Regulations of AÎ²PP Glycosylation Modification and Roles of

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Regulations of A#PP Glycosylation Modification and Roles of Glycosylation on the A#PP Cleavage in Alzheimer’s disease Peng-Fei Tao, and Hanchang Huang ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00574 • Publication Date (Web): 25 Feb 2019 Downloaded from http://pubs.acs.org on February 27, 2019

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ACS Chemical Neuroscience

Regulations of AβPP Glycosylation Modification and Roles of Glycosylation on the AβPP Cleavage in Alzheimer’s disease Peng-Fei Tao, Han-Chang Huang* Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University, Beijing, 100191, China

Running Title:

Glycosylation modification affects AβPP cleavage *Corresponding author: Han-Chang Huang, Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University, Beijing 100191, China. No. 197, Beitucheng West Road, Haidian District, Beijing, P. R. of China E-mail: [email protected] Phone: +8610-62004534

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Abstract: The presence of senile plaques in the grey matter of the brain is one of the major pathologic features of Alzheimer's disease (AD), and Amyloid-β (Aβ) is the main component of extracellular deposits of the senile plaques. Aβ derives from Amyloid-β precursor protein (AβPP) cleaved by β-secretase (BACE1) and γ-secretase, and the abnormal cleavage of AβPP is an important event leading to overproduction and aggregation of Aβ species. After translation, AβPP undergoes post-translational modifications (PTMs) including glycosylation and phosphorylation in the Endoplasmic reticulum (ER) and Golgi apparatus, and these modifications play an important role in regulating the cleavage of this protein. In this review, we summarized the research progress on the modification of glycosylation, especially O-GlcNAcylation and mucin-type O-linked glycosylation (also known as O-GalNAcylation), on the regulation of AβPP cleavage and on the influence of AβPP’s glycosylation in the pathogenesis of AD. Keywords: Alzheimer’s disease; Amyloid-β precursor protein (AβPP); Protein glycosylation; O-linked N-Acetyl-β-D-glucosamine (O-GlcNAc) transferase (OGT); O-GlcNAcase (OGA)

Introduction Alzheimer's diseases (AD) is a degenerative central nervous system (CNS) disease, and the clinical manifestations include memory loss, cognitive decline, and progressive decline in self-care ability 1-3. AD is characterized by neuronal loss and brain atrophy, and there are two neuropathological characteristics: senile plaques (SPs) and neurofibrillary tangles (NFTs). Senile plaques are the extracellular deposits that mainly contain Amyloid-β (Aβ), which is derived from its precursor—Amyloid-β protein precursor (AβPP). Neurofibrillary tangles are the paired helical filaments (PHF) of hyperphosphorylated microtubule-associated protein tau. So far, the exact pathogenesis of AD isn’t clear. The widely accepted "Amyloid cascade hypothesis" suggests that the deposition of Aβ in the brain is the crucial step that ultimately leads to the death of neurons and AD development. Studies have shown that abnormal aggregation of Aβ may lead to endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and the formation of intracellular NFTs 4, 5. Since AβPP is the precursor of Aβ, it is essential to elucidate the molecular mechanisms of gene expression and regulation, cellular trafficking, and cleavage pathways. Glycosylation modification of proteins is the process of covalent binding of oligosaccharides to the specific amino acid residues of proteins. Glycosylation modification of proteins is evolutionarily conservative, which take places in mammals, echinoderms, worms; and insects, protozoa, and certain types of fungi. According to the chemical bond formed between amino acid residue and the sugar, glycosylation modification of protein is classified as N-linked glycosylation (N-glycosylation) and O-linked glycosylation (O-glycosylation). The N-linked glycosylation modification of a protein mostly occurs in ER and is finished in Golgi apparatus while the O-linked glycosylation occurs at a later stage during protein processing and mostly happens in Golgi apparatus. N-linked glycosylation is formed between asparagine (Asn) residue and the first linked N-Acetyl-β-D-glucosamine (GlcNAc) of oligosaccharide chain. Studies have demonstrated 2 / 20


