The True Sweet Science
Researchers develop a taste for the study of
sugars
John Travis
"A spoonful of sugar helps the medicine go down,"
sings Julie Andrews in the popular Disney movie Mary
Poppins. The musical nanny doesn't seem to realize
that sometimes the sugar is the medicine. Consider the
blood-thinner heparin, the best-selling drug in the
world. This complex sugar, or carbohydrate, is a
reminder that there's more to life than DNA and the
proteins it encodes. Complex sugars coat almost every
cell in the body, as well as microbes that cause
disease. And many proteins acquire a covering of sugars.
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BRANCHING OUT. Glycoproteins
perform myriad roles for cells. In this
representation, the protein portions (brown) sit
within a cell membrane and branching chains of
simple sugars (green hexagons) extend into the
space between cells.
funhousefilms.com |
Although scientists once thought such sugars were
mere decoration, they now appear to play a vital role in
the function of glycoproteins (from glukus, the
Greek word for sweet). That influence can make a
difference in the body or in a drug-manufacturing plant.
The California-based pharmaceutical company Amgen, for
example, discards significant amounts of its yield of
red blood cell-boosting erythropoietin because improper
gylcosylation during its manufacture creates a less
effective drug.
As erythropoietin and heparin attest, the study of
sugars, a field known as glycobiology, can be big
business. Nonetheless, because of the difficulties
associated with studying sugars, it's only been in the
past decade or so that the discipline has grabbed the
attention of more than a small cadre of biologists and
chemists.
"Historically, people focused on DNA and proteins.
Sugars were a nuisance," notes Ram Sasisekharan, a
chemist at the Massachusetts Institute of Technology
(MIT).
Physicians were also slow to realize the importance
of sugars to human health. They now know that a variety
of birth defects, potentially fatal syndromes, and other
illnesses stem from problems with sugars.
"Glycobiology did not earn the respect it deserves
until the recent descriptions of several human
congenital diseases with defects in the glycosylation of
proteins," Harry Schachter of the University of Toronto
notes in the December 2001 Journal of Clinical
Investigation. Schachter says he expects that many
more such illnesses will soon be recognized. "It is
likely that we have seen only the tip of the iceberg,"
he contends.
Indeed, scientists have begun to reveal myriad roles
for complex sugars. One recent study, for example,
suggested that molecules in heparin's family are crucial
to the embryonic development of animals. Another found
that the coating of sugars on tumor cells helps
determine whether those cells will spread in the body.
Reflecting the desire of the scientific community to
push glycobiology forward, the National Institute of
General Medical Sciences last fall committed $34 million
over 5 years to certain areas of carbohydrate research.
This glycobiology renaissance springs from newly
developed tools and techniques that have enabled
investigators to more quickly decipher the complex
structures of sugars and to synthesize the molecules in
more-efficient ways.
"The technology has begun to fall in place," says
Sasisekharan.
Sugarcoated chips
Whereas the building blocks of DNA and proteins bond
in a few, predictable ways, there are many ways that two
simple sugar molecules may join. This flexibility
greatly increases the complexity of the resulting, more
complex sugars. For example, two glucose molecules can
join in almost a dozen ways, and three hexose molecules
can combine into several thousand distinct forms.
When different simple sugars are combined, the
possibilities seem endless. One family of carbohydrates
similar to heparin's uses 32 simple sugars in its
construction. Adding to the complexity, carbohydrates
can consist of a linear chain of sugars or contain
multiple chains branching off individual sugars.
Given all that, it would be surprising if scientists
could simply isolate a complex sugar from a tissue
sample, pop it into a machine, and quickly read the
sequence of simple sugars that constitute it. Although
such tools have long been available for sequencing the
building blocks of DNA and proteins, glycobiologists
haven't had the same luxury. They're making progress,
however.
Most sugar investigators are turning to various forms
of mass spectrometry, a technique in which complex
molecules are vaporized into ions. These are then sorted
and analyzed by mass, charge, and other characteristics.
