One strategy scientists are trying to slow the progression of Alzheimer’s is inhibiting the BACE1 enzyme that cleaves the amyloid precursor protein (APP). However, since BACE1 cuts a range of other substrates, side effects are a concern. One group now reports that changing the sugar coating on the enzyme’s surface could tweak its function just enough to reduce APP cleavage, while perhaps sparing other substrates. Researchers led by Naoyuki Taniguchi, RIKEN-Max Planck Joint Research Center, Wako, Japan, report in the January 15 EMBO Molecular Medicine that knocking out a sugar transferase responsible for the modification reduced Aβ production and pathology in mice. The authors propose that inhibiting this enzyme could treat AD, and may pose fewer risks than blocking BACE1.
“This paper gives us a window onto a new avenue that we have not looked at carefully before,” said Gopal Thinakaran, University of Chicago. “Sugar modification of an enzyme could have a profound influence on the outcomes for Alzheimer’s pathogenesis.”
Glycosylation is a common post-translational modification of proteins. Added sugars enable everything from proper protein folding to ligand-receptor interactions. The sugars themselves can be further modified. For instance, the enzyme glycosyltransferase GnT-III (GnT-III) adds the monosaccharide N-acetylglucosamine (GlcNAc) in between the branches of a sugar called an N-glycan. This monosaccharide becomes a bisecting GlcNAc, being inserted at the junction of a bifurcating sugar chain (see image at left). GnT-III appears to be the only enzyme that can pull off this modification. Previous studies have suggested that bisecting GlcNAcs suppress cancer (see Song et al., 2010). However, their role in the brain remains unclear. Taniguchi’s group previously found higher amounts of GnT-III in the brains of people with AD than normal controls (see Akasaka-Manya et al., 2010). Since APP and its secretases carry sugars, the scientists wondered whether GnT-III adds bisecting GlcNAc to any of them, and how that might influence Aβ production.
First author Yasuhiko Kizuka and colleagues tested whether sugars on γ-secretase, β-secretase, or APP were decorated with bisecting GlcNAcs. They immunoprecipitated these proteins from APP23 mouse brains and blotted them with E4-PHA, a plant lectin that recognizes the bisecting GlcNAc structure. They found that neither γ-secretase nor APP reacted much, but BACE1 did. In APP23 mice lacking GnT-III, E4-PHA failed to recognize bisecting GlcNAcs on BACE1, suggesting that the enzyme modifies the secretase.
This one small sugar modification had a big impact on AD pathology. APP23 mice with no GnT-III had half the APP BACE1 cleavage products, lower levels of soluble and insoluble Aβ40 and Aβ42, and fewer than half the plaques in their brains, compared with animals with the enzyme. They also navigated a Y maze as well as controls. Together, the results imply that AD pathology worsens when GnT-III adds a bisecting GlcNAc onto BACE1.
Could the same could be true in AD patients? Kizuka reported elevated E4-PHA reactivity with BACE1 in the temporal lobe samples taken from postmortem brains of people who had died with early stage or later-stage AD.
How does the added sugar change BACE1? In vitro, bisecting GlcNAc had no effect on how well BACE1 cleaved its substrates. However, it did seem to relocate some of the enzyme from early endosomes to lysosomes. In brains from 3- and 12-month-old APP23 mice with functional GnT-III, BACE1 co-localized more with APP in early endosomes. In animals that lacked Gnt-III, about 5 percent less BACE1 hung around in endosomes, instead turning up in lysosomes. This hints that bisecting GlcNAc slows trafficking of BACE1 to lysosomes, making it more likely to cleave Aβ in endosomes, wrote the authors. Removing GnT-III cleared the enzyme a bit faster from endosomes (see image below).
The authors are unsure how this happens, but propose that an as-yet-unidentified protein recognizes the bisecting GlcNAc structure and directs the enzyme along the lysosomal pathway.
Sorting BACE1: A bisecting GlcNAc (red circle, left) keeps BACE1 in the early endosome, where it cleaves APP. Loss of the bisecting GlcNAc sends BACE1 to lysosomes, reducing Aβ generation (right). [Image courtesy of Kizuka et al., 2015.]
