Rubber News - Science: Hunting for the elusive latex allergen


	Rubber News

Science: Hunting for the elusive latex allergen

Yeang Hoong Yeet

Sunday, March 23, 2003

Malaysian scientists have found the nastiest of latex allergens and aptly named it No 13. YEANG HOONG YEET, who led the research team, tells the story. EVERYDAY, some 50 million latex gloves are used all over the world, mainly in healthcare settings — clinics, emergency rooms, operating theatres. As a barrier against infection, natural rubber latex has no peer. Yet, despite its many qualities, its track record is not without blemish. Some proteins in rubber latex cause an allergy among sensitive people.

Certainly, latex allergy is uncommon in the general population, occurring in perhaps one person per 1,000. Among health care workers who don latex gloves habitually, it can rise to between three and 16 per cent.

Another high-risk group comprises children with spina bifida among whom more than 50 per cent can be allergic to latex. Although latex allergy is uncommon in Malaysia, the shadow cast by this problem among consumers in the West looms over the country's RM4.6 billion latex industry.

There has been little dispute that the allergic reaction is triggered by specific proteins. But which ones? Natural rubber latex contains hundreds of proteins but only a few are allergens (proteins that cause allergy).

Several laboratories in the US and Europe have been actively engaged in this task. Among latex producing countries, only Malaysia, through the Rubber Research Institute of Malaysia, is involved.

Today, the International Union of Immunological Societies (IUIS) recognises 13 latex allergens. They are assigned the WHO-IUIS names Hevb1 to Hevb13, numbered in chronological order of their identification. The RRIM identified and named Hevb2, Hevb3, Hevb4 and Hevb13.

This is the story of how RRIM researchers resolved a dispute in the scientific community over the latex allergen Hevb1 and how they unravelled the identities of two others — Hevb3 and Hevb13. There was no fast-track to answers and we would not have succeeded without the help of collaborators in Malaysia and abroad. And a bit of luck, of course.

Until 1993, no latex proteins had been pinpointed as allergens. Researchers and physicians took notice, therefore, when scientists in Germany reported that they knew the specific protein responsible for latex allergy. According to the report, the allergenic protein was the Rubber Elongation Factor (REF), a protein located on the membrane of rubber particles.

Barely had this result been published when a Canadian-American group of researchers raised doubts on the finding. Were the Germans correct? That same year, the Medical College of Wisconsin drew attention to the very high prevalence of latex allergy among spina bifida patients. This institution, a
pioneer in latex allergy research, found that two water-soluble proteins, 14kDa and 27kDa in size, were the main culprits. Was there a link between the 14kDa latex protein observed at Wisconsin and the REF that was a tantalising 14.6kDa in size? (Dalton is the measurement unit for molecular mass.) RRIM researchers decided to resolve the controversy. We were aware that natural rubber latex contains a complex mix of proteins and so the likelihood of other latex proteins co-purifying with REF could not be overlooked.

Since even small amount of protein allergens could trigger an allergenic reaction, these interloping proteins, rather than REF, might turn out to be the real offenders. We realised that the only certain way REF could be prepared absolutely free of contaminating latex proteins was not to get it from natural rubber latex.

We did, in fact, make use of latex as the starting material for the experiments, but what we isolated from the latex was not the REF protein, but the specific DNA that encoded REF. This DNA was genetically engineered into bacteria that then acted as a surrogate for the rubber tree and produced a "synthetic" REF protein.

Results showed that the two differing research groups were partially correct. REF was indeed an allergenic protein, but it was not the major villain.

Patients with spina bifida were found to be particularly allergic to REF but health care workers — by far the larger group of latexallergic patients — were considerably less sensitised to the protein.

While several recombinant latex allergens have since been successfully synthesised in laboratories worldwide, the REF was the first functional recombinant latex allergen produced.

Our work on REF showed it to be the 14kDa allergen of spina bifida patients encountered by the Medical College of Wisconsin. We next turned our attention to the second allergen affecting spina bifida patients. This was the 27kDa latex protein, the identity of which was a mystery.

We examined this protein and suspected it to be a protein that had been isolated earlier at RRIM, but had remained unpublished in scientific literature.

Like the REF, this was a rubber particle protein. This insoluble protein was confined mainly to the surface of the most minute rubber particles in latex. For this reason, it was called the Small Rubber Particle Protein (SRPP).

With participation from Universiti Sains Malaysia, the protein was shown to be allergenic to spina bifida patients. After further work, SRPP was assigned the WHO-IUIS allergen name Hevb3 upon RRIM's application.

Having established that Hevb3 was an allergen, the question remained whether or not this was the same protein touted by the Medical College of Wisconsin as the major cause of latex allergy among spina bifida patients. Our studies showed Hevb3 to be 22kDa in size, rather than the reported 27kDa.

