Sci. Aging Knowl. Environ., 14 January 2004
Vol. 2004, Issue 2, p. pe3
[DOI: 10.1126/sageke.2004.2.pe3]


Glycation Stimulates Amyloid Formation

Mark E. Obrenovich, and Vincent M. Monnier

The authors are in the Department of Pathology at Case Western Reserve University, Cleveland, OH 44106, USA. E-mail: vmm3{at} (V.M.M.)

Key Words: amyloid • glycation • protein aggregation • protein folding • {beta} sheet • cross-{beta} structure


One hallmark of aging is the irreversible accumulation of amyloid proteins in arteries and tissues throughout the body. A recent publication from the Dutch group of Gebbink and colleagues (1) provides new information about the process of amyloid formation. Amyloids are a class of insoluble proteinaceous substances (2) that are formed from soluble oligomers (3). Deposits of amyloid appear amorphous under light microscopy, but electron micrographs reveal that all types of amyloid are composed largely of a loose meshwork of linear nonbranching fibrils, 75 to 100 � in width and of indeterminable length (4).

Multiple proteins have the capacity to form amyloid deposits, which are associated with numerous pathological conditions (see, for example, Berezovska Perspective). Some amyloid protein aggregates are found in cellular inclusions, and others become evident as plaques, which may interact with other functional proteins to induce disease. Amyloid fibrillogenesis can involve proteins such as glycosaminoglycans (5) and serum amyloid P component (6), both of which are universal nonfibrillar constituents of amyloid. Additionally, amyloid deposits are known to contain apolipoprotein E (7), basement membrane components, and heparan sulfate proteoglycans, which contribute to the stability of these aggregates (8).

In general, amyloid forms when proteins with a largely {alpha}-helical structure lose their original conformation and are converted into a predominantly {beta}-sheet form, thereby increasing their propensity to form highly insoluble fibrillar aggregates. Amyloid fibrils display a characteristic quarternary structural element, the cross-{beta} conformation, which consists of packed {beta} sheets [reviewed in (9)]. Environmental and genetic factors are known to be involved in amyloid fibrillogenesis, but the mechanism by which this process occurs is poorly understood (see also "Picture This"). The research by Bouma et al. points to protein glycation as a key mechanism in the formation of the cross-{beta} structure of amyloid (1). Glycation is the result of chemical modifications by reducing sugars, oxoaldehydes, or oxidized ascorbic acid with amino groups of proteins, which results in the modification of proteins and leads to the formation of advanced glycation end products (AGEs) (see Monnier Perspective).

Detection of Amyloid

In order to diagnose amyloidosis, pathologists examine biopsies of tissue or organs, such as the liver, kidney, brain, rectum, tongue, and nerves. Even though amyloid proteins are often biochemically unrelated, in situ amyloid is recognizable by specific histological stains that can differentiate {beta}-sheet structure from nonfibrillar {alpha}-helical structures. This {beta}-sheet structure results in the marked affinity of all amyloids for the dye Congo red. Further, the cross-{beta} structure, which possesses different refractive indices with respect to light polarized in different directions, is detectable when stained with Congo red (10, 11). When amyloid-containing specimens are stained with Congo red, and viewed under plane-polarized light with a polarizing microscope, a classic apple-green birefringence is exhibited. These birefringent effects are a result of the change in dichroism, which is the preferential absorption of light in a in a specific direction, and are observed in optically inhomogeneous substances such as amyloid, which contain structural elements with a definite orientation rather than a random arrangement. An example of birefringent amyloid found in human vessels is shown in Fig. 1. Other dyes demonstrate a chromatic shift (change of color) in the presence of amyloid as well; for example, crystal violet stains red rather than violet, and amyloid becomes fluorescent with thioflavin T or thioflavin S (see also "Plaques Aglow").

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Fig. 1. Amyloid angiopathy in cerebral vascular tissue. (A) Hematoxylin and eosin stain viewed by light microscopy. The amyloid appears eosinophilic. (B) Congo red-stained section of same visual field as (A), taken with a polarizing microscope. (C) Congo red stain as in (B), with chromatic shift caused by changing the angle of the polarizing lens. The same amyloid material appears green or orange depending on the angle of the polarizer. Taken together, (B) and (C) show birefringence. [Credit: H. Akiyama/Tokyo Institute of Psychiatry]

Glycation and Amyloid Formation

Previous work has suggested links between protein aggregation and glycation [reviewed in (1)]. For example, amyloid aggregates often display AGE modifications. Amyloid lesions and glycated proteins also share common properties, such as the ability to bind to multiligand receptors such as RAGE (the AGE receptor). [For further information on amyloid and proteoglycan receptor binding, see (8).] To further investigate the potential role of glycation in amyloid formation, Bouma and colleagues (1) incubated purified albumin (a globular protein with a primarily {alpha}-helical structure) with glucose-6-phosphate and other glycating agents such as D(+)-glucose and glyoxylic acid for several weeks. In a search for evidence of {beta}-pleated sheet conformation, which would indicate a conformational change and imply amyloid formation, the glycated albumin preparations were analyzed by transmission electron microscopy, light microscopy (after Congo red and thioflavin T staining), circular dichroism, and x-ray diffraction. The group also used tissue plasminogen activator (tPA) as a novel detection tool, because tPA was recently shown to bind the cross-{beta} structure found in aggregated proteins or amyloid. These experiments indicated that glycated albumin aggregates; binds to Congo red, thioflavin T, and tPA; and contains substantial amounts of {beta}-sheet structure, unlike globular albumin. Glycated albumin was also shown to exhibit the cross-{beta} structure (Fig. 2). The authors conclude that glycation leads to the conversion of globular albumin to amyloid fibrils.