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that N-linked glycosylation plays important roles on protein folding ， stability, subcellular trafficking and extracellular secretion; protein N-glycosylation might also had a major influence on neurotransmission and synaptic plasticity 6-8. The disorder of N-linked glycosylation may involve in Colorectal cancer, Fetal alcohol syndrome, Parkinson's disease (PD), and AD 9-11. O-linked glycosylation is formed between serine/threonine (Ser/Thr) or tyrosine (Tyr) residues and the first linked sugar of oligosaccharide chain. The first linked sugar of oligosaccharide chain includes N-Acetyl-D-galactosamine, N-Acetyl-β-D-glucosamine; and Xylose, mannose, etc. The GalNAc-type O-linked glycosylation (O-GalNAcylation, also called mucin-type O-glycosylation) and the GlcNAc-type O-linked glycosylation (O-GlcNAcylation) are two major types of O-linked glycosylation. The mucin-type O-glycosylation is related to many physiological functions, such as modulating pro-protein’s processing and recognition and guiding the cell-cell interactions during tissue and organ development12-14. The membrane-associated mucin-1 (MUC1) is highly pressed and aberrantly O-glycosylated in most adenocarcinomas including breast and pancreatic cancers; therefore, the regulation on mucin-type O-glycosylation of this protein has long been considered as a strategy for the immunodiagnosis and the immunotherapy of adenocarcinomas 15. O-GlcNAcylation of proteins was firstly discovered in 1980s by Torres and Hart et al, and later it was found that protein O-GlcNAcylation exists in all the major compartments of the cell including membranes, cytoplasm, mitochondria, and nucleus16, 17. More than 1000 O-GlcNAcylation sits of proteins have been identified, which play critical roles in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression18, 19. As a potential therapeutic target, protein O-GlcNAcylation has been suggested to treat AD, PD, and other diseases related to cell apoptosis and autophagy20-22. There is growing evidence that protein glycosylation contributes to AD pathology since both SPs and NFTs are regulated by protein glycosylation. Studies show that up-regulation of N-glycosylation and O-glycosylation at specific sites of AβPP may regulate this protein’s trafficking and decreases Aβ production 23, 24; Pharmacological means to increase tau O-GlcNAcylation may also attenuate the formation of NFTs by decreasing tau phosphorylation25. In addition, it is suggested that abnormal protein O-GlcNAcylation leads to the energetic deficiency of mitochondria, the synaptic loss, and the neuronal damage 20, 26. In this review, we summarized the molecular mechanism on glycosylation modifications of AβPP and the roles of glycosylation modifications on the cleavage pathways of this protein.

1 An overview of AβPP 1.1 The expression and glycosylation of AβPP AβPP gene is located on human chromosome 21 (21q21.3), and this gene contains 18 exons. Due to the diversity of alternative splicing of immature AβPP mRNA, more than 10 kinds of AβPP transcripts are formed after transcription of AβPP gene, and therefore more than 10 kinds of AβPP isoforms are generated after translation of AβPP gene. Among these isoforms, AβPP770, AβPP751, and AβPP695 are the three major isoforms, and AβPP695 is mainly expressed in neurons 27, 28. After AβPP mRNA is translated into protein in the ER, the nascent AβPP undergoes post-translational modifications (PTMs) including glycosylation and phosphorylation in ER and 3 / 20



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Golgi apparatus 29, 30. The confirmed and the potential glycosylation sites of AβPP are listed in Fig.1 and table 1. As a type I trans-membrane glycoprotein, the mature AβPP is transported into plasma membrane 31, 32; further, the membrane-bound AβPP might undergo alternative cleavage pathways.

Fig. 1 the potential and confirmed glycosylation sites of AβPP695 and AβPP770 Tab. 1 the glycosylation sites of AβPP695 and AβPP770 Types of glycosylation

Clarity

AβPP695

AβPP770

N-linked glycosylation(GlcNAc…)

confirmed

Asn496, Asn46723, 33

Asn571, Asn54234

potential

Ser124

mucin-type

Thr291, Thr292,

O-glycosylation(GalNAc…)

Thr57635, Thr558, Thr577,

Thr633, Thr651,Thr652，

Thr588, Ser59236, 37

Thr663，Ser66736, 37

confirmed

O-GlcNAcylation(GlcNAc…)

potential

Thr291, Thr292, Thr576

O-glycosylation(Xylose…)

confirmed

Ser65637

O-glycosylation(HexNAc…)*

confirmed

Thr659, Tyr68136

Note: * the first linked sugar is still unknown to be which kind of hexose in HexNAc-type of O-glycosylation of AβPP. 4 / 20