To demonstrate that the proteins were identical, available studies were compared. The sequences from RRIM, Wisconsin and Genentech Inc (a US biotech company that stumbled on this protein in unrelated research) matched, demonstrating that the three proteins were one and the same. So, the molecular
weight of 27kDa originally ascribed to Hevb3 was wrong.

Even when the protein causing latex allergy in spina bifida patients was conclusively identified as Hevb3, there was still an unanswered question.

Hevb3 was an insoluble rubber particle membrane protein. Why was the protein detected in latex serum even after processes that should have separated out all solids? To answer this question, we sifted through previous work. The smallest rubber particles visible under the electron microscope are smaller than most bacteria.

During investigation, we found the protein on the surface of tiny rubber particles that escaped sedimentation even after being spun at high-speed.

The DNA that encodes Hevb3 was successfully cloned in 1999 through collaboration between RRIM, the University of Vienna and Kumho Life and Environmental Sciences Laboratory, Korea.

So, that explained much about latex allergies in the spina bifida group but not allergies plaguing a far bigger group of people — health care workers. One such major allergen was a 42-to-46kDa latex protein that had been frequently observed from the early years of latex allergy research.

In 1995, US scientists announced a 46kDa latex allergen similar to patatin, the major storage protein of potatoes. This protein was named Hevb7. Shortly after, scientists at the University of Groningen in the Netherlands uncovered a 43kDa protein from latex serum. This was also similar to patatin.

About this time, an RRIM post-graduate at the University of Hertfordshire, England, was investigating a 44kDa protein isolated from latex serum, which inhibits the tree's production of rubber. Again, similarity to patatin was observed. When the proteins isolated in the US, the Netherlands and England were
all shown to be allergenic, they were deemed to be Hevb7 or its variants.

The RRIM, together with the University of Austria, cloned the DNA encoding Hevb7 in 1998. The recombinant protein produced was also allergenic. All the pieces seemed to fall into place. With this, the mystery of the 42-to46kDa allergenic latex protein was declared solved. The file was closed.

But at the RRIM, we remained quietly concerned. Patients sensitive to natural or recombinant Hevb7 were far fewer than expected. Studies showed 12-32 per cent of latex allergic patients were sensitive to Hevb7.

On the other hand, more than 80 per cent reacted to the 42-to-46kDa protein detected in unpurified whole latex. It just didn't add up. Was Hevb7 a red herring? Was there still another unknown latex allergen, a far more potent one, lurking out there somewhere? Four years went by. During this time, five other latex allergens, Hevb8 to Hevb12, were registered with the IUIS. None were major
allergens. By this time, most researchers in the world thought that all the important latex allergens had been discovered. One laboratory that remained unconvinced was the German Research Institute for Occupational Medicine. The institute studied latex-allergic patients to find out which latex proteins each patient was sensitive to. As expected, most patients were sensitive to multiple proteins. But in a handful, their test results came back negative but still they had an allergic reaction to latex. BGFA realised that a major latex allergen was still "missing".

Meanwhile, back at the RRIM — a component of the Malaysian Rubber Board by 1998 — the decision was taken to dust off the old files on Hevb7. After many experiments on allergenic proteins sized 42 to 46kDa, we zoomed in on one. It was very similar to Hevb7 and reacted with Immunoglobulin E, the antibody humans send out when an allergen enters the body.

Was this yet another variant of Hevb7? Protein analyses at Yale University revealed a new protein of 43kDa similar to the Early Nodule Specific Protein (ENSP) of leguminous plants. We were cautiously optimistic that we might have an important protein in our hands.

But it had to be tested on a sizeable latexallergic population. Malaysia lacked sufficient numbers of such patients. So the protein was sent to the Johns Hopkins University School of Medicine (JHU) for tests using blood samples from latex allergic patients.

Next, the definitive test for allergenicity: the skin prick test. A joint study was set up with the US National Institute of Occupational Safety and Health (NIOSH).

When the results came in, both JHU and NIOSH told the same story. This was among the most allergenic of latex allergens.

When RRIM supplied a sample of the protein to BGFA in Germany, 87 per cent of its patients — including every one of the erstwhile negatives — reacted to the ENSP look-alike. BGFA found the "missing" latex allergen.

In 2002, the International Union of Immunological Societies accepted the RRIM/MRB's application to name the ENSP lookalike Hevb13. The RRIM isolated and cloned the complete DNA sequence encoding Hevb13 earlier this year.

# Yeang Hoong Yeet is head of Biotechnology and Strategic Research at RRIM. This article is an excerpt of his inaugural lecture to commemorate his admission as a Fellow of the Malaysian Academy of Science on March 22.

©New Straits Times (M) Berhad
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