Overall Sulfation of Heparan Sulfate from Pancreatic Islet β-TC3 Cells Increases Maximal Fibril Formation but Does Not Determine Binding to the Amyloidogenic Peptide Islet Amyloid Polypeptide

Islet amyloid, a pathologic feature of type 2 diabetes, contains the islet β-cell peptide islet amyloid polypeptide (IAPP) as its unique amyloidogenic component. Islet amyloid also contains heparan sulfate proteoglycans (HSPGs) that may contribute to amyloid formation by binding IAPP via their heparan sulfate (HS) chains. We hypothesized that β-cells produce HS that bind IAPP via regions of highly sulfated disaccharides. Unexpectedly, HS from the β-cell line β-TC3 contained fewer regions of highly sulfated disaccharides compared with control normal murine mammary gland (NMuMG) cells. The proportion of HS that bound IAPP was similar in both cell lines (∼65%). The sulfation pattern of IAPP-bound versus non-bound HS from β-TC3 cells was similar. In contrast, IAPP-bound HS from NMuMG cells contained frequent highly sulfated regions, whereas the non-bound material demonstrated fewer sulfated regions. Fibril formation from IAPP was stimulated equally by IAPP-bound β-TC3 HS, non-bound β-TC3 HS, and non-bound NMuMG HS but was stimulated to a greater extent by the highly sulfated IAPP-bound NMuMG HS. Desulfation of HS decreased the ability of both β-TC3 and NMuMG HS to stimulate IAPP maximal fibril formation, but desulfated HS from both cell types still accelerated fibril formation relative to IAPP alone. In summary, neither binding to nor acceleration of fibril formation from the amyloidogenic peptide IAPP is dependent on overall sulfation in HS synthesized by β-TC3 cells. This information will be important in determining approaches to reduce HS-IAPP interactions and ultimately prevent islet amyloid formation and its toxic effects in type 2 diabetes.     

A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.     

High-glucose-induced structural changes in the heparan sulfate proteoglycan, perlecan, of cultured human aortic endothelial cells

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [(35)S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [(3)H] glucosamine incorporation into GAG but not lower [(3)H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.     

Effects of Sulfate Ions on Alzheimer β/A4 Peptide Assemblies: Implications for Amyloid Fibril-Proteoglycan Interactions

To model the possible involvement of sulfated proteoglycans in amyloidogenesis, we examined the influence of sulfate ions, heparan, and Congo red on the conformation and morphology of peptides derived from the Alzheimer beta/A4 amyloid protein. The peptides included residues 11-28, 13-28, 15-28, and 11-25 of beta/A4. Negative-stain electron microscopy revealed a sulfate-specific tendency of the preformed peptide fibrillar assemblies of beta(11-28), beta(13-28), and beta(11-25), but not beta(15-28), to undergo extensive lateral aggregation and axial growth into "macrofibers" that were approximately 0.1-0.2 micron wide by approximately 20-30 microns long. Such effects were observed at low sulfate concentrations (e.g., 5-50 mM) and could not be reproduced under comparable conditions with Na2HPO4, Na2SeO4, or NaCl. Macrofibers in NaCl were only observed at 1,000 mM. At physiological ionic strength of NaCl, fibril aggregation was observed only with addition of sulfate ions at 5-50 mM. Selenate ions, by contrast with sulfate ions, induced only axial and not substantial lateral aggregation of fibrils. X-ray diffraction indicated that the original cross-beta peptide conformation remained unchanged; however, sulfate binding did produce an intense approximately 65 A meridional reflection not recorded with control peptides. This new reflection probably arises from the periodic deposition of the electron-dense sulfate along the (long) axis of the fibril. The sulfate binding could provide sites for the binding of additional fibrils that generate the observed lateral and axial aggregation. The binding of heparan to beta(11-28) also produced extensive aggregation, suggesting that in vivo sulfated compounds can promote macrofibers. The amyloid-specific, sulfonated dye Congo red, even in the presence of sulfate ions, produced limited aggregation and reduced axial growth of the fibrils. Therefore, electrostatic interactions are important in the binding of exogenous compounds to amyloid fibrils. Our findings suggest that the sulfate moieties of certain molecules, such as glycosaminoglycans, may affect the aggregation and deposition of amyloid fibrils that are observed as extensive deposits in senile plaques and cerebrovascular amyloid.     

