Fibulin-4 Accelerates Amyloid Formation by Binding with a Keratin 5 Peptide Fragment

Abstract

Keratins are the major amyloid fibril component in localized cutaneous amyloidosis. We analyzed the amyloid components in the skin of patients with localized cutaneous amyloidosis by immunohistochemical staining using antisera against extracellular matrix proteins and keratin 5 (K5). Fibulin-4 and K5 colocalized in the amyloid deposits. Using 14 synthetic peptides, we screened for amyloidogenic sequences in the C-terminal region of K5, including the α-helical rod domain and the tail domain. Two peptides stained with thioflavin T possessed a β-sheet structure and formed amyloid-like fibrils. Among the amyloidogenic peptides, a peptide KT5-6 (YQELMNTKLALDVEIATYRKLLEGE) derived from the α-helical rod domain of K5 specifically bound to fibulin-4. In addition, amyloid formation of KT5-6 was accelerated by fibulin-4. These results suggest that degraded fragments of K5 containing the KT5-6 sequence form amyloid fibrils with fibulin-4. The data further suggest that degraded fragments of K5 and fibulin-4 have the potential to initiate cutaneous amyloidosis.

Keywords: CD, circular dichroism; K5, keratin 5; K5/14, keratin 5/14; PLCA, primary localized cutaneous amyloidosis.

Figure 1

Keratin 5 and fibulin-4 were found in the amyloid deposits of Bowen’s carcinoma and lichen amyloidosis skin. (a) The skin from a Bowen’s carcinoma (patient 1) was stained with anti‒fibulin-4, anti‒keratin 5, and anti‒fibrillin-1 antibodies (the left column) together with thioflavin T. Bar = 50 μm. (b) Paraffin-embedded sections (patient 2, lichen amyloidosis) were stained with anti‒fibulin-4 (red) and anti‒keratin 5 (green, data not shown) and with anti‒keratin 5 and thioflavin T. Bar = 100 μm.

Figure 2

Fibulin-4 and keratin 5 were found in the amyloid deposits of lichen amyloidosis (patient 3). (a) Consecutive frozen sections were stained with anti-keratin 5 or with anti‒fibulin-4, and amyloid deposits were detected with thioflavin T. Each merged image is also shown. (b) The staining with anti‒fibulin-4 in the same section as the second row of a was shown at lower magnification. This image also shows the localization of fibulin-4 in normal human skin. Fibulin-4 localizes in elastic fibers and in the surrounding blood vessels, not only in the papil1ary dermis but also in the reticular dermis. Bar = 100 μm.

Figure 3

The amyloid deposits in lichen amyloidosis skin derived from patient 4. Keratin 5 and fibulin-4 colocalized in the amyloid deposits detected with thioflavin T; however, the staining with anti‒fibulin-4 was very weak. Bar = 50 μm. The fluorescence intensity along the dotted line was shown at the right panel showing the colocalization of thioflavin T and keratin 5/fibulin-4. S indicates the starting point of the measurement.

Figure 4

The amyloid deposits of familial PLCA skin derived from patient 5. Keratin 5 and fibulin-4 were found in the amyloid deposits. Bar = 50 μm. The fluorescence intensity along the dotted line was shown at the right panel showing the colocalization of thioflavin T and keratin 5/fibulin-4. S indicates the starting point of the measurement. PLCA, primary localized cutaneous amyloidosis.

Figure 5

Immunohistological analyses using the antibodies against elastic fiber components: elastin, fibrillin-1, versican, LTBP1, LTBP2, LTBP4, and fibulin-5. Skin sections were derived from patient 3. The merged pictures with thioflavin T staining were shown at the middle panel, and each negative control was shown at the right panel. Bar = 100 μm. The staining with anti-LTBP4 was shown also at higher magnification (bar = 100 μm) to show that LTBP4 was not accumulated in amyloid. LTBP1 and fibulin-5 were abnormally deposited in the amyloid, but other proteins were not. Skins from patients 4 and 5 were also stained, and data were similar (data not shown).

Figure 6

Physicochemical properties of peptides derived from the C-terminal region of keratin 5. (a) Localization of synthetic peptides in the keratin 5/14 molecule. (b) Dose-response curve for peptides measured with thioflavin T. Thioflavin T solution (50 μl, 20 μM in 0.2 mM glycine‒NaOH buffer [pH 8.5]) was added to the peptide solutions (50 μl, 3.125‒100 μM in H₂O) in a 96-well plate, and the fluorescent intensity was measured immediately at an excitation wavelength of 455 nm and emission wavelength at 490 nm. (c) CD spectra of keratin peptides. (d) Electron micrograph of keratin peptide amyloid-like fibrils. Bar = 100 nm. CD, circular dichroism; H₂O, water; NaOH, sodium hydroxide.

Figure 7

Binding of keratin peptides to fibulins. (a) Biotin KT5-6 binds to fibulin-4 but not to fibulin-5, whereas biotin KT5-8 does not bind to either fibulin. Results were expressed as means + SD. Comparison of mean values was performed using repeated measures one-way ANOVA and a Welch’s t-test. ∗P < 0.01, significantly different from biotin-KT5-8 on fibulin-4. (b) Biotin-KT5-6 binds to fibulin-4 in a dose-dependent manner but not fibulin-5, whereas none of the homologous KT5-6 peptides does bind to either fibulin. (c) Kinetic analysis of KT5-6 amyloid formation with fibulins. min, minute.

Figure 8

Possible mechanism of amyloidosis by keratin 5 and fibulin-4. First, keratin 5/14 in basal keratinocytes is degraded by enzymes, and then the degradation fragments move to the dermis. Next, the degradation fragments containing the KT5-6 sequence form amyloid-like fibrils with fibulin-4 on elastic fibers. Further accumulation of the degradation fragments enlarges amyloid-like fibrils and forms amyloid deposits.