Interestingly, knocking out GnT-III did not affect BACE1 processing of the two other substrates, full-length CHL1 and contactin-2, whereas BACE inhibitors did block processing of both. While these are only two of the many known BACE1 substrates, the authors think that inhibiting GnT-III may specifically limit APP processing by BACE1. GnT-III knockouts have few significant deficiencies, therefore GnT-III could present a better drug target than BACE1, the authors contend (see Orr et al., 2013). No GnT-III inhibitor exists yet, but Taniguchi said that he and collaborators are preparing to screen 200,000 compounds.
“This is the first paper to describe an N-glycosylation of BACE1 that modifies its function,” said Cheng-Xin Gong, New York State Institute for Basic Research, Staten Island. He pointed out that GnT-III adds bisecting GlcNAcs to a number of other proteins, hence cautions that inhibiting it might have unexpected consequences, as well (see Zhao et al., 2008; Isaji et al., 2010).
Gong expressed doubt that altered trafficking accounts for the entire mechanism, given the small differences in lysosomal BACE between APP23 mice and their Mgat3-knockout counterparts. GnT-III could add the bisecting GlcNAc to the substrate-binding site for APP, he suggested, raising BACE1 affinity for the precursor protein. Thinakaran agreed that the differences between endosome and lysosome co-localization were small, and is likewise unconvinced that a shift in trafficking accounts for the mechanism. However, he agrees that something links the bisecting GlcNAc to APP processing, possibly through effects on BACE1.
In terms of therapeutics, Thinakaran was pleased that GnT-III inhibition did not affect other BACE1 substrates, but wondered if blocking GnT-III during adulthood might cause problems that do not occur in mice that lack GnT-III from the get-go.—Gwyneth Dickey Zakaib
Research Models Citations
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- Akasaka-Manya K, Manya H, Sakurai Y, Wojczyk BS, Kozutsumi Y, Saito Y, Taniguchi N, Murayama S, Spitalnik SL, Endo T. Protective effect of N-glycan bisecting GlcNAc residues on beta-amyloid production in Alzheimer's disease. Glycobiology. 2010 Jan;20(1):99-106. PubMed.
- Orr SL, Le D, Long JM, Sobieszczuk P, Ma B, Tian H, Fang X, Paulson JC, Marth JD, Varki N. A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins. Glycobiology. 2013 Mar;23(3):363-80. Epub 2012 Oct 31 PubMed.
- Zhao Y, Sato Y, Isaji T, Fukuda T, Matsumoto A, Miyoshi E, Gu J, Taniguchi N. Branched N-glycans regulate the biological functions of integrins and cadherins. FEBS J. 2008 May;275(9):1939-48. Epub 2008 Apr 1 PubMed.
- Isaji T, Kariya Y, Xu Q, Fukuda T, Taniguchi N, Gu J. Functional roles of the bisecting GlcNAc in integrin-mediated cell adhesion. Methods Enzymol. 2010;480:445-59. PubMed.
- Agostinho P, Pliássova A, Oliveira CR, Cunha RA. Localization and Trafficking of Amyloid-β Protein Precursor and Secretases: Impact on Alzheimer's Disease. J Alzheimers Dis. 2015;45(2):329-47. PubMed.
- Taniguchi N, Korekane H. Branched N-glycans and their implications for cell adhesion, signaling and clinical applications for cancer biomarkers and in therapeutics. BMB Rep. 2011 Dec;44(12):772-81. PubMed.
- Kizuka Y, Kitazume S, Fujinawa R, Saito T, Iwata N, Saido TC, Nakano M, Yamaguchi Y, Hashimoto Y, Staufenbiel M, Hatsuta H, Murayama S, Manya H, Endo T, Taniguchi N. An aberrant sugar modification of BACE1 blocks its lysosomal targeting in Alzheimer's disease. EMBO Mol Med. 2015 Jan 15;7(2):175-89. PubMed.