The sulfate moieties of glycosaminoglycans are critical for the enhancement of beta-amyloid protein fibril formation

Our previous studies have demonstrated that perlecan and perlecan-derived glycosaminoglycans (GAGs) not only bind beta-amyloid protein (Abeta) 1-40 and 1-42, but are also potent enhancers of Abeta fibril formation and stabilize amyloid fibrils once formed. However, it was not determined which moieties in perlecan heparan sulfate GAG chains may be responsible for the observed effects and whether other GAGs were also capable of a similar enhancement of Abeta fibril formation as observed with perlecan GAGs. In the present study, thioflavin T fluorometry (over a 1-week period) was used to extend our previous studies and to test the hypothesis that the sulfate moiety is critical for the enhancing effects of heparin/heparan sulfate GAGs on Abeta 1-40 fibrillogenesis. This hypothesis was confirmed when removal of all sulfates from heparin (i.e., completely desulfated N-acetylated heparin) led to a complete loss in the enhancement of Abeta fibrillogenesis as demonstrated in both thioflavin T fluorometry and Congo red staining studies. On the other hand, removal of O-sulfate from heparin (i.e., completely desulfated N-sulfated heparin), and to a lesser extent N-sulfate (i.e., N-desulfated N-acetylated heparin), resulted in only a partial loss of the enhancement of Abeta 1-40 fibril formation. These studies indicate that the sulfate moieties of GAGs are critical for enhancement of Abeta amyloid fibril formation. In addition, other sulfated molecules such as chondroitin-4-sulfate, dermatan sulfate, dextran sulfate, and pentosan polysulfate all significantly enhanced (greater than twofold by 3 days) Abeta amyloid fibril formation. These latter findings indicate that deposition and accumulation of other GAGs at sites of Abeta amyloid deposition in Alzheimer's disease brain may also participate in the enhancement of Abeta amyloidosis.     

Characterization of the heparin binding site in the N-terminus of human pro-islet amyloid polypeptide: implications for amyloid formation

Islet amyloid deposits are a characteristic pathological hallmark of type 2 diabetes mellitus. Islet amyloid polypeptide (IAPP), also referred to as amylin, aggregates in the islet extracellular space to form amyloid deposits in up to 95% of patients with the disease. IAPP is stored with insulin in beta-islet cells and is processed in parallel by subtilisin-like prohormone convertases prior to secretion. There is indirect evidence that normal processing of the prohormone precursor, proIAPP, at the N-terminal cleavage site is defective in type 2 diabetes and results in secretion of an N-terminal extended proIAPP intermediate. The N-terminal flanking region of proIAPP is detected in amyloid deposits; however, the C-terminal flanking region is not. Immunohistochemical studies implicate the presence of the heparan sulfate proteoglycan (HSPG) perlecan in islet amyloid deposits, suggesting a role for HSPGs in mediating amyloid deposition in type 2 diabetes and implicating a binding domain in the N-terminus of proIAPP. Initial studies of proIAPP indicated that the HSPG binding region is contained within the first 30 residues. Here, we characterize the potential HSPG binding site of proIAPP in detail by analyzing a set of peptide fragments. Binding is tighter at low pH due to protonation of histidine residues. Deletion studies show that Arg-22 and His-29 play a role in binding. Reduction of the Cys-13 to Cys-18 disulfide leads to a noticeable decrease in binding. We demonstrate the ability of heparan sulfate to induce amyloid formation in N-terminal fragments of proIAPP. The oxidized peptide forms amyloid more rapidly than the reduced variant in the presence of heparan sulfate, but the reduced peptide ultimately forms more extensive amyloid deposits. The potential implications for islet amyloid formation in vivo are discussed.     

Heparin binds 8 kDa gelsolin cross-β-sheet oligomers and accelerates amyloidogenesis by hastening fibril extension

