pubmed.ncbi.nlm.nih.gov

Heparin accelerates gelsolin amyloidogenesis - PubMed

  • ️Sun Jan 01 2006

Heparin accelerates gelsolin amyloidogenesis

Ji Young Suk et al. Biochemistry. 2006.

Abstract

The chemical environment of the extracellular matrix may influence the tissue-selective deposition observed there in gelsolin amyloid disease. Previously, we have identified the proteases that generate the amyloidogenic fragments from the full-length gelsolin variants and demonstrated that heparin is capable of accelerating gelsolin amyloidogenesis. Herein, we identify the structural features of heparin that promote the 8 kDa disease-associated gelsolin fragments (residues 173-243) generated at the cell surface to form amyloid. In conjunction with electron microscopy analyses, our kinetic studies demonstrate that heparin efficiently accelerates the formation of gelsolin amyloid by enabling intermolecular beta-sheet formation. The use of heparin analogues reveals that sulfation is important in accelerating amyloidogenesis and that the extent of acceleration is proportional to the molecular weight of heparin. In addition, heparin accelerated aggregation at both early and late stages of amyloidogenesis. Dynamic light scattering coupled to size exclusion chromatography showed that heparin promotes the formation of soluble aggregates. Collectively, these data reveal that heparin templates fibril formation and affords solubility to the aggregating peptides through its sulfated structure. By extension, the biochemical results herein suggest that tissue-selective deposition characteristic of the gelsolin amyloidoses is likely influenced by the extracellular localization of distinct glycosaminoglycans.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Heparan sulfate (HS) and heparin have the same repeating disaccharide subunits of glucuronic acid (GlcA) or iduronic acid (IdoA) and glucosamine (GlcN) or N-acetylated glucosamine (GlcNAc) linked by an α-1,4 glycosidic linkage with various sulfation patterns (box). IdoA is formed by epimerization of GlcA at the C5 position. In both heparin and HS, these subunits can have variable N-acetylation and N- or O- sulfation at C2 or C6 positions. HS has fewer sulfates, is less negatively charged, and has a more heterogeneous structure than heparin. Chondroitin sulfate A (ChonA) contains 4-O-sulfo GalNAc; dermatan sulfate (DS) also known as chondroitin sulfate B (ChonB) contains IdoA, 4-O-sulfo GalNAc; chondroitin sulfate C (ChonC) contains 6-O-sulfo GalNAc; chondroitin sulfate D (ChonD) contains 2-O-sulfo IdoA and 6-O-sulfo GalNAc; chondroitin sulfate E (ChonE) contains 4, 6-O-sulfo GalNAc. Hyaluronic acid is a copolymer of GlcA and GlcNAc and is the only GAG that is not sulfated and is not linked to a core protein Keratan sulfate contains galactose units (Gal) instead of hexuronic acid units. R1 indicates that the substituent can be either a hydrogen (-H) or a sulfo group (-SO3-). R2 indicates that the the substituent can be either a hydrogen (-H), a sulfo group (-SO3-) or an acetyl group (-COCH3). Negatively charged groups are shown in red (-COO-, SO3-).

Figure 2
Figure 2

TfT monitoring of D187N gelsolin173-243 (10 μM) amyloidogenesis (37 °C, pH 7, 5 s of shaking every 10 min) with (colored lines) or without (black line) GAGs. Heparin (Hep), heparin sulfate (HS), chondroitin A (ChonA), dermatan sulfate (DS), chondroitin C (ChonC), chondroitin D (ChonD), chondroitin E (ChonE), keratan sulfate (KS) and hyaluronic acid (HA) were added at the initiation of aggregation. Increasing the GAG concentrations (0.05 - 5 μM) increased the rates of the reactions (5 μM data shown). The data is representative of 5 to 6 independent experiments, each in triplicates. The GAGs alone did not affect the TfT fluorescence.

Figure 3
Figure 3

CD-spectra of D187N gelsolin173-243 (36 μM) samples aggregated alone (A) or with 5 μM heparin (Hep) (B) (37 °C, pH 7, constant rocking at 30 cycles/min) were taken at the indicated time points. The CD spectra minima are observed at 215-220 nm for a cross β-sheet structure. The transition of the structure towards a β-sheet rich structure was more pronounced in samples with heparin. (C) The fraction of structural transition completed for each time point was calculated as the ratio of ellipticity at 220 nm to its maximum value observed and normalized. (D) TfT fluorescence of samples were taken from the CD samples and diluted (see experimental procedures). Samples in the presence of heparin exhibited increased TfT fluorescence compared to D187N gelsolin173-243 samples aggregated alone. The data is representative of at least 5 to 6 independent experiments. Analysis of heparin alone (∼5 μM) did not lead to a CD signal at 220 nm.

