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A breakthrough in understanding the sugar biology of multicellular organisms

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<div data-thumb=”https://scx1.b-cdn.net/csz/news/tmb/2023/a-breakthrough-in-unde.jpg” data-src=”https://scx2.b-cdn.net/gfx/news/hires/2023/a-breakthrough-in-unde.jpg” data-sub-html=”CMT activity is not divalent metal ion dependent. a, Schematic of CMT-mediated tryptophan C-mannosylation of secretory and transmembrane proteins in the endoplasmic reticulum (ER). Nascent polypeptide chains (pink line) containing the WxxW/C sequon (pink boxes) are mannosylated by CMT using Dol-P-Man (mannosyl group depicted in green) as donor substrate, thereby forming the depicted C-glycosidic bond. Glycopeptides are subsequently folded and secreted via the Golgi apparatus. b, In vitro C-mannosylation reaction using purified CMT CeDPY19. Tricine–SDS–PAGE was used to separate fluorescently labeled acceptor peptide upon mannosylation or unmodified, n = 1 independent replicates. c, LC–MS analysis of in vitro C-mannosylation reaction, demonstrating the attachment of a single hexose to the fluorescently labeled acceptor peptide, n = 1 independent replicates. d, Tricine–SDS–PAGE analysis of in vitro C-mannosylation reaction in presence of the divalent metal ions MnCl2 and MgCl2 as well as in the absence of divalent metal ions and with CeDPY19 preincubated with the metal ion chelator EDTA, demonstrating that CMT activity is unaffected by the absence of divalent metal ions, n = 1 independent replicates. Credit: Nature Chemical Biology (2023). DOI: 10.1038/s41589-022-01219-9″>
CMT activity is not divalent metal ion dependent. a, Schematic of CMT-mediated tryptophan C-mannosylation of secretory and transmembrane proteins in the endoplasmic reticulum (ER). Nascent polypeptide chains (pink line) containing the WxxW/C sequon (pink boxes) are mannosylated by CMT using Dol-P-Man (mannosyl group depicted in green) as donor substrate, thereby forming the depicted C-glycosidic bond. Glycopeptides are subsequently folded and secreted via the Golgi apparatus. b, In vitro C-mannosylation reaction using purified CMT CeDPY19. Tricine–SDS–PAGE was used to separate fluorescently labeled acceptor peptide upon mannosylation or unmodified, n = 1 independent replicates. c, LC–MS analysis of in vitro C-mannosylation reaction, demonstrating the attachment of a single hexose to the fluorescently labeled acceptor peptide, n = 1 independent replicates. d, Tricine–SDS–PAGE analysis of in vitro C-mannosylation reaction in presence of the divalent metal ions MnCl2 and MgCl2 as well as in the absence of divalent metal ions and with CeDPY19 preincubated with the metal ion chelator EDTA, demonstrating that CMT activity is unaffected by the absence of divalent metal ions, n = 1 independent replicates. Credit: Nature Chemical Biology (2023). DOI: 10.1038/s41589-022-01219-9

In multicellular organisms, there are three types of protein glycosylation. N-glycosylation, O-mannosylation and C-mannosylation. All of these processes take place in the endoplasmic reticulum, and in all of them enzymes attach sugar residues to specific sites in newly forming protein.

While N- and O-glycosylation are well studied, the third form, C-mannosylation of tryptophan side chains, has long been a mystery to researchers. Although 20 percent of all secretory proteins, as well as , are affected by it, it was unclear until recently what the change was for, how the specific sequences are recognized and how the associated reaction is chemically possible at all.

In an , researchers from ETH Zurich, the Walter and Eliza Hall Institute of Medical Research (WEHI) in Australia, the University of Chicago and the University of Bern have now elucidated the structure and function of the responsible enzyme, ‘tryptophan C-mannosyltransferase’ (CMT). The corresponding study was published in the latest issue of the journal Nature Chemical Biology.

