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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.

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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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ETH Zurich

 

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A breakthrough in understanding the sugar biology of multicellular organisms (2023, January 16)
retrieved 16 January 2023
from https://phys.org/news/2023-01-breakthrough-sugar-biology-multicellular.html

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B.C.’s economy, health care and housing to be the focus of throne speech: Eby

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VICTORIA — British Columbia’s economy will be a key focus for politicians as they return to the legislature for the spring sitting.

Premier David Eby says economists are predicating a “global slowdown and potentially recession” and his government is focused on keeping the economy strong by building trade relationships and supporting businesses.

The session starts with a speech from the throne today, which Eby says will outline the government’s key priorities of health care, housing, public safety and the economy.

However, Eby won’t be there for the start of the session.

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He’ll be travelling to Ottawa where Prime Minister Justin Trudeau is hosting a first ministers’ meeting to try to work out an agreement with the provinces and territories for increased funding for health care.

B.C. house leader Ravi Kahlon says the government has plans to introduce more than two dozen pieces of legislation during the session, which is set to conclude in May.

The government will table its budget at the end of the month.

This report by The Canadian Press was first published Feb. 6, 2023.

 

The Canadian Press

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Health

Canada Facing Difficult Battle with Mental Health Struggles

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Mental health is an important health issue that affects us all, and unfortunately, it’s an issue that is rarely discussed openly. According to the World Health Organization, approximately 20% of Canadians will experience a mental illness.

This makes mental health one of the most pressing issues facing Canadians today. Let’s take a closer look at why mental health is such an important issue in Canada.

 

The Need for Better Mental Health Care

In Canada, access to quality mental health care can be costly and difficult to obtain. Many Canadians are unaware of what services are available or how to access them due to a lack of public education about mental health.

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Additionally, there is still a stigma attached to seeking help for mental illness, which can make it difficult for those who need help to get it. As a result, many people cannot access the care they need in order to live happy and healthy lives.

This deficiency can have severe consequences; untreated mental illness can lead to increased risk for suicidal behavior, substance abuse, homelessness, unemployment, and other serious problems.

Additionally, research shows that early diagnosis and treatment can help prevent long-term complications and reduce the overall costs associated with mental health treatment.

Mental health services are especially important for marginalized populations such as Black Canadians, Latinx individuals, LGBTQ+ individuals, immigrants, and individuals with low incomes who have been underserved when it comes to healthcare access.

These communities often experience higher rates of poverty and discrimination which results in an even greater need for quality mental health services but also fewer resources available to them.

Given these facts, it is clear that there is a great urgency for better access to mental health services. To make meaningful progress towards addressing this issue we must first focus on breaking down barriers such as stigma against seeking help as well as lack of information about available services among vulnerable populations.

Furthermore, a greater investment must be made into training more providers so there are enough qualified professionals available who understand how to provide culturally competent care.

Particularly when working with traditionally underserved communities – while also ensuring accessibility through reduced cost or free options for those with limited insurance coverage or financial resources.

 

Mental Illness as an Invisible Disease

Unlike physical illnesses, mental illnesses are often invisible and difficult to diagnose. This makes it difficult for those living with a mental illness to get the help they need as well as understand what they are going through.

It also means that many people do not realize the severity of mental illnesses and the impact they have on the lives of those living with them until they experience it firsthand or hear stories from someone who has gone through similar struggles.

Mental illness affects more people than most realize. It can be difficult to comprehend the depth of mental health disorders, as they are often invisible and misunderstood. Mental illness is a disease, yet it can remain hidden while still having a profound effect on a person’s life.

 

The Impact on Society

Mental illness has far-reaching effects on society as a whole. Untreated mental illness can lead to substance abuse, homelessness, unemployment, and even suicide in some cases.

All of these have ripple effects throughout our communities, from increased crime rates and lower productivity at work to higher healthcare costs and fewer resources available for those in need, making this an issue that affects us all regardless of our personal situations.

We need to create a friendly environment in which those with invisible diseases feel comfortable sharing their stories and seeking help without fear of judgment or rejection.

Mental illness should not be ignored; rather it should be treated with respect and understanding just like any other type of medical condition.

By recognizing the reality of invisible diseases such as depression, anxiety, PTSD, bipolar disorder, and more we can begin to create a world where everyone gets the help they need regardless of whether or not their condition is visible on the surface.

With understanding comes empathy, and empathy leads us toward meaningful change for ourselves and our communities alike.

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Health

Five things to know about health-care talks Tuesday between Trudeau, premiers

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OTTAWA — On Tuesday in Ottawa, Canada’s 13 premiers and Prime Minister Justin Trudeau will sit around the same table in person for the first time since COVID-19 hoping to find a path toward a new long-term health-care funding deal.

Both sides are optimistic a deal will emerge but there are some big divides to overcome, including how much more money Ottawa is willing to put on the table, and how much accountability the provinces are willing to put up in return.

