Cleavage of rabbit myelin basic protein by thrombin

Area of Science:

• Biochemistry
• Proteomics
• Enzymology

Background:

• Myelin basic protein (BP) is a key component of the myelin sheath.
• Understanding protein cleavage sites is crucial for studying protein function and degradation.
• Thrombin is a serine protease with known cleavage specificities.

Purpose of the Study:

• To investigate the cleavage sites of rabbit myelin basic protein (BP) by thrombin.
• To determine how varying reaction conditions (enzyme-substrate ratio, substrate concentration, incubation time) affect thrombin's cleavage specificity on BP.
• To correlate cleavage patterns with the known enzymatic specificity of thrombin.

Main Methods:

• Incubation of rabbit myelin basic protein (BP) with thrombin under three distinct sets of conditions.
• Quantitative analysis of cleavage products to identify specific cleavage sites (Arg-X bonds).
• Comparison of cleavage patterns across different species and within specific peptide fragments.

Main Results:

• Under low enzyme/substrate ratio and short incubation, thrombin specifically cleaved rabbit BP at the Arg(95)-Thr(96) bond, a conserved site across species.
• Increased enzyme/substrate ratio, substrate concentration, and incubation time led to additional cleavages at Arg(43)-Phe(44), Arg(128)-Ala(129), Arg(31)-His(32), and Arg(63)-Thr(64) bonds.
• Detailed analysis of peptide (96-168) revealed cleavage at Arg(128)-Ala(129) and partial cleavage at unmethylated Arg(105)-Gly(106), Arg(111)-Phe(112), Arg(150)-Leu(151), and Arg(160)-Ser(161) bonds.
• Cleavage at Arg(128)-Ala(129) and Arg(160)-Ser(161) suggests potential beta-turns preceding these sites.

Conclusions:

• Thrombin's cleavage of myelin basic protein (BP) is highly dependent on reaction conditions.
• The Arg(95)-Thr(96) bond represents a primary, highly specific cleavage site for thrombin in BP.
• Cleavage patterns provide insights into protein structure, including potential secondary structures like beta-turns, and thrombin's substrate specificity.
• Hydrophobic regions, such as the Phe-Phe(87-88) sequence, appear resistant to thrombin cleavage in BP.