CMT is a member of the category C (GT-C) glycosyltransferase enzymes, one of the three glycosyltransferase superfamilies. The most prominent member is oligosaccharyltransferase (OST), which is responsible for N-glycosylation.

Similar to the OST, the CMT also recognizes highly specific sequences in proteins, with the difference, however, that in mammals four different CMTs occur simultaneously, which also recognize different protein sequences.

Sugars help immuno-receptors to the cell surface

Only in recent years, the necessary tools, such as special antibodies and mass spectrometry test methods, were developed in order to be able to investigate the extent of C-mannosylation. It was shown that this process occurs almost exclusively where cell-cell communication is essential, especially in cytokine receptors of the immune system and adhesion GPCRs. The latter serve as “sensory antennae” for growing neurons that make their way through the brain.

“The topic is red-hot, especially for our understanding of the cell-cell communication of the immune system,” explains Kaspar Locher, Professor of Structural Biology at ETH Zurich: “Signaling molecules such as cytokines direct the immune response during an infection. While these and their associated receptors have been intensively studied for decades, it has long been neglected that C-mannosylation determines whether a cytokine receptor reaches the to exert its function.”

“With our insights into the structure of the enzymes involved, we now have a near-complete understanding of how C-mannosylation gets to these receptors,” adds study first author Joël Bloch, former senior scientist in Locher’s group.

Tailor-made molecular construction kit

The ETH researchers succeeded in producing the CMT enzyme in its pure form. With the help of chemists from WEHI (AUS) and the University of Bern, they built customized molecules that mimic CMT-specific protein sequences and sugar substrates. This allowed them to test the enzyme for its specific properties in the test tube for the first time.

The researchers quickly realized that the enzyme chemistry of CMT must be novel and completely different from that of OST. “In such a case, we can only find out the mechanism of an enzyme using high-resolution structural elucidation. The problem, however, was that CMT could not be crystallized until now and had too little mass for cryo-EM, because this technique is particularly difficult to apply to proteins below 100 kDa,” Locher explains.

Antibody enables high-resolution electron microscopy

A trick finally brought the breakthrough: In collaboration with researchers from the University of Chicago, the ETH scientists produced a synthetic antibody that binds specifically to the CMT. This antibody increased the mass of the enzyme so much that its structure could be elucidated with the help of cryo-EM. With the help of the cryo-EM structures, the group led by Kaspar Locher was finally able to decipher how the different CMT variants recognize different protein sequences.

Based on these insights, the researchers could now predict more precisely which proteins in humans carry the modification. From this, they hope to be able to capture the ‘C-mannosyl proteome’ in the near future.

By deciphering the peptide binding mechanism of CMTs, the researchers also hope to make progress in the production of CMT-specific inhibitors. Such molecules could contribute to advances in drug production, such as those to combat the malaria pathogen Plasmodium falciparum, which has its own CMT and needs it to attach to the host.

The sequence and organ specificity of the human CMT variant CMT2 could also be used, as it plays a key role in sperm development. The new findings could therefore be used to develop CMT2 inhibitors as contraceptives for men.

A novel enzyme mechanism

Another enigma for scientists was the enzymatic mechanism of CMT. This creates a unique carbon-carbon bond between protein and sugar. Using a custom-made CMT inhibitor molecule, the scientists were able to “capture” CMT in the middle of a glycosyl transfer reaction and elucidate a cryo-EM structure of it.

This allowed them to visualize the CMT reaction mechanism: a previously unknown form of electrophilic aromatic substitution enabled by precisely arranged side chains. Such insights could contribute to the development of designer enzymes that catalyze bonds between carbon atoms.

Evolutionarily conserved protective mechanism in glycosyltransferases

With a total of four different structures of the CMT, the scientists succeeded for the first time in visualizing a practically complete catalytic cycle of an enzyme of the GT-C superfamily.