The premiers have been asking for a new deal for more than two years. Trudeau kept punting until the COVID-19 crisis was largely over.

That time has come.

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Trudeau has been clear a deal is not going to be finished this week. But here’s a snapshot of how we got to this point, and what they’re going to be talking about.

Money, Money, Money, Money

This year Canada expected to transfer almost $88 billion to the provinces and territories for health, education, social supports and equalization. The Canada Health Transfer, or CHT, is $45.2 billion, or 51 per cent of that.

In their 2022-23 budgets, the provinces collectively forecast to spend $203.7 billion on health care. Ottawa’s transfer accounts for 22 per cent of that. The provinces want that increased to 35 per cent, which would mean $26 billion more this year alone.

“There’s been continual demands for an increase in the CHT although I’ve never seen quite as large a demand for an increase as this one,” said Gregory Marchildon, a professor emeritus at the Institute of Health Policy, Management and Evaluation at the University of Toronto.

Trudeau intends to put an offer on the table Tuesday. It will not be an immediate increase of $26 billion, but Ottawa has been silent on where it will land.

While it has existed in its current form only since 2004, some sort of federal health transfer dates from 1957, when Ottawa offered 50-50 funding for health care to provinces that agreed to provide public hospital services based on national standards.

It has evolved and changed at least five times since then, including splitting the federal share between cash and a transfer of tax points — when the federal government cut its income tax rates and the provinces could raise their own in exchange.

In 1995, then-finance minister Paul Martin, desperate to turn around Canada’s debt problems, slashed the health and social transfer by 20 per cent, followed by a 15 per cent cut in 1996. Some provinces have said their health systems have never recovered.

In 2004, a new deal was reached between the premiers and Martin, who by then was prime minister, to see the Canada Health Transfer increased six per cent a year for a decade.

The Conservatives under prime minister Stephen Harper kept that in place, but told the provinces that in 2017-18, the CHT increase would be based on a three-year average of economic growth, but with a minimum increase of at least three per cent.

Trudeau and the Liberals have maintained that.

With economic growth, the annual CHT increase has averaged five per cent since 2017-18.

Over the last 10 years, the CHT has increased 67 per cent, to $45 billion from about $27 billion in 2012-13.

An attempt in 2016 to negotiate a new CHT deal mostly failed, resulting in one-on-one agreements between Ottawa and the provinces and territories to share $11.5 billion over 10 years, beginning in 2017-18, to improve mental-health and home care.

Angling for Accountability

In the split jurisdictional world Canada’s governments live in, provinces are the ones who control health-care delivery. So for the most part, the federal government helps fund it and the provinces get to say how it’s spent.

The Canada Health Act, passed in 1984, sets out the guiding principles for recipients of the Canada Health Transfer, including that health-care systems must be universally accessible. Failing to abide by the principles can, and has, resulted in Ottawa clawing back some transfers.

Trudeau has made clear any increase to federal health transfers must be met with provincial accountability to show results. The federal government has been frustrated at the lack of accountability from provinces over transfers for health care made during COVID-19.

It is adamant that will not be the case with a new funding deal, and is looking at a combination of an annual increase to the CHT and separate deals to target specific problem areas, like health-care worker retention and training, access to family doctors, surgical backlogs, and data collection and sharing.

The 2017 deals on mental-health and home care will be a bit of a model. Those deals saw Ottawa promise $11.5 billion over 10 years for the two areas, but in exchange provinces had to agree to a common set of principles and goals, and to report results.

The Canadian Institute for Health Information was tapped to help collect and publish data. The most recent report in December is still laden with gaps and incomplete data. The reports note it will take time for the reporting to lead to change, and that provinces need to harmonize their data collection in order to better compare statistics across provincial lines.

Marchildon said one of the biggest problems for the federal government in demanding accountability is that measuring health outcomes is difficult, and hard targets are rare.

It’s all about the numbers

Of course, it’s difficult to measure progress if you’re not keeping track.

Data — or the lack of it — is a long-standing weakness of Canada’s federalized system, with 13 separate health-care systems working alongside one another but not necessarily in tandem.

In his first public overture to open negotiations with provinces on health funding in November, Health Minister Jean-Yves Duclos told provincial health ministers the federal government would increase the Canada Health Transfer if provinces agree to work together on a “world-class health data system for Canada.”

“It is the foundation for understanding what we’re doing, who’s receiving services, whether we’re making improvements,” said Kim McGrail, a professor with the University of British Columbia School of Population and Public Health.

McGrail was one of several experts the federal government tasked with reporting on what a “world-class health data system” would look like in Canada.

Gaps in Canada’s data tripped up the national health responses in dozens of different ways during the pandemic, from tracking the number of COVID-19 cases to reporting adverse effects from vaccines.

The same is true of tracking surgical backlogs and other information about how well, or not, the health system is working.