Glycosaminoglycans (GAGs) are highly sulfated linear polysaccharides prevalent in the extracellular matrix, and they associate with virtually all amyloid deposits in vivo. GAGs accelerate the aggregation of many amyloidogenic peptides in vitro, but little mechanistic evidence is available to explain why. Herein, spectroscopic methods demonstrate that GAGs do not affect the secondary structure of the monomeric 8 kDa amyloidogenic fragment of human plasma gelsolin. Moreover, monomerized 8 kDa gelsolin does not bind to heparin under physiological conditions. In contrast, 8 kDa gelsolin cross-β-sheet oligomers and amyloid fibrils bind strongly to heparin, apparently because of electrostatic interactions between the negatively charged polysaccharide and a positively charged region of the 8 kDa gelsolin assemblies. Our observations are consistent with a scaffolding mechanism whereby cross-β-sheet oligomers, upon formation, bind to GAGs, accelerating the fibril extension phase of amyloidogenesis, possibly by concentrating and orienting the oligomers to more efficiently form amyloid fibrils. Notably, heparin decreases the 8 kDa gelsolin concentration necessary for amyloid fibril formation, likely a consequence of fibril stabilization through heparin binding. Because GAG overexpression, which is common in amyloidosis, may represent a strategy for minimizing cross-β-sheet oligomer toxicity by transforming them into amyloid fibrils, the mechanism described herein for GAG-mediated acceleration of 8 kDa gelsolin amyloidogenesis provides a starting point for therapeutic strategy development. The addition of GAG mimetics, small molecule sulfonates shown to reduce the amyloid load in animal models of amyloidosis, to a heparin-accelerated 8 kDa gelsolin aggregation reaction mixture neither significantly alters the rate of amyloidogenesis nor prevents oligomers from binding to GAGs, calling into question their commonly accepted mechanism.     

Inhibition of amyloidosis using low-molecular-weight heparins

BACKGROUND: Amyloid diseases are characterized by the tissue deposition of extracellular proteinaceous material, which results in organ dysfunction and death. Colocalization of heparan sulfate (HS) proteoglycans to amyloid deposits suggests that they may be an early event in amyloid formation and play an important role in fibril formation. Structural analysis has demonstrated that HS interacts with amyloidogenic proteins resulting in structural changes that allow for an increase in beta-sheet content, possibly enhancing fibrillogenesis. Recent studies have shown that small-molecule anionic sulfonates or sulfates can arrest inflammation-associated (AA) amyloid induction. MATERIALS AND METHODS: In the present study, we examine the effect of low-molecular-weight heparins (LMWHs) on the development of amyloid in the mouse model of AA amyloid. In addition, in vitro fibril formation assays were performed to determine the effect of LMWHs on fibrillogenesis. RESULTS: Injection of mice with clinically relevant doses of LMWHs (enoxaparin and dalteparin) demonstrated a reduction in AA amyloid deposition. These compounds were capable of arresting the progression of AA amyloid and eventually resulting in regression of the amyloid deposits. In vitro analysis indicated that LMWHs prevented AA and Abeta peptide fibril formation by impeding the structural changes necessary for fibril formation. CONCLUSIONS: Our findings suggest that the LMWHs may provide beneficial effects against the development of amyloidoses, including Alzheimer's disease.     

Role of heparan sulfate proteoglycans in cell-cell signaling in Drosophila.

Heparan sulfate proteoglycans (HSPGs) are abundant molecules associated with the cell surface and extracellular matrix, and consist of a protein core to which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached. Although these molecules have been the focus of intense biochemical studies in vitro, their biological functions in vivo were unclear until recently. We have undertaken an in vivo functional study of HSPGs in Drosophila. Our studies, as well as others, demonstrate the critical roles of HSPGs in several major signaling pathways, including ibroblast growth factor (FGF), Wnt, Hedgehog (Hh) and TGF-beta. Our results also suggest that specific HS GAG chain modifications, as well as specific HSPG protein cores, are involved in specific signaling pathways.     

Identification and tissue-specific distribution of sulfated glycosaminoglycans in the blood-sucking bug Rhodnius prolixus (Linnaeus)

We have previously characterized heparan sulfate (HS) as the major ovarian sulfated glycosaminoglycan (GAG) in females of Rhodnius prolixus, while chondroitin sulfate (CS) was the minor component. Using histochemical procedures we found that GAGs were concentrated in the ovarian tissue but not found inside the oocytes. Here, we extend our initial observations of GAG expression in R. prolixus by characterizing these molecules in other organs: the fat body, intestinal tract, and the reproductive tracts. Only HS and CS were found in the three organs analyzed, however CS was the major GAG species in these tissues. We also determined the compartmental distribution of GAGs in these organs by histochemical analysis using 1,9-dimethylmethylene blue, and evaluated the specific distribution of CS within both male and female reproductive tracts by immunohistochemistry using an anti-CS antibody. We also determined the GAG composition in eggs at days 0 and 6 of embryonic development. Only HS and CS were found in eggs at day 6, while no sulfated GAGs were detected at day 0. Our results demonstrate that HS and CS are the only sulfated GAG species expressed in the fat body and in the intestinal and reproductive tracts of Rhodnius male and female adults. Both sulfated GAGs were also identified in Rhodnius embryos. Altogether, these results show no qualitative differences in the sulfated GAG composition regarding tissue-specific or development-specific distribution.     