In 1999, for example, Sasisekharan and his colleagues
described a new mass spectrometry strategy to sequence
the sugars in heparin's family, called the heparan
sulfates. These sugars coat cells and are also an
integral part of the matrix of proteins and other
molecules that occupy the space between cells. Among
their other roles, heparan sulfates help regulate the
release of growth factors sequestered in this matrix.
Sasisekharan's team has employed the new strategy to
study updated versions of the drug heparin. Traditional
heparin has been used since the 1930s to prevent blood
clots that lead to strokes or heart disease. The drug
has side effects, however. So, to retain the
blood-thinning properties but avoid the problems,
scientists have recently made experimental drugs out of
fragments of heparin.
The sequence of sugars of one of the less effective
of these so-called low-molecular-weight heparins was
different from what scientists had thought. This work
revealed that the fragment was missing some of the
heparin-molecule region that's key to the drug's
anticoagulant properties.
Sasisekharan notes that his group's technique may
help manufacturers ensure the purity of heparin and its
derivatives. These companies haven't had an easy way
until now to check whether the sugar molecules they were
making were uniform in structure from one production
batch to the next, he says.
Considering how difficult it is to take complex
sugars apart for analysis, it's not surprising that
glycobiologists have also had a hard time synthesizing
complex sugars in a precise way. There are several
laboratory strategies for building a carbohydrate out of
sugars. The most natural and efficient way is to exploit
the same enzymes that cells use to construct their
complex sugar molecules.
"The problem is we don't have all the enzymes," says
Peter H. Seeberger of MIT.
With their incomplete set of molecular tools,
scientists and manufacturers have had to synthesize
carbohydrates through a series of complicated chemical
reactions. Seeberger and his colleagues recently
developed a more efficient, enzyme-free strategy for
making complex sugars to order. In the Feb. 23, 2001
Science, they describe using a technique called
solid-phase synthesis to create carbohydrates with as
many as 12 simple-sugar building blocks. "Some of those
structures used to take 6 months to a year to make. We
can make them in about 2 days now," notes Seeberger.
Chi-Huey Wong of the Scripps Research Institute in La
Jolla, Calif., and his colleagues have developed their
own automated technique for enzyme-free carbohydrate
synthesis. They recently tested it by building a
six-sugar compound that sits on the surface of some
tumor cells. Other scientists are testing whether this
carbohydrate can stir the immune system to fight off
cancer.
Although chemists have made great strides in
carbohydrate synthesis recently, Wong cautions that it
will be several more years before biologists will easily
make any complex sugar they might want to study.
Learning to decipher the structures of sugars and to
synthesize them efficiently is important, but scientists
are also eager to learn with what substances these
molecules interact. That's where another technological
advance comes into play—the carbohydrate chip. It's not
a starchy snack but a glass slide or other material onto
which researchers attach an array of hundreds to
thousands of sugar-containing molecules. With such
chips, scientists can learn what proteins or other
molecules in tissue or fluid samples naturally interact
with the sugars. Biologists have previously created
chips with arrays of proteins or DNA strands to identify
protein-protein interactions or to measure the activity
of genes in a cell or tissue (SN: 3/8/97, p. 144).
Several research groups are developing carbohydrate
chips. In the March Nature Biotechnology, Denong
Wang of Columbia University and his colleagues describe
a simple method for building such instruments. Without
ruining the carbohydrate array's ability to interact
with other substances, Wang's group found they could
affix thousands of complex sugars to glass slides coated
with the carbohydrate nitrocellulose. Such carbohydrate
chips should be much more stable than those bearing
proteins or DNA.
"You can produce a [carbohydrate] chip and use it for
many years. There's no special handling required," says
Wang.