In the process, they uncovered an astonishing mechanism: The sugar substrates of the CMT are complex to produce due to their lipid binding and are therefore particularly valuable. As it turned out, the CMT initially binds them in a non-reactive protected binding pocket. Only when the protein or peptide to be modified docks onto the CMT is the sugar substrate shifted by a peptide sensor and brought into a highly reactive state.

The scientists assume that this mechanism is evolutionarily conserved in GT-C enzymes and prevents valuable substrate molecules from being prematurely consumed. “Having uncovered the general architecture of GT-C enzymes three years ago, we now have a holistic understanding of their enzyme chemistry. It is another milestone in glycobiology,” explains Locher.

More information:
Joël S. Bloch et al, Structure, sequon recognition and mechanism of tryptophan C-mannosyltransferase, Nature Chemical Biology (2023). DOI: 10.1038/s41589-022-01219-9

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Health Canada approves updated Moderna COVID-19 vaccine

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TORONTO – Health Canada has authorized Moderna’s updated COVID-19 vaccine that protects against currently circulating variants of the virus.

The mRNA vaccine, called Spikevax, has been reformulated to target the KP.2 subvariant of Omicron.

It will replace the previous version of the vaccine that was released a year ago, which targeted the XBB.1.5 subvariant of Omicron.

Health Canada recently asked provinces and territories to get rid of their older COVID-19 vaccines to ensure the most current vaccine will be used during this fall’s respiratory virus season.

Health Canada is also reviewing two other updated COVID-19 vaccines but has not yet authorized them.

They are Pfizer’s Comirnaty, which is also an mRNA vaccine, as well as Novavax’s protein-based vaccine.

This report by The Canadian Press was first published Sept. 17, 2024.

Canadian Press health coverage receives support through a partnership with the Canadian Medical Association. CP is solely responsible for this content.

The Canadian Press. All rights reserved.

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These people say they got listeria after drinking recalled plant-based milks

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TORONTO – Sanniah Jabeen holds a sonogram of the unborn baby she lost after contracting listeria last December. Beneath, it says “love at first sight.”

Jabeen says she believes she and her baby were poisoned by a listeria outbreak linked to some plant-based milks and wants answers. An investigation continues into the recall declared July 8 of several Silk and Great Value plant-based beverages.

“I don’t even have the words. I’m still processing that,” Jabeen says of her loss. She was 18 weeks pregnant when she went into preterm labour.

The first infection linked to the recall was traced back to August 2023. One year later on Aug. 12, 2024, the Public Health Agency of Canada said three people had died and 20 were infected.

The number of cases is likely much higher, says Lawrence Goodridge, Canada Research Chair in foodborne pathogen dynamics at the University of Guelph: “For every person known, generally speaking, there’s typically 20 to 25 or maybe 30 people that are unknown.”

The case count has remained unchanged over the last month, but the Public Health Agency of Canada says it won’t declare the outbreak over until early October because of listeria’s 70-day incubation period and the reporting delays that accompany it.

Danone Canada’s head of communications said in an email Wednesday that the company is still investigating the “root cause” of the outbreak, which has been linked to a production line at a Pickering, Ont., packaging facility.

Pregnant people, adults over 60, and those with weakened immune systems are most at risk of becoming sick with severe listeriosis. If the infection spreads to an unborn baby, Health Canada says it can cause miscarriage, stillbirth, premature birth or life-threatening illness in a newborn.

The Canadian Press spoke to 10 people, from the parents of a toddler to an 89-year-old senior, who say they became sick with listeria after drinking from cartons of plant-based milk stamped with the recalled product code. Here’s a look at some of their experiences.

Sanniah Jabeen, 32, Toronto

Jabeen says she regularly drank Silk oat and almond milk in smoothies while pregnant, and began vomiting seven times a day and shivering at night in December 2023. She had “the worst headache of (her) life” when she went to the emergency room on Dec. 15.

“I just wasn’t functioning like a normal human being,” Jabeen says.