High affinity interactions between the Alzheimer's beta-amyloid precursor proteins and the basement membrane form of heparan sulfate proteoglycan

High affinity interactions were studied between the basement membrane form of heparan sulfate proteoglycan (HSPG) and the 695-, 751-, and 770-amino acid Alzheimer amyloid precursor (AAP) proteins. Based on quantitative analyses of binding data, we identified single binding sites for the HSPG on AAP-695 (Kd = 9 x 10(-10) M), AAP-751 (Kd = 10 x 10(-9) M), and AAP-770 (Kd = 9 x 10(-9) M). It is postulated that the "Kunitz" protease inhibitor domain which is present in AAP-751 and -770 reduces the affinity of AAPs for the HSPG through steric hindrance and/or conformational alteration. HSPG binding was inhibited by heparin and dextran sulfate, but not by dermatan or chondroitin sulfate. HSPG protein core, obtained by heparitinase digestion, also bound to the beta-amyloid precursor proteins with high affinity, indicating that the high affinity binding site is constituted by the polypeptide chain rather than the carbohydrate moiety. The effects of various cations on these interactions were also studied. Our results suggest that specific interactions between the AAP proteins and the extracellular matrix may be involved in the nucleation stages of Alzheimer's disease type amyloidogenesis.     

Sulfated glycosaminoglycans (GAG) in the developing mouse brain : Quantitative aspects on the metabolism of total and individual sulfated GAG in vivo

Sulfation and desulfation of total glycosaminoglycans (GAG) as well as of chondroitin sulfates (A + C), dermatan sulfate, and heparan sulfate were quantified in the developing cerebrum and cerebellum of mice by labeling with [35S]sulfate combined with chases started 24 hr after [35S]sulfate injection. In both the developing cerebrum and cerebellum, the rate of biosynthesis of total sulfated GAG was highest shortly after birth (2 days), decreased sharply thereafter, and reached a plateau after 14 days. The biosynthetic activities of chondroitin sulfates and heparan sulfate decreased sharply up to 14 days and retained constant levels afterward. By contrast, the rates of biosynthesis of dermatan sulfate increased up to 14 days. The biodegradation rates of total sulfated GAG as well as of chondroitin sulfates, heparan sulfate, and dermatan sulfate were strongly correlated with the corresponding rates of biosynthesis during the first 2 postnatal weeks. Total and individual sulfated GAG showed high degradation rates resulting in half-life times of a few hours up to 1 1/2 days. Thus sulfated GAG are synthesized in excess and the actual net content seems to be co-regulated to a high degree by lysosomal degradation. In both brain parts, a proportional increase of the sulfated GAG content vs the total GAG content from 40% at birth to 90% at 28 days was observed. Since during development heparan sulfate and dermatan sulfate manifested a relative increase in their daily net synthesis besides a decrease of chondroitin sulfates, a developmental increase of the sulfate groups linked to GAG is evidenced. This molecular differentiation resulting in microenvironmental changes may be of high functional significance.     

Differences in the apical and basolateral pathways for glycosaminoglycan biosynthesis in Madin-Darby canine kidney cells

Serglycin with a green fluorescent protein tag (SG-GFP) expressed in epithelial Madin-Darby canine kidney cells is secreted mainly (85%) into the apical medium, but the glycosaminoglycan (GAG) chains on the SG-GFP protein core secreted basolaterally (15%) carry most of the sulfate added during biosynthesis (Tveit et al. (2005) J. Biol. Chem., 280, 29596-29603). Here we report further differences in apical and basolateral GAG synthesis. The less intensely sulfated chondroitin sulfate (CS) chains on apically secreted SG-GFP are longer than CS chains attached to basolateral SG-GFP, whereas the heparan sulfate (HS) chains are of similar lengths. When the supply of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is limited by chlorate treatment, the synthesis machinery maintains sulfation of HS chains on basolateral SG-GFP until it is inhibited at 50 mM chlorate, whereas basolateral CS chains lose sulfate already at 12.5 mM chlorate and become longer. Apically, incorporation of 35S-sulfate into CS is reduced to a lesser extent at higher chlorate concentrations than basolateral CS, although apical CS is less intensely sulfated than basolateral CS in control cells. Similar to what was found for basolateral HS, sulfation of apical HS was not reduced at chlorate concentrations below 50 mM. Also, protein-free, xyloside-based GAG chains secreted basolaterally are more intensely sulfated than their apical counterpart, supporting the view that separate apical and basolateral pathways exist for GAG synthesis and sulfation. Introduction of benzyl beta-d-xyloside (BX) to the GAG synthesis machinery reduces the apical secretion of SG-GFP dramatically and also the modification of SG-GFP by HS.     

