The muscle protein titin has long been the titan of the molecular world. Built of more than 34,000 amino acids and weighing in at a hefty 3.7 megadaltons, titin has long been considered the largest protein in the world—until now.
A new group of toxin-making enzymes has been discovered inside the algae Prymnesium parvum, one of which—christened PKZILLA-1—dwarfs titin by more than 10,000 amino acids. P. parvum uses the monstrous PKZILLAs to manufacture a toxin that it secretes into the water, posing a threat to fish and human health alike.
“Godzilla is big, marine and spews toxins,” marine biochemist Bradley Moore, Ph.D., of the University of California, San Diego (UCSD), told Fierce Biotech in an interview. “PKZILLA does all three of those things.”
Biochemist Timothy Fallon, Ph.D., is mainly interested in giant toxic molecules produced by single-celled aquatic organisms called dinoflagellates, which are a common cause of harmful algal blooms known as red tides. But dinoflagellates have massive genomes, which makes them difficult to study. Working with Moore, Fallon turned to P. parvum as a simpler study system because it has a much smaller genome but produces a similar toxin, called prymnesin.
“Because it's got a small genome, it could be a model system for these other toxins that are quite impactful in human health that so far have been hard to make a lot of progress on,” Fallon, also at UCSD, told Fierce Biotech in an interview.
But in order for P. parvum to be a model for other harmful algae, Fallon needed to find out what molecular machinery it uses to make its toxins. “In order for it to be a model system, you first have to solve the problem, right?” he said.
Prymnesin is a polyketide, built from a chain of ketone and methylene groups. Fallon scoured the genome of P. parvum for polyketide synthesis (PKS) genes to try to find the builder of prymnesin, but automated processes for identifying genes within genomes can be error-prone.
To be sure he didn’t miss anything, Fallon manually ran potential PKS gene segments through databases to confirm their identity and hit a stroke of good luck.
“It just so happened that the first one that I searched was what we now call PKZILLA-1,” he said. “When you look at it in a genome browser, it's enormous.”
Bits and pieces of the PKZILLAs were scattered throughout the sequence data, so Fallon painstakingly assembled the genes over time. Once he had the genes, he could translate them into the proteins they code for—a puzzle he finally completed several years ago.
“I sent an email to [Moore], being like, 'this is a very long protein,'” Fallon recalled. “And I just pasted the whole protein into the email.”
The next step was confirming that the enormous PKZILLA genes are actually turned into proteins in living cells. The researchers freeze-dried the algae and chopped up all their proteins into fragments using enzymes. Sequencing those fragments revealed that at least 75% of PKZILLA-1 and 76% of PKZILLA-2 are in fact turned into protein. This approach doesn’t allow them to confirm that PKZILLAs exist as single, massive proteins, but Fallon is confident that they do.
“Since we think it's a single-gene model, single transcript, single peptide or protein, [we think] that it's going to be contiguous,” he said. “But we don't get nail-in-the-coffin evidence from bottom-up proteomics alone.” If it's confirmed to be contiguous, this would make PKZILLA-1, at 45,212 amino acids long, the largest confirmed protein on Earth.
Further evidence that PKZILLAs exist as single contiguous proteins comes from their function. Biochemist Vikram Shende, Ph.D., also of UCSD, analyzed the proteins and identified the chemical reactions that different sections of it would produce. “These two giant proteins, you can almost read them like a book,” Moore said. “The coolest thing is being able to, as a chemist, read that sequence and draw a chemical structure of what each of these enzymes is doing.” The molecule that Shende predicted PKZILLA makes is strikingly similar to prymnesin, supporting the idea that the giant toxin is indeed made by these equally giant proteins.
“The toxins are huge, so it does not surprise me that the enzymes required to produce these compounds are also huge,” Elisabeth Varga, Ph.D., a chemist at the University of Veterinary Medicine, Vienna who was not involved with the study, told Fierce Biotech in an email. Varga hopes the finding will help scientists understand why P. parvum makes the toxins in the first place.
“It must require a lot of energy to produce these huge molecules, so there has to be an advantage in producing them,” Varga said. “When we know which genes are required to produce prymnesins, we can study when the expression is triggered and under which circumstances.”
Varga is currently working to uncover how different types of prymnesin work, and why some are more toxic than others.
Understanding the methods algae use to make colossal molecules can not only help us address harmful algal blooms, Moore said but also help us learn how to build new types of molecules ourselves.
“Being able to manipulate cells to become cell factories to make materials, that is a direction I think our community is going,” he said. “What can we learn from the hundreds of millions of years of evolution where nature has been at this game, to be able to learn how to make complex chemicals?”