Figure 4
Figure 4

(A) Heparin derivatives synthesized with indicated sulfate substitutions; desulfated heparin (deS Hep), N-desulfated heparin (N-deS Hep), fully O-sulfated-N-acetylated heparin (fullyO-S, N-Ac Hep), 2-O-desulfated iduronic acid heparin (2-deS IdoA Hep). ‘*’ indicates positions where variable N-acetylation occurs. (B) TfT monitoring of D187N gelsolin173-243 aggregation (10 μM) (37 °C, pH 7, 5 s of shaking every 10 min) in the presence or absence of the synthetic heparin derivatives (5 μM).

Figure 5
Figure 5

Synthesized heparin molecules (A) (0.1 mg/mL) having 4 saccharide units (dp4), 8 saccharide units (dp8), 12 saccharide units (dp12), 20 saccharide units (dp20), as well as a 5 kDa low molecular weight heparin (LMW Heparin), and 12-19 kDa heparin (heparin) were added to the D187N gelsolin173-243 (10 μM) aggregation reaction to discern their influence. (B) TfT assays were performed (37 °C, pH 7, constant rocking) to monitor the role of aggregation. TEM image of D187N gelsolin173-243 fibrils formed in the presence of dp4 (C) and 12-19 kDa heparin (D). Contrasts of the images shown were optimized using the auto-level function of Adobe Photoshop. Original images are presented in the supporting information.

Figure 6
Figure 6

(A) TfT analysis of D187N gelsolin173-243 (10 μM) aggregation was performed (37 °C, pH 7, 5 s of shaking every 10 min) and the averages of triplicate samples are shown (n = 4). The aggregation process was accelerated when heparin was added at the initiation of aggregation. When heparin was added at 43 h, a continuous increase in the TfT fluorescence was detected in contrast to D187N gelsolin173-243 aggregated alone. TEM images of D187N gelsolin173-243 aggregated at 43 h (B), and at 90 h (C). (D) D187N gelsolin173-243 aggregated with heparin present from the start; image taken at 43 h. (E) Aggregated sample at 90 h after heparin is introduced at 43 h. Contrasts of the images shown were optimized using the auto-level function of Adobe Photoshop. Original images are presented in the supporting information.

Figure 7
Figure 7

Overlaid gel filtration UV absorption traces (280 nm) of D187N gelsolin173-243 (36 μM) aggregation time course (37 °C, pH 7, constant rocking) in the absence of added GAGs (A), with HS (B) and with heparin (C) obtained at 0, 12, 23, 37 and 88 h. The monomer peak and the soluble aggregate peak (molar mass derived from light-scattering) are indicated. Fractions of monomers (D) and soluble aggregates (E) were calculated from UV absorption. The mass balance is poor in A (no aggregate peak growing in) because the aggregates are removed by the centrifugation/filtration step prior to FPLC injection.

Similar articles

Cited by

References

    1. Sekijima Y, Wiseman RL, Matteson J, Hammarstrom P, Miller SR, Sawkar AR, Balch WE, Kelly JW. The biological and chemical basis for tissue-selective amyloid disease. Cell. 2005;121:73–85. - PubMed
    1. Kiuru S. Gelsolin-related familial amyloidosis, Finnish type (FAF), and its variants found worldwide. Amyloid. 1998;5:55–66. - PubMed
    1. Kiuru-Enari S, Somer H, Seppalainen AM, Notkola IL, Haltia M. Neuromuscular pathology in hereditary gelsolin amyloidosis. J Neuropathol Exp Neurol. 2002;61:565–71. - PubMed
    1. Kiuru-Enari S, Keski-Oja J, Haltia M. Cutis laxa in hereditary gelsolin amyloidosis. Br J Dermatol. 2005;152:250–7. - PubMed
    1. Chen CD, Huff ME, Matteson J, Page L, Phillips R, Kelly JW, Balch WE. Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca(2+) stabilization. EMBO J. 2001;20:6277–87. - PMC - PubMed

Publication types

MeSH terms

Substances