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1.2 The transport and the cleavage pathways of AβPP As mentioned above, AβPP is generated in the ER, and the nascent AβPP subsequently undergoes PTMs including N-linked glycosylation and O-linked glycosylation in ER and Golgi apparatus. Then the mature AβPP is transported into plasma membrane where the major part of AβPP is cleaved through the non-amyloidogenic or the amyloidogenic pathway (Fig. 2). On one hand, the membrane-bound AβPP can be cleaved by α-secretase at the peptide bond between 612nd and 613rd amino acid residues in AβPP695 (corresponding to 687thand 688th in AβPP770), producing a soluble N-terminal fragment (sAPPα) and a C-terminal fragment (CTFα, also called C83). The sAPPα is further released to outside of plasma membrane while CTFα is cleaved by γ-secretase, generating P3 peptides (Aβ17-40 or Aβ17-42) and the AβPP intracellular domain (AICD) 38, 39. Since the cleavage site of α-secretase on AβPP is located at the amino acid residues within Aβ sequences, there is no Aβ’s release from AβPP upon cleavage by this secretase. So, this cleavage pathway is called the non-amyloidogenic pathway 5, 40, 41. On the other hand, AβPP is firstly cleaved by β-site AβPP-cleaving enzyme 1 (BACE1, also known as β-secretase) at the peptide bond between 596th and 597th amino acid residues in AβPP695 (corresponding to 671st and 672nd in AβPP770), generating a soluble N-terminal fragment sAPPβ and a C-terminal fragment CTFβ (CTFβ, also called C99), and subsequently the CTFβ is degraded by γ-secretase to produce Aβ1-40 or Aβ1-42 and AICD. This cleavage pathway is called amyloidogenic pathway 28, 42-44. Meanwhile, a part of AβPP in the plasma membrane is rapidly internalized through clathrin-dependent endocytosis and transported to the early endosomal compartment, in which BACE1 mainly exists 45. So, in the early endosomal compartment, AβPP is mainly cleaved through amyloidogenic pathway. Interestingly, the AβPP in the early endosomal compartment might be further transported to trans-Golgi network (TGN) by the retromer, and the AβPP in the TGN is dynamically delivered back to the plasma membrane46.

Fig. 2 the transport and cleavage of AβPP The nascent AβPP in the ER is subsequently undergoes PTMs including N-linked glycosylation and O-linked glycosylation in ER and Golgi apparatus. Then the mature AβPP is transported into plasma membrane. The major part of membrane-bound AβPP is cleaved through non-amyloidogenic or amyloidogenic pathway. Meanwhile, a part of AβPP in the plasma membrane is rapidly internalized through clathrin-dependent endocytosis and transported to the early endosome and might further be delivered to the trans-Golgi network by retromer. The cleavage of AβPP in plasma membrane and early endosomal compartment is occurred through non-amyloidogenic 5 / 20



or amyloidogenic pathway. In non-amyloidogenic pathway, AβPP cleaved by α-secretase and γ-secretase, producing sAPPα, P3 (Aβ17-40 or Aβ17-42), and AICD. In amyloidogenic pathway, AβPP is cleaved by BACE1 and γ-secretase, generating sAPPβ, Aβ1-40 or Aβ1-42, and AICD. How the intracellular Aβ is delivered to the outside of the cell is still controversial.

In recent years, there is a third pathway on the cleavage of AβPP, which suggests that AβPP is cleaved by η-secretase at the site between 504th and 505th amino acid residues in AβPP695, generating a soluble N-terminals AβPPη fragment and a C-terminal CTFη fragment. Then the CTFη is further cleaved by α-secretase, producing a peptide of Aη-α 47. The Aη-α may attenuate neuronal activity and long-term potentiation (LTP) 47. Studies have shown that the activity of η-secretase and the generation of Aη-α may be enhanced by inhibiting the lysosome protein degradation and the activity of BACE1 48. However, the underlying mechanism that η-secretase participates in cleavage pathway of AβPP is not completely understood.

1.3 The physiological functions of AβPP The physiological function of AβPP is not well elucidated yet. On one hand, study has recommended that AβPP mediates the process of synaptic dysfunction triggered by Aβ 49. AβPP facilitates the Aβ oligomer binding to the synapses, and the knockout of AβPP gene prevents this binding as well as the synaptic dysfunctions triggered by Aβ. On the other hand, AβPP may play an important role in synaptic repairing, neurons survival, and the improvement of synaptic plasticity. The knockout of AβPP gene causes the damages of the LTP and the spatial learning capacity of rats50. Therefore, AβPP seems to play complicated physiological functions. Since AβPP is a glycoprotein, the glycosylation modification may play a key role on AβPP’s physiological functions.

