Re-creating coagulation in a lab
When considering modern medicine’s fight against infections, a horseshoe crab is likely not the first thing that comes to mind. However, the dwindling number of these ancient arthropods is cause for concern in the biomedical industry.
Bacterial lipopolysaccharide, or LPS, is a toxic molecule that can cause life-threatening infectious reactions in humans. Horseshoe crab hemolymph (a blood equivalent) is extremely sensitive to LPS, coagulating in response to even trace amounts. Due to this property, hemolymph is used in the Limulus test, a critical tool to ensure medical devices and drugs are free of LPS contamination. Unfortunately, the harvesting of hemolymph pits medical and conservation interests against each other.
Shun-Ichiro Kawabata, a researcher at Kyushu University in Japan, seeks a solution to this conflict. “(The) raw materials of Limulus test are totally dependent on the limited natural resource,” he said. “As an alternative approach, we have been doing studies to develop a next-generation Limulus test using recombinant (engineered) proteins.”
Recent research Kawabata and his colleagues published in the Journal of Biological Chemistry represents an important step toward a hemolymph-free Limulus test.
In their study, Kawabata’s team focused on the main catalytic pathway of hemolymph coagulation: three zymogens (inactive enzyme precursors) called proC, proB and proCE. In the presence of LPS, proC is activated into an enzyme called alpha-chelicerase C that converts proB into chelicerase B; chelicerase B in turn activates proCE into the clotting enzyme that coagulates the hemolymph. Kawabata previously engineered functional proC and proB; in this new research, his lab also made a functional proCE without the use of hemolymph for the first time.
“We have overcome several difficulties in preparing these recombinant proteins, and now we have (all) three recombinants: proC, proB, and proCE,” Kawabata said. Using these three proteins to reconstitute the coagulation cascade, the team discovered that specific regions and amino acids in proB and proCE are key for activation. Now they are using their set of recombinant zymogens and these recent data to improve on nature’s designs.
“We are pushing this work forward to prepare more effective and stable recombinants of the protease zymogens applicable for the detection of LPS,” Kawabata said.
Challenges remain before a fully synthetic hemolymph substitute can be used for the Limulus test. While the proteins developed by the team represent the core components, three proteins alone cannot mimic hemolymph perfectly. “Some cofactors, environmental proteins or some preservatives must be indispensable,” Kawabata said.
It is also important to ensure that the cascade cannot be triggered by other environmental substances, something that will require further testing and optimization.
Although questions remain, Kawabata and his team already are working with companies in Japan “to make a more sensitive and convenient test … and ensure a continuous supply of the best materials,” he said.
In time, the proteins developed in the Kawabata lab could bring medical and conservation concerns into harmony, protecting the horseshoe crab population while providing a powerful tool to prevent infections.
Enjoy reading ASBMB Today?
Become a member to receive the print edition four times a year and the digital edition weekly.
Learn moreGet the latest from ASBMB Today
Enter your email address, and we’ll send you a weekly email with recent articles, interviews and more.
Latest in Science
Science highlights or most popular articles
Gene-mutation pathway discovery paves way for targeted blood cancers therapies
A new study by researchers at the universities of Texas and Chicago explains the enzymatic activity that’s needed for tumor suppression in leukemias and other cancers.
Candy binges can overload your gut microbiome
While most Halloween candies contain lots of sugar, some are better for your gut microbiome than others.
Water rescues the enzyme
“Sometimes you must bend the rules to get what you want.” In the case of using water in the purification of calpain-2, it was worth the risk.
Virtual issue celebrates water in ASBMB journals
Read a dozen gold open-access articles covering exciting research about the society’s 2024 Molecule of the year.
There are worse things in the water than E. coli
E. coli levels determined whether Olympic swimmers could dive into the Seine this past summer. But are these bacteria the best proxy for water contamination?
Biobots arise from the cells of dead organisms
Given the right conditions, certain types of cells are able to self-assemble into new lifeforms after the organism they were once part of has died.