To test their invention, the investigators created a
chip out of the sugar molecules and glycoproteins that
normally coat certain microbes. The researchers showed
that when they exposed this chip to a blood sample,
antibodies would stick to the sugars of specific
microbes. Such a chip, says Wang, could serve as a quick
diagnostic test for active infections.
Medical sweet spots
As scientists become more adept at making and
analyzing carbohydrates, they should learn what these
complex molecules do inside and outside cells. It's
already clear that sugars mediate many forms of
cell-to-cell interactions. For example, immune cells
bristle with many different sugars. When a tissue
suffers an infection or injury, nearby blood vessels
make sugar-binding proteins that grab onto some of the
immune-cell sugars and guide the cells to the damaged
area.
Sugars also help explain how a string of amino acids
consistently folds into a protein's three-dimensional
shape—one of the more enduring mysteries in biology.
Studies have revealed that sugars can regulate when a
newly minted protein interacts with so-called chaperone
molecules, which help it fold.
The sugars "actually help a protein to fold," says
Raymond Dwek, director of the Oxford University
Glycobiology Institute. "That is one of the most
significant discoveries in glycobiology."
Sugars are also now seen as crucial players in the
growth of an embryo. In the Jan. 18 Journal of
Biological Chemistry, Scott Saunders of Washington
University School of Medicine in St. Louis and his
colleagues report that a protein called Noggin binds to
heparan sulfate proteoglycans, molecules combining
heparan sulfate and a protein. Noggin normally governs
development by inactivating proteins that diffuse
through an embryo and establish its body plan. From his
group's work, Saunders concludes that heparan sulfates
indirectly influence how the embryo develops.
That finding may be of more than academic interest.
Saunders studies kids with Simpson-Golabi-Behmel
syndrome, a rare disease that has been traced to a
defect in the production of heparan sulfate
proteoglycans. Children with the syndrome typically
develop an enlarged head or body and fused fingers or
toes. They also experience certain childhood cancers
more often than normal.
Cancer researchers, too, are increasingly paying
attention to sugars. It's been known for a long time
that the sugar coating of a cell usually changes when
the cell becomes cancerous. It isn't clear, however,
whether that's a byproduct of the cancerous
transformation or a factor that helps the tumor.
In the Jan. 22 Proceedings of the National Academy
of Sciences, Sasisekharan and his colleagues offer
evidence that some heparan sulfate fragments promote the
growth and spread of tumors, while others inhibit those
processes. In mice carrying melanoma or lung cancer
cells, the investigators tested the effects of two
enzymes, each of which cleaves heparan sulfates in a
different spot. When the researchers injected one enzyme
into the rodents, the cancers grew and spread more
readily than normal. The other enzyme, however, produced
the opposite result. Cancer growth and spread was
inhibited.
The researchers obtained similar results when they
injected mice with the heparan sulfate fragments
themselves, rather than the enzymes. The results are
intriguing, says Sasisekharan, especially since there
have been some hints in the tests of heparin that the
blood thinner may reduce mortality in cancer patients.
Infectious disease is another area of human health
that has come to the attention of glycobiologists. Two
recently approved drugs for the flu, for example,
inhibit a viral enzyme that strips a sugar off a
glycoprotein on the surface of cells. In doing so, the
drugs prevent influenza viruses from infecting cells.
Dwek and his colleagues are also developing antiviral
agents that exploit the role of sugars in protein
folding. They've identified compounds that inhibit one
of the cellular enzymes that removes sugars from a new,
unfolded protein. In infected cells, the compounds
appear to prevent the proper folding of viral proteins
without harming the cells. The researchers hope this
year to begin testing the drugs on people infected with
hepatitis virus.
To Dwek, who coined the term glycobiology more than a
decade ago, the increasing scientific and medical
interest in the field is welcome. He says, "It's a boom
time, as far as I'm concerned."
References:
Dwek, R.A., et al. 2002.
Targeting glycosylation as a therapeutic approach.
Nature Reviews Drug Discovery 1(January):65-75.