2 AβPP N-linked glycosylation and its regulation The process of N-linked glycosylation of a protein starts in endoplasmic reticulum and is finished in Golgi apparatus (Fig.3). In endoplasmic reticulum, the β-linkage glycosidic bond is formed after the biochemical reactions between the nascent proteins and dolichol-linked precursor oligosaccharide. During this process, the amino group (-NH2) of the Asn residue in Asn-X-Ser/Thr motif (X can be any residue other than proline) is combined with the precursor oligosaccharide (in animal cells N-Acetyl-β-D-glucosamine is the first sugar binding with Asn) through covalent bond. In endoplasmic reticulum and Golgi apparatus, the precursor oligosaccharide chain bound on proteins is further modified, after the N-linked glycosylation modification, a mature protein is delivered to the plasma membrane. The dynamic balance of N-linked glycosylation of a protein is regulated by oligosaccharyltransferase (OST) and glycosidases 51, 52.

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Fig.3 the biosynthesis of N-linked glycosylation of proteins Studies found that the amino acid residues of Asn467 and Asn496 are the two N-linked glycosylation sites of AβPP695 23, 33(Tab. 1). The levels of Aβ1-40 and Aβ1-42 are significantly reduced when the level of AβPP N-linked glycosylation is elevated in the ER 33, 53. Regulator of calcineurin 1(RCAN1), a subunit of OST, can stabilize OST by interacting with ribophorin I (RPNI), a substrate-specific enhancer of OST, and then enhances the level of N-linked glycosylation of membrane proteins in the ER 29, 54, 55. However, it is worth noting that the RCAN1 enhances both the level of AβPP N-linked glycosylation and the activity of β-secretase and γ-secretase; therefore, Aβ is up-regulated on the contrary when RCAN1 is over activated 29. Since N-linked glycosylation modification of a protein occurs in the ER, where varieties of proteins are synthesized and undergoes diversified post-translational modifications, it is important that the structure of other proteins is not affected when the level of AβPP N-linked glycosylation is increased by treatment with drugs or endogenous substances.

3 AβPP O-linked glycosylation and its regulation O-linked glycosylation is the attachment of a reducing monosaccharide to an oxygen atom in a Ser or a Thr residue of a protein, and these reducing monosaccharides include N-Acetyl-D-galactosamine (GalNAc), N-Acetyl-β-D-glucosamine (GlcNAc), and so on. As mentioned above, according to the difference on the reducing sugar, there are several kinds of protein O-linked glycosylation; mucin-type O-glycosylation (GalNAc-type O-linked glycosylation) and GlcNAc-type O-glycosylation (O-GlcNAcylation) are the two main types of O-linked glycosylation of proteins. UDP-GalNAc: polypeptide N-α-Acetylgalactosaminyltransferase (GalNAc-T), which belongs to the family of glycosyltransferases (GTFs), is responsible for mucin-type 7 / 20



O-glycosylation modification of proteins, and it can catalyze a protein to generate a single glycosidic bond GalNAc α-Ser/Thr 56. There are 20 different isoforms of GalNAc-T, and most of these isoforms consist of a catalytic domain in N-terminal and a ricin-like lectin domain in C-terminal containing three potential carbohydrate Ser/Thr-O-GalNAc-binding sites. GalNAc-T isoforms are categorized as peptide- and glycopeptide-preferring subfamilies, and most of peptide-preferring isoforms readily glycosylate peptide substrates while the glycopeptide-preferring isoforms commonly display nearly sole activities against Ser/Thr-O-GalNAc containing glycopeptides. The peptide/glycopeptide speciﬁcity of the peptide-/glycopeptide-preferring isoforms of GalNAc-T predominantly exists in their catalytic domain but may be further regulated by remote lectin domain in C-terminal 57. A study had indicated that injection of Aβ25–35 in the hippocampal CA1 region of rats induces mucin-type O-glycosylation of hippocampal proteins and subsequently results in neuroinﬂammation and neuronal death 58. O-GlcNAcylation is a new type of protein glycosylation modification, in which the O-linked β-N-Acetylglucosamine is formed between N-Acetyl-β-D-glucosamine and Ser/Thr residue under the catalysis of O-linked N-Acetyl-β-D-glucosamine transferase (OGT), and this process mainly occurs in the Golgi apparatus 59. In contrast, N-Acetyl-β-D-glucosamine is removed from glycosylated proteins under the catalysis of O-GlcNAcase (OGA). Therefore, there is a dynamic balance between gaining and loss of O-GlcNAcylation for a protein, and the level of O-GlcNAcylation can be regulated by altering the activity of OGT and OGA 60. Glucose metabolism impairment in the brain is closely related to cognitive impairment, and it is also thought to be one of the pathological features of early AD 61, 62. Researchers have found that the excessively low level of glucose uptake in the hippocampus and the cingulate gyrus leads to the decline of cognitive ability of AD mice 63, 64. Improving the utilization of glucose in the hippocampus can inhibit Aβ aggregation and improve the ability of spatial learning and memory in the transgenic mice of AβPP/PS1 61, 65.The level of O-linked glycosylation may be affected by glucose metabolism; therefore, it is a potential strategy in AD therapy to reduce cognitive impairment and AD development by targeting glucose metabolism.

