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Amino-acid sequence of human and bovine brain myelin proteolipid protein (lipophilin) is completely conserved
Abstract:
Proteolipid protein (PLP) was isolated from white matter of human brain by chloroform/methanol extraction and further purified by chromatography. Performic acid oxidation yielded a product homogeneous in NaDodSO4-polyacrylamide electrophoresis with a molecular mass of 30 kDa. The carboxymethylated PLP was chemically cleaved with cyanogen bromide into four fragments: CNBr I 22-24 kDa, CNBr II 5 kDa, CNBr III 1.4 kDa and CNBr IV 0.7 kDa. HBr/dimethylsulfoxide cleavage at tryptophan residues released four fragments: Trp I 14-16 kDa, Trp II 2.0 kDa, Trp III 5 kDa and Trp IV 7 kDa. Hydrophilic fragments were enriched in 50% formic acid (CNBr II, III, IV and Trp II and III), whereas hydrophobic peptides precipitated from this solvent were CNBr I, Trp I and IV. The fragments were separated by gel filtration with 90% formic acid as solvent and finally purified by gel permeation HPLC (Si 60 and Si 100) for automated liquid and solid-phase Edman degradation. Large fragments were further cleaved with different proteinases (trypsin, V8-proteinase, endoproteinase Lys-C and thermolysin). We used an improved strategy in the sequencing of the human proteolipid protein compared with our approach to the structural elucidation of bovine brain PLP. The amino-acid sequence of human PLP contains 276 residues, the same as found in bovine proteolipid protein. The two sequences proved to be identical. The possible importance of the conservative structure of this integral membrane protein is discussed.
Insights
Researchers sequenced human brain proteolipid protein (PLP), an integral membrane protein. The human PLP sequence was identical to bovine PLP, highlighting its conserved structure.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Proteolipid protein (PLP) is a major component of the myelin sheath in the central nervous system.
- Understanding the structure of PLP is crucial for comprehending myelin formation and disorders.
- Previous studies elucidated the structure of bovine brain PLP.
Purpose of the Study:
- To determine the amino acid sequence of human brain proteolipid protein (PLP).
- To compare the human PLP sequence with that of bovine PLP.
- To investigate the structural conservation of this integral membrane protein.
Main Methods:
- Isolation and purification of human brain PLP using chloroform/methanol extraction and chromatography.
- Chemical cleavage of PLP with cyanogen bromide and HBr/dimethylsulfoxide.
- Fragment separation by gel filtration and HPLC, followed by automated Edman degradation for sequencing.
- Further enzymatic cleavage of large fragments using various proteinases.
Main Results:
- Human PLP was purified and characterized, yielding a 30 kDa product after oxidation.
- Chemical and enzymatic fragmentation produced distinct peptide fragments.
- The determined amino acid sequence of human PLP consists of 276 residues.
- The human PLP sequence was found to be identical to the previously determined bovine PLP sequence.
Conclusions:
- The amino acid sequence of human proteolipid protein has been fully elucidated.
- The identical sequences of human and bovine PLP indicate a highly conserved structure.
- The conserved structure suggests significant functional importance for PLP as an integral membrane protein.