“Data informs every part of the way we think about health,” McGrail said, which includes the health of individual patients.

Canadians who move from one province to another can’t easily access their records because the technology isn’t compatible.

It’s a problem that exists even within provinces, as incompatible technology makes records inaccessible between hospitals and clinics.

“We need those technology systems to be able to talk to one another, to be able to to move data back and forth or to send messages back and forth in some way,” she said.

It’s an expensive problem to fix. Just last week, Nova Scotia government signed a $365-million contract to bring new electronic health-care records to the province, which may or may not be compatible with other provincial systems.

McGrail said investments will pay off if important information about the health of Canadians stops falling through the cracks.

The expert panel delivered a report last year that will likely serve as a road map for improving data sharing in Canada. It includes 31 recommendations, starting with provinces, territories and the federal government agreeing on a shared national vision for health data.

Ontario and Quebec have indicated a willingness to work with Ottawa on data, though other provinces have been less firm about it.

Aging gracefully

Provincial leaders have been able to agree with Ottawa on the need to reform Canada’s long-term care homes, though exactly how to accomplish that is still up for debate.

Duclos has said helping Canadians “age with dignity” is one of Ottawa’s priorities for a new health-care deal, and long-term care plays a major role in that.

So does home care, and the 2017 bilateral deals already began to advance improvements on that front.

Long-term care is an entirely different story.

The pandemic cast a glaring light on the dismal conditions in care homes across the country, when COVID-19 outbreaks led to thousands of deaths and inhumane living conditions for seniors. The military and the Red Cross were summoned to help.

In the early months of the pandemic, Canada had the worst record for COVID-19-related deaths in long-term care of the world’s wealthy countries.

Meanwhile, residents were isolated from the outside world and workers struggled to provide basic care and ensure dignity.

Experts and advocates say the problems long predate the pandemic, and have gone largely ignored until now.

“Given the devastation that we’ve seen in the COVID-19 pandemic and the impacts on our health-care system … we’re seeing this unprecedented moment where finally there’s some hope of collaboration,” said Dr. Amit Arya, a palliative care physician and founder of Doctors for Justice in Long-Term Care, which advocates for an overhaul of Ontario’s long-term care system.

Governments are now scrambling to improve the conditions, as the number of people who need specialized care grows every year and the number of workers willing to provide that care dwindles.

Several provinces have already announced plans to increase the number of hours of care residents receive per day and build new spaces for the growing number of seniors who are living longer with more serious cognitive and physical impairments.

The federal government created a $1 billion “safe long-term care fund” during the pandemic to help pay for immediate infection prevention and control measures to stop the spread of the virus.

The government also set aside $3 billion to help provinces bring homes in line with national standards for the design and operation of long-term care, though specific agreements with provinces haven’t yet been signed to deliver that money.

Those standards were publicly released last week but are unlikely to factor into the health-care talks.

Still, there is plenty of work that needs to be done if provinces have a hope of meeting the standards, especially when it comes to the workforce.

“I think we’re stepping into a crisis,” said Dr. Joseph Wong, the founder of Yee Hong Centre for Geriatric Care, the largest non-profit nursing home in the country.

He said Canada will need upwards of 100,000 new personal support workers to provide care over the next 10 to 15 years in order to provide adequate care to residents.

“It is a time bomb,” he said.

Essential Workers

The same could be said of the health system at large.

None of the lofty goals of the federal or provincial politicians will be possible if they don’t find a way to persuade workers to stay in hospitals, clinics and long-term care centres across Canada, said Linda Silas, president of the Canadian Federation of Nurses Unions.

“They don’t have the staff to do the job,” she said.

Staff shortages have been the common theme among some of the most serious issues underlying the public-health crisis in Canada.

Dozens of emergency rooms have been forced to close temporarily or reduce hours because there weren’t enough staff to treat urgent injuries and illnesses. The Canadian Medical Association estimates nearly five million Canadians don’t have a family doctor. And hundreds of thousands of Canadians are sitting on wait-lists for backlogged surgeries and diagnostic tests.

Health unions and professional associations want a national strategy to keep doctors, nurses and personal support workers in their jobs as well as train new staff to bolster their ranks.

Silas said after years of burnout and moral distress over not being able to care for their patients properly, nurses in particular have said, “I’ve had enough.”

Nurses in Ontario have also balked at a law limiting pay increases to one per cent a year.

Data from the Canadian Institute for Health Information shows that because of new graduates, the supply of nurses is still growing. However, many have chosen not to take full-time positions, and existing staff are increasingly eyeing early retirement, Silas said.

The heavier demands of the job since the pandemic, combined with fewer and fewer people to do the work, has created what even the federal health minister calls a crisis.

“We need to stop the bleed,” Silas said.

This report by The Canadian Press was first published Feb. 5, 2023.

 

Mia Rabson and Laura Osman, The Canadian Press

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