3.1 Glucose metabolism and O-GlcNAcylation regulation on protein Glucose is transported to the cytosol by glucose transporter (GLUT) proteins. A part of (about 2-5%) glucose undergoes hexosamine biosynthetic pathway (HBP) to generate uridine 5'-diphosphate-N-Acetylglucosamine (UDP-GlcNAc) (Fig. 4). Thus, the level of UDP-GlcNAc is affected by the utilization of intracellular glucose 66.

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HO

OHHO

O HO

OH

glucose

HK HO

Glycogen synthesis

OH

OH P O OH

O

O

HO OH glucose-6-phosphate

G6P-I O

Glycolysis

HO NH2

H2N

OH

O OH P O OH

HO OH fructose-6-phosphate

OH

GFAT

O O Glutamine H2N

OH

O

OH P

O O HO

O P OH OH N N HO O H2N OH OH N O O O O N P P O O

HN

O

O

N OH H

N H

S O

O

HO

HBP

O O P P O O OH OH OH

O N

OH

glucosamine-6-phosphate

Acetyl-CoA O

OH

OH

uridine diphosphate

uridine diphosphate N-acetylglucosamine (UDP-GlcNAc)

Fig. 4 the biosynthetic process of UDP-GlcNAc A majority of glucose is used for glycogen synthesis and glycolysis after glucose was converted into glucose-6-phosphate (G6P) by hexokinase (HK) and further into fructose-6-phosphate (Fru-6P) by glucose-6-phosphate isomerase (G6P-I). A part of Fru-6P is channeled into the hexosamine biosynthetic pathway (HBP) after Fru-6P is converted into glucosamine-6-phosphate (GlcN-6P) by the catalysis of glutamine: fructose-6-phosphate amidotransferase (GFAT). After acetylation and subsequent uridylation, GlcN-6P is transformed into UDP-GlcNAc, the donor substrate for protein O-GlcNAcylation. UDP-GlcNAc is the donor of glycosyl group and the only substrate of OGT for a protein’s O-GlcNAcylation. The enzyme 9 / 20



OGT has an extremely high affinity to UDP-GlcNAc (Km=545 nM) 67. A very low concentration of UDP-GlcNAc can trigger the OGT-mediated reaction of protein O-GlcNAcylation, and the O-GlcNAcylation level of various proteins could be improved by increasing the concentration of UDP-GlcNAc 68. Therefore, the UDP-GlcNAc is likely to be an important mediator for regulating the level of O-GlcNAcylation. The level of protein O-GlcNAcylation is enhanced in various tissues of hyperglycemia patients 69. Studies have found that even a low concentration of supplemented glucosamine in cultured cells could significantly improve the level of O-GlcNAcylation through bypassing the rate-determining step of HBP (transformation of fructose-6-phosphate into glucosamine-6-phosphate catalyzed by glutamine: fructose-6-phosphate amidotransferase (GFAT)); therefore, HBP is considered to be the main pathway that determining the level of O-GlcNAcylation in cells 70, 71. However, it’s not a simple positive correlation between protein O-GlcNAcylation level and activity of HBP pathway. For example, the O-GlcNAcylation level of peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α (PGC-1α), a key regulator of energy metabolism, reaches the top level when the concentration of glucose arrives to 5 mM, and subsequently the O-GlcNAcylation level is down along with the increase of glucose concentration72. Studies show that AD models both in vitro and in vivo exhibited a marked reduction in global O-GlcNAcylation levels, which was strongly correlated with disruption of the mitochondrial network, damage of mitochondrial bioenergetic function, and loss of cell viability20, 26. At present, it is not clear whether glucose metabolism alters the cleavage pathway of AβPP by regulating the level of O-GlcNAcylation of this protein or the other associated proteins. For what it’s worth, the analysis on the effects of glucose metabolism in both physiological and pathological conditions on protein O-GlcNAcylation in the nervous system can help us to establish a long-term effective prevention strategy for AD.