Told she was dehydrated, Jabeen was given fluids and a blood test and sent home. Four days later, she returned to hospital.

“They told me that since you’re 18 weeks, there’s nothing you can do to save your baby,” says Jabeen, who moved to Toronto from Pakistan five years ago.

Jabeen later learned she had listeriosis and an autopsy revealed her baby was infected, too.

“It broke my heart to read that report because I was just imagining my baby drinking poisoned amniotic fluid inside of me. The womb is a place where your baby is supposed to be the safest,” Jabeen said.

Jabeen’s case is likely not included in PHAC’s count. Jabeen says she was called by Health Canada and asked what dairy and fresh produce she ate – foods more commonly associated with listeria – but not asked about plant-based beverages.

She’s pregnant again, and is due in several months. At first, she was scared to eat, not knowing what caused the infection during her last pregnancy.

“Ever since I learned about the almond, oat milk situation, I’ve been feeling a bit better knowing that it wasn’t something that I did. It was something else that caused it. It wasn’t my fault,” Jabeen said.

She’s since joined a proposed class action lawsuit launched by LPC Avocates against the manufacturers and sellers of Silk and Great Value plant-based beverages. The lawsuit has not yet been certified by a judge.

Natalie Grant and her seven year-old daughter, Bowmanville, Ont.

Natalie Grant says she was in a hospital waiting room when she saw a television news report about the recall. She wondered if the dark chocolate almond milk her daughter drank daily was contaminated.

She had brought the girl to hospital because she was vomiting every half hour, constantly on the toilet with diarrhea, and had severe pain in her abdomen.

“I’m definitely thinking that this is a pretty solid chance that she’s got listeria at this point because I knew she had all the symptoms,” Grant says of seeing the news report.

Once her daughter could hold fluids, they went home and Grant cross-checked the recalled product code – 7825 – with the one on her carton. They matched.

“I called the emerg and I said I’m pretty confident she’s been exposed,” Grant said. She was told to return to the hospital if her daughter’s symptoms worsened. An hour and a half later, her fever spiked, the vomiting returned, her face flushed and her energy plummeted.

Grant says they were sent to a hospital in Ajax, Ont. and stayed two weeks while her daughter received antibiotics four times a day until she was discharged July 23.

“Knowing that my little one was just so affected and how it affected us as a family alone, there’s a bitterness left behind,” Grant said. She’s also joined the proposed class action.

Thelma Feldman, 89, Toronto

Thelma Feldman says she regularly taught yoga to friends in her condo building before getting sickened by listeria on July 2. Now, she has a walker and her body aches. She has headaches and digestive problems.

“I’m kind of depressed,” she says.

“It’s caused me a lot of physical and emotional pain.”

Much of the early days of her illness are a blur. She knows she boarded an ambulance with profuse diarrhea on July 2 and spent five days at North York General Hospital. Afterwards, she remembers Health Canada officials entering her apartment and removing Silk almond milk from her fridge, and volunteers from a community organization giving her sponge baths.

“At my age, 89, I’m not a kid anymore and healing takes longer,” Feldman says.

“I don’t even feel like being with people. I just sit at home.”

Jasmine Jiles and three-year-old Max, Kahnawake Mohawk Territory, Que.

Jasmine Jiles says her three-year-old son Max came down with flu-like symptoms and cradled his ears in what she interpreted as a sign of pain, like the one pounding in her own head, around early July.

When Jiles heard about the recall soon after, she called Danone Canada, the plant-based milk manufacturer, to find out if their Silk coconut milk was in the contaminated batch. It was, she says.

“My son is very small, he’s very young, so I asked what we do in terms of overall monitoring and she said someone from the company would get in touch within 24 to 48 hours,” Jiles says from a First Nations reserve near Montreal.

“I never got a call back. I never got an email”

At home, her son’s fever broke after three days, but gas pains stuck with him, she says. It took a couple weeks for him to get back to normal.

