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Analysis of core region from egg white lysozyme forming amyloid fibrils.

Tokunaga Y, Sakakibara Y, Kamada Y, Watanabe K, Sugimoto Y - Int. J. Biol. Sci. (2013)

Bottom Line: The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C.A substantial number of fibrils were generated under this pH condition and incubation period.Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Bioscience The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065 Japan.

ABSTRACT
Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.

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Structure of HEWL and location of the K peptide region. A. Illustration by ribbon representation. Red color represents the K peptide region with the side chain of tryptophan 62. B. Illustration by space-filling model. Red and yellow show tryptophan 62 and other amino acids of the K peptide, respectively. Green and dark violet illustrate tryptophan 63 and other amino acids except the K peptide region. Amino acids in the K peptide are largely buried in the molecule.
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Figure 1: Structure of HEWL and location of the K peptide region. A. Illustration by ribbon representation. Red color represents the K peptide region with the side chain of tryptophan 62. B. Illustration by space-filling model. Red and yellow show tryptophan 62 and other amino acids of the K peptide, respectively. Green and dark violet illustrate tryptophan 63 and other amino acids except the K peptide region. Amino acids in the K peptide are largely buried in the molecule.

Mentions: Lysozyme is a ubiquitous enzyme present in many organisms that attacks the peptidoglycan cell wall of certain microorganisms, selectively hydrolyzing the β-1,4 glycosidic linkages between the N-acetylmuramic acid and N-acetylglucosamine 9. Hen egg white lysozyme (HEWL), which belongs to the c-type class of lysozymes 10, has two domains called α and β; the former has four α-helices (A to D) and the latter consists mainly of an antiparallel β-sheet. A long loop and enzyme active site are located in a cleft that is formed between these two domains (Figs. 1A and B) 11. HEWL is known to form amyloid fibrils under denaturing conditions in organic solvent, detergent, chemical denaturant or acid pH with high temperature 12-14. The properties of such fibrils and the process of fibrillation have been studied in considerable detail 15,16. It is generally accepted that the formation of amyloid fibrils is implicated in particular regions of protein as well as the entire protein 17. Verma et al. verified that most point mutations have a significant effect on human lysozyme dynamics, many of which lead to local unfolding and trend to form amyloid fibrils 18. We recently found during analysis of the interaction between ovalbumin and HEWL that a peptide from HEWL has the ability of self-fibrillation, and designated it as the K peptide; we think that the region might work as a core for the amyloidogenesis of HEWL 19. This peptide, corresponding to the residues 54-62 (GILQINSRW) of HEWL and being located in the hydrophobic cluster 3 20-22, contains tryptophan 62, which is thought to be crucial to forming fibrils 21,23,24. It is shorter than the previously reported fibril-forming peptide of 16 amino acids from the residues 49-64 (GSTDYGILQINSRWWC) of HEWL 25. The K peptide region is found as a pocket in the junction connecting the α- and β-domains, and is probably buried partially within the lysozyme molecule (Figs. 1A and B). Here, we reconfirmed the self-fibrillation ability of the K peptide and compared it to those of some K peptide mutants. In each of these mutants, a certain residue was replaced by another amino acid. For the aggregation and fibrillation of HEWL, not only the length of the K peptide region but also the presence of aromatic amino acids, especially at the 62nd position, is assumed to be critical.


Analysis of core region from egg white lysozyme forming amyloid fibrils.

Tokunaga Y, Sakakibara Y, Kamada Y, Watanabe K, Sugimoto Y - Int. J. Biol. Sci. (2013)

Structure of HEWL and location of the K peptide region. A. Illustration by ribbon representation. Red color represents the K peptide region with the side chain of tryptophan 62. B. Illustration by space-filling model. Red and yellow show tryptophan 62 and other amino acids of the K peptide, respectively. Green and dark violet illustrate tryptophan 63 and other amino acids except the K peptide region. Amino acids in the K peptide are largely buried in the molecule.
© Copyright Policy
Related In: Results  -  Collection

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Figure 1: Structure of HEWL and location of the K peptide region. A. Illustration by ribbon representation. Red color represents the K peptide region with the side chain of tryptophan 62. B. Illustration by space-filling model. Red and yellow show tryptophan 62 and other amino acids of the K peptide, respectively. Green and dark violet illustrate tryptophan 63 and other amino acids except the K peptide region. Amino acids in the K peptide are largely buried in the molecule.
Mentions: Lysozyme is a ubiquitous enzyme present in many organisms that attacks the peptidoglycan cell wall of certain microorganisms, selectively hydrolyzing the β-1,4 glycosidic linkages between the N-acetylmuramic acid and N-acetylglucosamine 9. Hen egg white lysozyme (HEWL), which belongs to the c-type class of lysozymes 10, has two domains called α and β; the former has four α-helices (A to D) and the latter consists mainly of an antiparallel β-sheet. A long loop and enzyme active site are located in a cleft that is formed between these two domains (Figs. 1A and B) 11. HEWL is known to form amyloid fibrils under denaturing conditions in organic solvent, detergent, chemical denaturant or acid pH with high temperature 12-14. The properties of such fibrils and the process of fibrillation have been studied in considerable detail 15,16. It is generally accepted that the formation of amyloid fibrils is implicated in particular regions of protein as well as the entire protein 17. Verma et al. verified that most point mutations have a significant effect on human lysozyme dynamics, many of which lead to local unfolding and trend to form amyloid fibrils 18. We recently found during analysis of the interaction between ovalbumin and HEWL that a peptide from HEWL has the ability of self-fibrillation, and designated it as the K peptide; we think that the region might work as a core for the amyloidogenesis of HEWL 19. This peptide, corresponding to the residues 54-62 (GILQINSRW) of HEWL and being located in the hydrophobic cluster 3 20-22, contains tryptophan 62, which is thought to be crucial to forming fibrils 21,23,24. It is shorter than the previously reported fibril-forming peptide of 16 amino acids from the residues 49-64 (GSTDYGILQINSRWWC) of HEWL 25. The K peptide region is found as a pocket in the junction connecting the α- and β-domains, and is probably buried partially within the lysozyme molecule (Figs. 1A and B). Here, we reconfirmed the self-fibrillation ability of the K peptide and compared it to those of some K peptide mutants. In each of these mutants, a certain residue was replaced by another amino acid. For the aggregation and fibrillation of HEWL, not only the length of the K peptide region but also the presence of aromatic amino acids, especially at the 62nd position, is assumed to be critical.

Bottom Line: The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C.A substantial number of fibrils were generated under this pH condition and incubation period.Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry and Bioscience The United Graduate School of Agricultural Sciences, Kagoshima University, Kagoshima 890-0065 Japan.

ABSTRACT
Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.

Show MeSH
Related in: MedlinePlus