3.2 OGT regulates O-GlcNAcylation modification of AβPP The enzyme OGT catalyzes the addition of a single N-Acetyl-β-D-glucosamine to Ser or Thr residues of intracellular proteins. The OGT protein is a heterotrimer consisting of two 110-kDa subunits and one 78-kDa subunit. The 110-kDa subunit includes a domain that contains 13 tetratricopeptide repeats (TPR) at N-terminal domain and a catalytic core with a binding site of UDP-GlcNAc at C-terminal domain 73, 74. After S-nitrozation, the catalytic activity of OGT is strongly inhibited up to more than 80% of native OGT, and denitrosylation promotes the catalytic activity of this enzyme, which might lead to the excessive O-GlcNAcylation of downstream proteins on the contrary 75. Therefore, the denitrosylation of OGT could be used as an important way to improve the level of protein O-GlcNAcylation. Liu et al 66 silenced the OGT gene with small hairpin RNA, resulting in the reduction of O-GlcNAcylation level of tau protein but the increase of phosphorylation level of this protein. The aggregated tau protein doesn’t seem to undergo O-GlcNAcylation at all in the brain of AD patients 76. Notably, both serine and threonine residues are also the phosphorylation sites of both AβPP and tau protein77. Therefore, the post-transcriptional modification of AβPP on O-GlcNAcylation and on phosphorylation shows a competitive relationship as the same as tau protein does; inhibiting phosphorylation of AβPP may increase the level of O-GlcNAcylation of this protein.

3.3 OGA regulates O-GlcNAcylation modification of AβPP 10 / 20


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OGA belongs to the family of glycoside hydrolase, and its N-terminal domain has a hydrolytic enzyme activity, while the C-terminal region displays a histone acetyltransferase activity 78, 79. Some microRNAs (miRNAs), which regulate gene expression by inhibiting the translation of target mRNAs, might regulate the expression of OGA gene. For example, miRNA-539 significantly inhibits the expression of OGA gene and increases the level of protein O-GlcNAcylation in the myocardial cells of newborn rats 80. However, it remains to be further explored whether miRNA-539 can regulate OGA expression and further improve O-GlcNAcylation level of AβPP in neurons. The chemical PugNAc, a potent OGA inhibitor, elevates the level of AβPP O-GlcNAcylation in the HeLa cells overexpressing human AβPP of Swedish mutation 46. Chun, Y. S. et al 24 mutated respectively three predicted O-GlcNAcylation sites of AβPP695 into alanine (Thr291Ala, Thr292Ala and Thr576Ala) and expressed these three mutations of AβPP stably in HeLa cells, respectively. These AβPP695 mutants all reduce the level of O-GlcNAcylation on these proteins, and the mutant with Thr576Ala almost completely inhibits the alteration of O-GlcNAcylation level when cells were treated with the OGA inhibitor PugNAc. These results indicate that Thr291, Thr292 and Thr576 might be the endogenous modified sites of AβPP O-GlcNAcylation and that Thr576 is not only the main endogenous modified site but also the key site regulated by OGA chemical inhibitors. Therefore, Thr576 residue in AβPP695 is an important site for the regulation of AβPP cleavage, and O-GlcNAcylation of AβPP695 at Thr576 is likely to be a new therapeutic target for AD treatment.

3.4 The effect of O-linked glycosylation on AβPP cleavage Although the physiological function of AβPP is still a controversial topic, AβPP seems to play diverse roles on the cell survival and cell damage. The knockout of AβPP gene may be negative for brain development. Therefore, more and more researches focus on the regulation of AβPP cleavage pathways to suppress the generation of Aβ. Protein O-GlcNAcylation level in the brain extract of AD patients is about 22% lower than that in age controls 81. After translation, AβPP undergoes various post-translational modifications including glycosylation, and these modifications might affect AβPP’s translocation and cleavage. Therefore, modification on O-linked glycosylation of AβPP may also be an important target for AD therapy.

