reducing conditions. The polypeptides were visualized by Coomassie staining and the fluorescence was analyzed using an imaging system.