Structural characterization of heparan sulfate and chondroitin sulfate of syndecan-1 purified from normal murine mammary gland epithelial cells. Common phosphorylation of xylose and differential sulfation of galactose in the protein linkage region tetrasaccharide sequence

Syndecan-1, present on the surfaces of normal murine mammary gland epithelial cells, is a transmembrane hybrid proteoglycan, which bears glycosaminoglycan (GAG) side chains of heparan sulfate (HS) and chondroitin sulfate (CS). Purified syndecan-1 ectodomains were analyzed for disaccharide composition and the GAG-protein linkage region after digestion with bacterial lyases. The HS chains contained predominantly a nonsulfated unit with smaller proportions of two monosulfated, two disulfated, and a trisulfated unit, whereas CS chains were demonstrated for the first time to bear GlcUA-GalNAc(4-O-sulfate) as a major component as well as GlcUA-GalNAc, GlcUA-GalNAc(6-O-sulfate), and an E disaccharide unit GlcUA-GalNAc(4,6-O-disulfate) as minor yet appreciable components. Two kinds of linkage region tetrasaccharides, GlcUA-Gal-Gal-Xyl and GlcUA-Gal-Gal-Xyl(2-O-phosphate), were found for the HS chains in a molar ratio of 55:45. In marked contrast, an additional sulfated tetrasaccharide, GlcUA-Gal(4-O-sulfate)-Gal-Xyl, was demonstrated only for the CS chains, and the unmodified phosphorylated and sulfated components were present at a molar ratio of 55:26:19. The present study thus provided conclusive evidence for the hypothesis that 4-O-sulfation of Gal is peculiar to CS chains in contrast to the phosphorylation of Xyl, which is common to both HS and CS chains. These modifications may be required for biosynthetic maturation of the linkage region tetrasaccharide sequence, which is a prerequisite for creating the repeating disaccharide region of GAG chains and/or biosynthetic selective chain assembly of CS and HS chains.     

Identification and distribution of chondroitin sulfate in the three electric organs of the electric eel, Electrophorus electricus (L.)

The electrogenic tissue of the electric eel Electrophorus electricus (L.) is distributed in three well-defined electric organs, the Main electric organ, Sach's organ and Hunter's organ. Sulfated glycosaminoglycan (GAG) composition was characterized in the three electric organs of the electric eel. Sulfated GAGs were analyzed in the electric organs using metachromatic staining, biochemical analysis including electrophoresis before and after specific enzymatic or chemical degradations, and immunostaining with an antibody against chondroitin sulfate (CS). Our results showed in the three electric organs that CS was the main sulfated GAG species detected, accompanied by small and diminutive amounts of CS/dermatan sulfate hybrid chains and heparan sulfate (HS), respectively. However, HS was not detected in the Sach's organ. CS was predominantly detected in the innervated membrane face of the electroplaques in the three electric organs. Our findings extend previous observations on the GAG composition in the electric organs of E. electricus and provide new information regarding the tissue distribution and location of CS.     

Embryonic lung morphogenesis in organ culture: experimental evidence for a proteoglycan function in the extracellular matrix

The lung rudiment, isolated from mid-gestation (11 day) mouse embryos, can undergo morphogenesis in organ culture. Observation of living rudiments, in culture, reveals both growth and ongoing bronchiolar branching activity. To detect proteoglycan (PG) biosynthesis, and deposition in the extracellular matrix, rudiments were metabolically labeled with radioactive sulfate, then fixed, embedded, sectioned and processed for autoradiography. The sulfated glycosaminoglycan (GAG) types, composing the carbohydrate component of the proteoglycans, were evaluated by selective GAG degradative approaches that showed chondroitin sulfate PG principally associated with the interstitial matrix, and heparan sulfate PG principally associated with the basement membrane. Experiments using the proteoglycan biosynthesis disrupter, beta-xyloside, suggest that when chondroitin sulfate PG deposition into the ECM is perturbed, branching morphogenesis is compromised.     