3.4.1 The effect of mucin-type O-linked glycosylation on AβPP cleavage Keiko Akasaka-Manya et al 82 transfected three genes of GalNAc-T family (GALNT1, GALNT4 and GALNT6) into the HEK293T cells to overexpress GalNAc-T1, GalNAc-T4 and GalNAc-T6 isomers respectively, and they found that all the three GalNAc-Ts show the regulatory activity to AβPP cleavage pathways, but GalNAc-T6 shows the most prominent activity. GalNAc-T1 and GalNAc-T4 only reduced Aβ1-40 generation while GalNAc-T6 significantly reduced both generation of Aβ1-40 and Aβ1-42. These results indicate that mucin-type O-linked glycosylation catalyzed by GalNAc-Ts plays a significantly role in AβPP post-translational modification, and the overexpressed GalNAc-Ts improve AβPP mucin-type O-linked glycosylation and inhibit Aβ generation. However, it is not clear that the specific mechanism on inhibiting Aβ generation by improving the level of AβPP mucin-type O-linked glycosylation. The mucin-type O-linked glycosylation of a protein may not only affect its conformation but also 11 / 20



affect its transport and localization in the cell 83, 84. The expression of α-secretase and β-secretase is also slightly altered in the cells transfected with GalNAc-Ts, but the activities of α-secretase and β-secretase are not significantly changed 82. Therefore, the mucin-type O-linked glycosylation of AβPP catalyzed by GalNAc-Ts may change its conformation, reduce the sensitivity of this protein to BACE1, and then affect the production of Aβ. In addition, mucin-type O-linked glycosylation catalyzed by GalNAc-Ts may also have an inhibitory effect on AβPP transport to endosome and further to trans-Golgi network, which changes both amyloidogenic and non-amyloidogenic pathways and sequentially affects the generation of Aβ. It has been reported that the site-specific mucin-type O-linked glycosylation of a membrane-bound protein is related to the cleavage of its ectodomain. The O-linked glycosylation of TNF-α regulated by the GalNAc-T2 isoform regulates the extracellular shedding of a 17-kDa C-terminal fragment mediated by ADAM17, which has an activity of α-secretase for AβPP 85. The mucin-type O-linked glycosylation of AβPP might affect the protein’s cleavage by α-, β-, and γ-secretases. The mucin-type O-glycans are attached at Ser124, Thr291, Thr292; Thr558, Thr576, and Thr577; Ser588 and Ser592 in the AβPP695 (as shown in Tab.1) 35-37. Ser124 is necessary for AβPP’s mucin-type O-linked glycosylation modification and trafficking to the secretory pathway, and the substitution of Ser124 to Cys124 diminishes AβPP cleavage by both α-secretase and β-secretase32. In HEK293T cells, the Thr577 residue of AβPP695 is preferentially O-GalNAcylated by GalNAc-T6 whereas Thr576 may be O-GalNAcylated by other GalNAc-Ts instead of GalNAc-T6, GalNAc-T4 or GalNAc-T182.

3.4.2 The effect of O-GlcNAcylation on AβPP cleavage O-GlcNAcylation might not only affect the translocation of AβPP but also play a role on the cleavage of this protein. On one hand, the transport of AβPP695 into cell membrane is reduced when the Thr576 residue was replaced with Ala, which is the potential site for O-GlcNAcylation 24. On the other hand, studies have shown that AβPP’s O-GlcNAcylation inhibits its endocytosis and improves its cleavage via non-amyloidogenic pathway, preventing the over-generation of Aβ 46, 86. Furthermore, sAPPα, the cleavage production of AβPP via non-amyloidogenic pathway, could improve the level of transthyretin and consequently prevents the hyperphosphorylated tau and hippocampal damage induced by Aβ 87. There is increasing evidence indicating that O-GlcNAcylation on AβPP has an effect on AβPP cleavage. O-GlcNAcylation of AβPP at different sites might play a different role on the pathway of AβPP cleavage. When cells were treated with OGA inhibitor PugNAc, which results in significant increase of O-GlcNAcylation at Thr576 residue of AβPP695, the level of membrane-bound AβPP is increased and the non-amyloidogenic pathway of AβPP cleavage is up-regulated along with more sAPPα generation but less generation of sAPPβ and Aβ 46. Jacobsen, K. T. et al 86 got the same results that AβPP O-GlcNAcylation promoted non-amyloidogenic pathway and reduced the generation of Aβ in neuron-like cells by small interfering RNA (siRNA) targeting with OGA and OGT. The globally cytosolic protein O-GlcNAcylation had been reported to increase in AD brain compared with age-matched controls, which may be due to the decreased expression of OGA; the protein level of OGA in AD brain is significantly decreased up to 75% of 12 / 20


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that in age-matched control while OGT protein level is unchanged 88. Therefore, OGA might be the potential target in the regulation on the pathway of AβPP cleavage.