Abstract.
Liu, D., et al. 2002. Tumor
cell surface heparan sulfate as cryptic promoters or
inhibitors of tumor growth and metastasis.
Proceedings of the National Academy of Sciences
99(Jan. 22):568-573. Abstract available at http://www.pnas.org/cgi/content/abstract/99/2/568.
Paine-Saunders, S., . . . and S.
Saunders. 2002. Heparan sulfate proteoglycans retain
noggin at the cell surface. Journal of Biological
Chemistry 277(Jan. 18):2089-2096. Abstract available
at http://www.jbc.org/cgi/content/abstract/277/3/2089.
Plante, O.J., E.R. Palmacci, and P.H.
Seeberger. 2001. Automated solid-phase synthesis of
oligosaccharides. Science 291(Feb. 23):1523-1527.
Abstract available at http://www.sciencemag.org/cgi/content/abstract/291/5508/1523.
Schachter, H. 2001. The clinical
relevance of glycobiology. Journal of Clinical
Investigation 108(Dec. 1):1579-1582. Available at http://www.jci.org/cgi/content/full/108/11/1579.
Sears, P., and C.-H. Wong. 2001.
Toward automated synthesis of oligosaccharides and
glycoproteins. Science 291(March 23):2344-2350.
Abstract available at http://www.sciencemag.org/cgi/content/abstract/291/5512/2344.
Venkataraman, G., . . . and R.
Sasisekharan. 1999. Sequencing complex polysaccharides.
Science 286(Oct. 15):537-542. Abstract available
at http://www.sciencemag.org/cgi/content/abstract/286/5439/537.
Wang, D., et al. 2002.
Carbohydrate microarrays for the recognition of
cross-reactive molecular markers of microbes and host
cell. Nature Biotechnology 20(March):275-281.
Abstract available at http://dx.doi.org/10.1038/nbt0302-275.
Further Readings:
Axford, J. 2001. The impact of
glycobiology on medicine. Trends in Immunology
22(May):237-239. Abstract.
Baum, L.G. 2002. Developing a taste
for sweets. Immunity 16(January):5-8. Summary
available at http://www.immunity.com/cgi/content/abstract/16/1/5/.
Kiessling, L.L., and C.W. Cairo.
2002. Hitting the sweet spot. Nature
Biotechnology 20(March):234-235.
Travis, J. 1997. Chips ahoy.
Science News 151(March 8):144-145.
Zitzmann, N., et al. 1999.
Imino sugars inhibit the formation and secretion of
bovine viral diarrhea virus, a pestivirus model of
hepatitis C virus: Implications for the development of
broad spectrum anti-hepatitis virus agents.
Proceedings of the National Academy of Sciences
96(Oct. 12):11878-11882. Abstract available at http://www.pnas.org/cgi/content/abstract/96/21/11878.
Sources:
Raymond A. Dwek
Glycobiology
Institute
Department of Biochemistry
University of
Oxford
South Parks Road
Oxford OX1 3QU
United
Kingdom
Ram Sasisekharan
Division of
Bioengineering and Environmental Health
Massachusetts
Institute of Technology
Cambridge, MA 02139
Scott Saunders
Department of
Molecular Biology and Pharmacology
Washington
University School of Medicine
St. Louis, MO 63110
Harry Schachter
Department of
Structural Biology and Biochemistry
University of
Toronto
Toronto, ON M5G 1X8
Canada
Peter H. Seeberger
Department of
Chemistry
Massachusetts Institute of
Technology
Cambridge, MA 02139
Denong Wang
Functional Genomics
Division
Columbia Genome Center
College of
Physicians and Surgeons
Columbia University
1150
St. Nicholas Avenue
New York, NY 10032
Chi-Huey Wong
Department of
Chemistry
Skaggs Institute for Chemical
Biology
Scripps Research Institute
10550 North
Torrey Pines Road
La Jolla, CA 92037
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