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Protein pathologies: Amyloid guided by glycans

Functional Glycomics (11 November 2010) | doi:10.1038/fg.2010.36

Mutational analysis and tracking of glycosylated isoforms suggest a functional role for glycans in amyloid processing.

Amyloid-β (Aβ) peptides have been closely linked to Alzheimer's disease, but identifying the mechanism of pathogenicity, and even which species is most important in the process, has proved difficult. In addition, although it is now common to study amyloid deposition associated with neurons, Aβ is also deposited in the blood vessels of the brain. By turning their attention to the relevant brain microvascular endothelial cells (BMECs), Taniguchi and colleagues now identify an alternate Aβ precursor, and its glycosylation status, as having an unexpected role in the process.

Amyloid precursor protein (APP) has three alternative isoforms: APP695 is primarily expressed in neurons, whereas APP751 and APP770 are expressed in several cell types. In addition to the APP695 sequence, APP751 contains the Kunitz-type protease inhibitor (KPI) domain, whereas APP770 includes the KPI domain in tandem with an OX2 domain. Each sequence can be cleaved by β-secretase and other enzymes to yield the disease-associated peptides Aβ40 and Aβ42.

Reporting in the Journal of Biological Chemistry, the authors first describe western blotting and other experiments that indicate that BMECs express more APP than do neurons. Furthermore, neurons express only APP695, as expected, whereas BMECs express a substantial amount of APP770. The western blot analysis also revealed a high-molecular-weight band in the BMEC sample; as previous work has suggested that APP might be glycosylated, the authors used a series of enzymatic treatments and lectin pull-down assays to identify N-glycan chains, including high mannose or hybrid glycans, and sialylated O-glycans as likely APP modifications. As only APP770 seemed to contain these glycan modifications, the authors further investigated the OX2 domain, which is unique to this isoform. Mutation of each serine or threonine residue in this region identified Thr353 as a probable point of attachment for an O-glycan, but mutation of four additional sites outside this domain, guided by previous research, was required to completely abrogate O-glycosylation.

These results demonstrated that APP770 is modified by several sugar chains, but what is the role of these carbohydrates? Although several glycoforms could be detected in the cell lysate, the authors detected only a single processed sequence in the cell media. The molecular weight of this processed 'sAPP' showed that the polypeptide was derived from the high-molecular-weight APP770; this origin was explained by a combination of biotinylation experiments indicating that, although both glycosylated and unmodified APP770 can reach the cell surface during normal trafficking, only the glycosylated form was subsequently endocytosed, where it would be exposed to cleavage enzymes. Finally, the authors examined the biological relevance of these studies by confirming APP770 expression (and cleavage products thereof) in cerebral cortex tissue samples.

It remains to be seen how these findings apply to neuronal APP, and what role the Aβ peptides from endothelial cells have in disease pathology as compared to the neuronally derived sequences. However, these results raise exciting new questions about the presence and function of glycans in amyloid processing and deposition. In particular, the authors point out that the O-glycan sites are predicted to be in unstructured regions of the protein, which may spur new hypotheses in cellular internalization and trafficking.

Catherine Goodman

Original research paper

  1. Kitazume, S. et al. Brain endothelial cells produce amyloid β from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form. J. Biol. Chem. (15 October 2010) doi: 10.1074/jbc.M110.144626 | Article