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In vitro assembly and structure of trichocyte keratin intermediate filaments: a novel role for stabilization by disulfide bonding.

Wang H, Parry DA, Jones LN, Idler WW, Marekov LN, Steinert PM - J. Cell Biol. (2000)

Bottom Line: In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins.We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF.Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from "microfibrils." These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.

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HPLC fractionation of DST–cross-linked CNBr/trypsin cleavage products of trichocyte IF oligomers. Peptide profiles are from the following experiments: (A) unreacted trichocyte chains, (B) DST–cross-linked eight-molecule oligomer from reduced proteins showing 30 peaks used for analyses, (C) oxidized one-molecule oligomer showing numerous shifted peaks due to disulfide bond formation, (D) oxidized four-molecule oligomer showing 10 new peaks used for analyses, and (E) oxidized four-molecule oligomer followed by DST–cross linking showing 25 new peaks used for analyses.
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Figure 6: HPLC fractionation of DST–cross-linked CNBr/trypsin cleavage products of trichocyte IF oligomers. Peptide profiles are from the following experiments: (A) unreacted trichocyte chains, (B) DST–cross-linked eight-molecule oligomer from reduced proteins showing 30 peaks used for analyses, (C) oxidized one-molecule oligomer showing numerous shifted peaks due to disulfide bond formation, (D) oxidized four-molecule oligomer showing 10 new peaks used for analyses, and (E) oxidized four-molecule oligomer followed by DST–cross linking showing 25 new peaks used for analyses.

Mentions: Large-scale cross-linking reactions of expressed trichocyte keratins (≈50 μg/ml in H buffer) were performed, followed by recovery of four sizes of oligomers for CNBr/trypsin cleavage. Fig. 6 shows HPLC chromatograms of resolved peptides. In comparison to unreacted chains (Fig. 6 A), 30 peaks were recovered that contained DST–cross-linked peptides. Of these, five common peaks were due to intramolecular cross linking across the heterodimer molecule, 12 peaks arose in the two-molecule oligomer, another six in the four-molecule oligomer, and seven more in the four-molecule oligomer. Amino acid analyses and protein sequencing for up to 12 Edman degradation cycles allowed identification of the Lys residues involved in each case. Almost all Lys residues on the rod domains of both the types Ia and IIa chains were used, and often with different partners. Most of these were located in identical positions as were found in previous cross-linking studies of cytokeratins vimentin and α-internexin (Steinert et al. 1993a,Steinert et al. 1993b,Steinert et al. 1993c; Steinert et al. 1999), which thereby adds validity to this database. 18 peaks could be assigned confidently to either one of three modes of alignment between antiparallel molecules—A11 (7), A22 (5), or A12 (6)—described previously for other IF types (see Table and Fig. 8). A total of seven other cross links (Fig. 6 B, 9, 12, 16, 22, 23, 27, and 30) may be due to linkages between molecules four rows apart in a two-dimensional surface lattice and will not be discussed further here.


In vitro assembly and structure of trichocyte keratin intermediate filaments: a novel role for stabilization by disulfide bonding.

Wang H, Parry DA, Jones LN, Idler WW, Marekov LN, Steinert PM - J. Cell Biol. (2000)

HPLC fractionation of DST–cross-linked CNBr/trypsin cleavage products of trichocyte IF oligomers. Peptide profiles are from the following experiments: (A) unreacted trichocyte chains, (B) DST–cross-linked eight-molecule oligomer from reduced proteins showing 30 peaks used for analyses, (C) oxidized one-molecule oligomer showing numerous shifted peaks due to disulfide bond formation, (D) oxidized four-molecule oligomer showing 10 new peaks used for analyses, and (E) oxidized four-molecule oligomer followed by DST–cross linking showing 25 new peaks used for analyses.
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Related In: Results  -  Collection

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Figure 6: HPLC fractionation of DST–cross-linked CNBr/trypsin cleavage products of trichocyte IF oligomers. Peptide profiles are from the following experiments: (A) unreacted trichocyte chains, (B) DST–cross-linked eight-molecule oligomer from reduced proteins showing 30 peaks used for analyses, (C) oxidized one-molecule oligomer showing numerous shifted peaks due to disulfide bond formation, (D) oxidized four-molecule oligomer showing 10 new peaks used for analyses, and (E) oxidized four-molecule oligomer followed by DST–cross linking showing 25 new peaks used for analyses.
Mentions: Large-scale cross-linking reactions of expressed trichocyte keratins (≈50 μg/ml in H buffer) were performed, followed by recovery of four sizes of oligomers for CNBr/trypsin cleavage. Fig. 6 shows HPLC chromatograms of resolved peptides. In comparison to unreacted chains (Fig. 6 A), 30 peaks were recovered that contained DST–cross-linked peptides. Of these, five common peaks were due to intramolecular cross linking across the heterodimer molecule, 12 peaks arose in the two-molecule oligomer, another six in the four-molecule oligomer, and seven more in the four-molecule oligomer. Amino acid analyses and protein sequencing for up to 12 Edman degradation cycles allowed identification of the Lys residues involved in each case. Almost all Lys residues on the rod domains of both the types Ia and IIa chains were used, and often with different partners. Most of these were located in identical positions as were found in previous cross-linking studies of cytokeratins vimentin and α-internexin (Steinert et al. 1993a,Steinert et al. 1993b,Steinert et al. 1993c; Steinert et al. 1999), which thereby adds validity to this database. 18 peaks could be assigned confidently to either one of three modes of alignment between antiparallel molecules—A11 (7), A22 (5), or A12 (6)—described previously for other IF types (see Table and Fig. 8). A total of seven other cross links (Fig. 6 B, 9, 12, 16, 22, 23, 27, and 30) may be due to linkages between molecules four rows apart in a two-dimensional surface lattice and will not be discussed further here.

Bottom Line: In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins.We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF.Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Skin Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Intermediate filaments (IF) have been recognized as ubiquitous components of the cytoskeletons of eukaryotic cells for 25 yr. Historically, the first IF proteins to be characterized were those from wool in the 1960s, when they were defined as low sulfur keratins derived from "microfibrils." These proteins are now known as the type Ia/type IIa trichocyte keratins that constitute keratin IF of several hardened epithelial cell types. However, to date, of the entire class of >40 IF proteins, the trichocyte keratins remain the only ones for which efficient in vitro assembly remains unavailable. In this paper, we describe the assembly of expressed mouse type Ia and type IIa trichocyte keratins into IF in high yield. In cross-linking experiments, we document that the alignments of molecules within reduced trichocyte IF are the same as in type Ib/IIb cytokeratins. However, when oxidized in vitro, several intermolecular disulfide bonds form and the molecular alignments rearrange into the pattern shown earlier by x-ray diffraction analyses of intact wool. We suggest the realignments occur because the disulfide bonds confer substantially increased stability to trichocyte keratin IF. Our data suggest a novel role for disulfide bond cross linking in stabilization of these IF and the tissues containing them.

Show MeSH