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Fig. 2. Diagram of cross-{beta} structures found in amyloid fibrils formed from AGE-modified albumin. [Reprinted from (1) with permission]

Of significance in Bouma's work is the finding that albumin forms amyloid irrespective of the glycating agent. Given that different glycating agents react more or less avidly and specifically with their particular substrates, including amino acids, it is plausible that the rate, extent, and preference of this process would depend on the major glycating species involved. The finding that amyloid formation is independent of the glycating agent is not entirely unexpected. Nevertheless, it does have an impact on our understanding of the basic science and might lead to the use of more minimally necessary or specific glycating agents in research and help elucidate the mechanism that underlies these modifications.

Further, it also is very plausible that once proteins become glycated at their exposed lysine residues, clearance by the ubiquitin-proteasome system would be impaired (see Gray Review). In this system, ubiquitin is covalently attached to lysine residues, a modification that targets proteins to the proteasome for degradation. Thus, one could imagine how accumulation of proteins as aggregates or as depositions or inclusions in tissues might occur after glycation, because ubiquitination on lysine residues might be impeded. In support of this notion, impairments in the ubiquitin-proteasome system are known to occur with aggregation of proteins (12). Over time, these aggregates might adopt further {beta}-sheet conformation and cross-{beta} structure as a result of glycation processes.

Previous research documented that AGEs can contribute to the insolubility of tissue proteins and even can induce fibril formation of the neuronal-specific Alzheimer-type amyloid, amyloid-{beta} (13). However, this study is the first to implicate glycation as a general mechanism for conformational change--specifically, for inducing the formation of the cross-{beta} structure in fibrillogenesis. The fact that an element of carbonyl stress (the action of reactive carbonyls such as glucose, methylglyoxal, and ascorbic acid oxidation products) is required for this process suggests that appropriate inhibitors of oxidative stress and glycation might be able to delay or prevent amyloidogenesis. Further study will be required to reproduce these findings under incubation conditions that more closely mimic a physiological system.

Amyloid and Disease

Glycated proteins are associated with a variety of conditions, including diabetes, atherosclerosis, end-stage renal disease, and cataracts. Formation of glycated amyloid is likely associated with biological toxicity, as evidenced by the presence of inflammatory cells in the vicinity of amyloid deposits (14). Eventually the surrounding cells undergo atrophy. Whether amyloidogenesis is a protective compensatory process that occurs in response to an underlying disease-inducing insult, leading thereby to simple lesion formation, or whether it is the cause of disease itself, amyloid eventually impairs tissue function (15). Amyloid formation is well documented in neurodegenerative diseases such as amyotrophic lateral sclerosis, Parkinson's disease, Huntington's disease, transmissible spongiform encephalopathies, Creutzfeldt-Jacob disease (CJD) (including the new variant form), prion disease PrPsc, Gerstmann-Str�ussler-Scheinker (GSS) syndrome, fatal familial insomnia (FFI), and Alzheimer's disease (and see "Detangling Alzheimer's Disease"), which regularly includes amyloid-{beta}/A4 deposition in the brain and in the cerebrovasculature (16-19) (Fig. 3). Patients with CJD, GSS, and FFI carry autosomal dominant amyloidogenic mutations in the prion protein gene (20). Thus, in these diseases, amyloidosis and conversion to the {beta}-sheet conformation occur regardless of the presence of an infectious agent.

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Fig. 3. Amyloid in a neurodegenerative disease, as revealed by thioflavin S staining. A neurofibrillary tangle is shown in (A) and a senile plaque is shown in (B). [Credit: P. Gambetti/Case Western Reserve University]

With regard to the aging process, the discovery that glycation predisposes proteins to amyloid formation will undoubtedly lead to new efforts to find methods to prevent protein glycation. Such methods have been recently reviewed (21, 22). Additionally, methods that prevent the potential seeding process that catalyzes fibril formation (a nucleation-dependent process similar to crystal formation in the chemistry lab) will be needed. Thus, it is hoped that exploration of the mechanism of AGE formation and the role of AGEs in degenerative diseases eventually will result in novel therapeutics for such diseases, as well as methods of protecting against amyloid formation in general.

January 14, 2004
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Citation: M. E. Obrenovich, V. M. Monnier, Glycation Stimulates Amyloid Formation. Sci. Aging Knowl. Environ. 2004 (2), pe3 (2004).

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