Role of Glycosaminoglycan Sulfation in the Formation of Immunoglobulin Light Chain Amyloid Oligomers and Fibrils

Primary amyloidosis (AL) results from overproduction of unstable monoclonal immunoglobulin light chains (LCs) and the deposition of insoluble fibrils in tissues, leading to fatal organ disease. Glycosaminoglycans (GAGs) are associated with AL fibrils and have been successfully targeted in the treatment of other forms of amyloidosis. We investigated the role of GAGs in LC fibrillogenesis. Ex vivo tissue amyloid fibrils were extracted and examined for structure and associated GAGs. The GAGs were detected along the length of the fibril strand, and the periodicity of heparan sulfate (HS) along the LC fibrils generated in vitro was similar to that of the ex vivo fibrils. To examine the role of sulfated GAGs on AL oligomer and fibril formation in vitro, a κ1 LC purified from urine of a patient with AL amyloidosis was incubated in the presence or absence of GAGs. The fibrils generated in vitro at physiologic concentration, temperature, and pH shared morphologic characteristics with the ex vivo κ1 amyloid fibrils. The presence of HS and over-O-sulfated-heparin enhanced the formation of oligomers and fibrils with HS promoting the most rapid transition. In contrast, GAGs did not enhance fibril formation of a non-amyloidogenic κ1 LC purified from urine of a patient with multiple myeloma. The data indicate that the characteristics of the full-length κ1 amyloidogenic LC, containing post-translational modifications, possess key elements that influence interactions of the LC with HS. These findings highlight the importance of the variable and constant LC regions in GAG interaction and suggest potential therapeutic targets for treatment.     

Sulfated glycosaminoglycans accelerate transthyretin amyloidogenesis by quaternary structural conversion

Glycosaminoglycans (GAGs), which are found in association with all extracellular amyloid deposits in humans, are known to accelerate the aggregation of various amyloidogenic proteins in vitro. However, the precise molecular mechanism(s) by which GAGs accelerate amyloidogenesis remains elusive. Herein, we show that sulfated GAGs, especially heparin, accelerate transthyretin (TTR) amyloidogenesis by quaternary structural conversion. The clustering of sulfate groups on heparin and its polymeric nature are essential features for accelerating TTR amyloidogenesis. Heparin does not influence TTR tetramer stability or TTR dissociation kinetics, nor does it alter the folded monomer-misfolded monomer equilibrium directly. Instead, heparin accelerates the conversion of preformed TTR oligomers into larger aggregates. The more rapid disappearance of monomeric TTR in the presence of heparin likely reflects the fact that the monomer-misfolded amyloidogenic monomer-oligomer-TTR fibril equilibria are all linked, a hypothesis that is strongly supported by the light scattering data. TTR aggregates prepared in the presence of heparin exhibit a higher resistance to trypsin and proteinase K proteolysis and a lower exposure of hydrophobic side chains comprising hydrophobic clusters, suggesting an active role for heparin in amyloidogenesis. Our data suggest that heparin accelerates TTR aggregation by a scaffold-based mechanism, in which the sulfate groups comprising GAGs interact primarily with TTR oligomers through electrostatic interactions, concentrating and orienting the oligomers, facilitating the formation of higher molecular weight aggregates. This model raises the possibility that GAGs may play a protective role in human amyloid diseases by interacting with proteotoxic oligomers and promoting their association into less toxic amyloid fibrils.     

硫酸肝素蛋白多糖在疾病中的作用

硫酸肝素蛋白多糖广泛分布于动物组织的细胞膜和细胞外基质,对于机体发育和维持生理平衡至关重要.聚糖链硫酸肝素特有的分子结构使得这类大分子复合物具有多种生物功能,这些功能主要通过与蛋白质配体的结合实现.细胞表面的硫酸肝素蛋白多糖介导多种细胞活性因子与其受体的结合,参与信号转导的过程.硫酸肝素蛋白多糖也是细胞间质的重要组成部分,与胶原蛋白一起维持间质结构的稳定.肝素酶通过降解硫酸肝素从而调节细胞因子的活性和细胞间质的微环境.因此,揭示硫酸肝素的分子结构及其功能是生物学的一个重要研究方向.然而,由于硫酸肝素结构复杂,且不均一,使得这个领域的研究发展相对缓慢.不过,随着分析手段的提高和完善,国际上对于硫酸肝素结构与功能的报道迅速增加,同时国内对于硫酸肝素的研究也逐步受到重视.关于硫酸肝素的生理功能最近已有几篇比较全面的综述.此综述主要介绍硫酸肝素在病变中的作用,旨在探讨利用硫酸肝素和肝素酶作为靶标,研发预防和治疗这些疾病药物的可能性.