<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 membrane proteins, are affected by it, it was unclear until recently what the change was for, how the specific protein sequences are recognized and how the associated enzyme reaction is chemically possible at all.
In an international collaboration, 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 cell surface 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
Citation:
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
This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no
part may be reproduced without the written permission. The content is provided for information purposes only.
HALIFAX – The Nova Scotia government says it could be months before it reveals how many people are on the wait-list for a family doctor.
The head of the province’s health authority told reporters Wednesday that the government won’t release updated data until the 160,000 people who were on the wait-list in June are contacted to verify whether they still need primary care.
Karen Oldfield said Nova Scotia Health is working on validating the primary care wait-list data before posting new numbers, and that work may take a matter of months. The most recent public wait-list figures are from June 1, when 160,234 people, or about 16 per cent of the population, were on it.
“It’s going to take time to make 160,000 calls,” Oldfield said. “We are not talking weeks, we are talking months.”
The interim CEO and president of Nova Scotia Health said people on the list are being asked where they live, whether they still need a family doctor, and to give an update on their health.
A spokesperson with the province’s Health Department says the government and its health authority are “working hard” to turn the wait-list registry into a useful tool, adding that the data will be shared once it is validated.
Nova Scotia’s NDP are calling on Premier Tim Houston to immediately release statistics on how many people are looking for a family doctor. On Tuesday, the NDP introduced a bill that would require the health minister to make the number public every month.
“It is unacceptable for the list to be more than three months out of date,” NDP Leader Claudia Chender said Tuesday.
Chender said releasing this data regularly is vital so Nova Scotians can track the government’s progress on its main 2021 campaign promise: fixing health care.
The number of people in need of a family doctor has more than doubled between the 2021 summer election campaign and June 2024. Since September 2021 about 300 doctors have been added to the provincial health system, the Health Department said.
“We’ll know if Tim Houston is keeping his 2021 election promise to fix health care when Nova Scotians are attached to primary care,” Chender said.
This report by The Canadian Press was first published Sept. 11, 2024.
ST. JOHN’S, N.L. – Newfoundland and Labrador‘s chief medical officer is monitoring the rise of whooping cough infections across the province as cases of the highly contagious disease continue to grow across Canada.
Dr. Janice Fitzgerald says that so far this year, the province has recorded 230 confirmed cases of the vaccine-preventable respiratory tract infection, also known as pertussis.
Late last month, Quebec reported more than 11,000 cases during the same time period, while Ontario counted 470 cases, well above the five-year average of 98. In Quebec, the majority of patients are between the ages of 10 and 14.
Meanwhile, New Brunswick has declared a whooping cough outbreak across the province. A total of 141 cases were reported by last month, exceeding the five-year average of 34.
The disease can lead to severe complications among vulnerable populations including infants, who are at the highest risk of suffering from complications like pneumonia and seizures. Symptoms may start with a runny nose, mild fever and cough, then progress to severe coughing accompanied by a distinctive “whooping” sound during inhalation.
“The public, especially pregnant people and those in close contact with infants, are encouraged to be aware of symptoms related to pertussis and to ensure vaccinations are up to date,” Newfoundland and Labrador’s Health Department said in a statement.
Whooping cough can be treated with antibiotics, but vaccination is the most effective way to control the spread of the disease. As a result, the province has expanded immunization efforts this school year. While booster doses are already offered in Grade 9, the vaccine is now being offered to Grade 8 students as well.
Public health officials say whooping cough is a cyclical disease that increases every two to five or six years.
Meanwhile, New Brunswick’s acting chief medical officer of health expects the current case count to get worse before tapering off.
A rise in whooping cough cases has also been reported in the United States and elsewhere. The Pan American Health Organization issued an alert in July encouraging countries to ramp up their surveillance and vaccination coverage.
This report by The Canadian Press was first published Sept. 10, 2024.
All orders are protected by SSL encryption – the highest industry standard for online security from trusted vendors.
Bizarre Sunlight Loophole Melts Belly Fat Fast! is backed with a 60 Day No Questions Asked Money Back Guarantee. If within the first 60 days of receipt you are not satisfied with Wake Up Lean™, you can request a refund by sending an email to the address given inside the product and we will immediately refund your entire purchase price, with no questions asked.