“In hindsight, I should have taken him (to the hospital) but we just tried to see if we could nurse him at home because wait times are pretty extreme,” Jiles says, “and I don’t have child care at the moment.”

Joseph Desmond, 50, Sydney, N.S.

Joseph Desmond says he suffered a seizure and fell off his sofa on July 9. He went to the emergency room, where they ran an electroencephalogram (EEG) test, and then returned home. Within hours, he had a second seizure and went back to hospital.

His third seizure happened the next morning while walking to the nurse’s station.

In severe cases of listeriosis, bacteria can spread to the central nervous system and cause seizures, according to Health Canada.

“The last two months have really been a nightmare,” says Desmond, who has joined the proposed lawsuit.

When he returned home from the hospital, his daughter took a carton of Silk dark chocolate almond milk out of the fridge and asked if he had heard about the recall. By that point, Desmond says he was on his second two-litre carton after finishing the first in June.

“It was pretty scary. Terrifying. I honestly thought I was going to die.”

Cheryl McCombe, 63, Haliburton, Ont.

The morning after suffering a second episode of vomiting, feverish sweats and diarrhea in the middle of the night in early July, Cheryl McCombe scrolled through the news on her phone and came across the recall.

A few years earlier, McCombe says she started drinking plant-based milks because it seemed like a healthier choice to splash in her morning coffee. On June 30, she bought two cartons of Silk cashew almond milk.

“It was on the (recall) list. I thought, ‘Oh my God, I got listeria,’” McCombe says. She called her doctor’s office and visited an urgent care clinic hoping to get tested and confirm her suspicion, but she says, “I was basically shut down at the door.”

Public Health Ontario does not recommend listeria testing for infected individuals with mild symptoms unless they are at risk of developing severe illness, such as people who are immunocompromised, elderly, pregnant or newborn.

“No wonder they couldn’t connect the dots,” she adds, referencing that it took close to a year for public health officials to find the source of the outbreak.

“I am a woman in my 60s and sometimes these signs are of, you know, when you’re vomiting and things like that, it can be a sign in women of a bigger issue,” McCombe says. She was seeking confirmation that wasn’t the case.

Disappointed, with her stomach still feeling off, she says she decided to boost her gut health with probiotics. After a couple weeks she started to feel like herself.

But since then, McCombe says, “I’m back on Kawartha Dairy cream in my coffee.”

This report by The Canadian Press was first published Sept. 16, 2024.

Canadian Press health coverage receives support through a partnership with the Canadian Medical Association. CP is solely responsible for this content.

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B.C. mayors seek ‘immediate action’ from federal government on mental health crisis

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VANCOUVER – Mayors and other leaders from several British Columbia communities say the provincial and federal governments need to take “immediate action” to tackle mental health and public safety issues that have reached crisis levels.

Vancouver Mayor Ken Sim says it’s become “abundantly clear” that mental health and addiction issues and public safety have caused crises that are “gripping” Vancouver, and he and other politicians, First Nations leaders and law enforcement officials are pleading for federal and provincial help.

In a letter to Prime Minister Justin Trudeau and Premier David Eby, mayors say there are “three critical fronts” that require action including “mandatory care” for people with severe mental health and addiction issues.

The letter says senior governments also need to bring in “meaningful bail reform” for repeat offenders, and the federal government must improve policing at Metro Vancouver ports to stop illicit drugs from coming in and stolen vehicles from being exported.

Sim says the “current system” has failed British Columbians, and the number of people dealing with severe mental health and addiction issues due to lack of proper care has “reached a critical point.”

Vancouver Police Chief Adam Palmer says repeat violent offenders are too often released on bail due to a “revolving door of justice,” and a new approach is needed to deal with mentally ill people who “pose a serious and immediate danger to themselves and others.”

This report by The Canadian Press was first published Sept. 16, 2024

The Canadian Press. All rights reserved.

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