4 Problems and prospects The dyshomostasis on global glycosylation of proteins may affect learning and memory ability of AD mice, and the glycosylation modification of AβPP in AD pathology may further moderate cleavage pathway of this protein 89. Therefore, AβPP glycosylation is becoming an important factor to explore the pathogenesis of AD. It is necessary to further clarify the specific glycosylation sites when investigating the role of glycosylation on the AβPP cleavage pathway. Compared with the change on the expressing level of a protein, the change on abundance of O-GlcNAc peptide could be altered more. At present, a total of 1850 O-GlcNAc peptides covering 1094 O-GlcNAcylation sites are identified from 530 proteins in the human brain, and 131 O-GlcNAc peptides covering 81 proteins were altered in AD brains compared with age controls90. The altered O-GlcNAcylation proteins belong to several structural and functional categories, including synaptic proteins, cytoskeleton proteins, and memory-associated proteins; all these proteins are closely related with AD’s occurring. However, it is still a big challenge to explore the molecular characteristics of glycosylated protein in the level of proteomics due to low stoichiometry and stability of glycosylation modification. Protein glycosylation is a highly dynamic and labile modification. The traditional mass spectrometry fragmentation technology, such as collision induced dissociation (CID), is difficult to analyze this post-translational modification, and such a modification on a protein is often lost before fragmentation of the peptide backbone. Therefore, it is difficult to determinate the localization of the glycosylation site by conventional CID technology. Recently, a new fragmentation technique of electron transfer dissociation (ETD) can preserve the labile PTMs including glycosylation of a protein, making it clear easier to identifying the class of PTMs and the localization of glycosylation sites 91. By combining chemical analysis and various mass spectrometric approaches including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS with stepping-energy CID and ETD, Stavenhagen K. et al 92 performed an in-depth site-specific N- and O-linked glycosylation analysis on human C1-inhibitor, a highly glycosylated plasma glycoprotein. They identified 10 mucin-type O-linked glycosylation sites, carrying mainly core1-type O-glycan, and 6 N-linked glycosylation sites on C1-inhibitor. Numerous studies have shown that AβPP play an important role in AD pathogenesis. Using advanced glycosylation analysis technology, the future researches should focus more on the regulatory mechanism of AβPP in the respect of intracellular transport and cleavage pathway mediated by protein glycosylation. Such researches could provide us a substantial foundation to deeply understanding and preventing AD.

Acknowledgments This study was supported by the National Natural Science Foundation of China (Grant No. 31471587), Premium Funding Project for Academic Human Resources Development in Beijing Union 13 / 20



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University (Grant No. BPHR2018CZ02), and the Scientific Research Project from Facing Characteristic Discipline of Beijing Union University (Grant No. KYDE40201703).

Conflict of Interest The authors have no conflict of interest to report.

Author Contributions This manuscript was written by Mr. Peng-Fei Tao, who is a postgraduate student, and professor Han-Chang Huang, who is the supervisor of Tao. As the corresponding author, professor Huang designed the main theme of the manuscript and the guidelines of the sections and the paragraphs. Mr. Tao finished the writing of the first draft, and professor Huang revised the manuscript thoroughly. The figures and the table was also finished by Mr. Tao under the guidance of professor Huang.

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Table of Contents Graphic

Brief Summary The nascent AβPP in the ER is subsequently undergoes PTMs including N-linked glycosylation and O-linked glycosylation in ER and Golgi apparatus. The mature AβPP is transported into plasma membrane while immature AβPP might be undergone hydrolysis in the cytosol. The major part of membrane-bound AβPP is cleaved through non-amyloidogenic or amyloidogenic pathway. Meanwhile, a part of AβPP in the plasma membrane is rapidly internalized through endocytosis and transported to the early endosome and might further be delivered to the trans-Golgi network by retromer. The abnormal glycosylation of AβPP might affects the cleavage of this protein. Increasing of amyloidogenic cleavage of AβPP results in the over-production of Aβ, which is a main molecule of the senile plaques.

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Regulations of AÎ²PP Glycosylation Modification and Roles of

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