Scientists Describe a New Way of Making Glycoproteins
By Jason Socrates
Bardi
A team of investigators at The Scripps Research Institute
and its Skaggs Institute for Chemical Biology has developed
a new way of making glycoproteins—proteins with carbohydrates
(sugars) attached.
Methods for making glycosylated proteins are important to
scientists who want to understand the role of carbohydrates
in protein structure and function, since the human body contains
many heavily glycosylated proteins, including antibodies,
hormones, and immune system proteins like cytokines and interleukins.
These methods are also of interest to doctors, since pharmaceuticals
are often heavily glycosylated proteins (e.g., erythropoietin,
which is useful for treating anemia, cancer, and AIDS).
"In the future, you will see more and more proteins [coming
forth] as drug candidates, mainly because of advances in genomic
research," says Chi-Huey Wong, Ph.D, who is Ernest W. Hahn
Professor and Chair in Chemistry at Scripps Research, "and
most of these proteins have sugars on them."
Led by Professor Wong, Professor Peter G. Schultz, Ph.D.,
who holds the Scripps Family Chair in Chemistry at Scripps
Research, and Scripps Research Associate Zhiwen Zhang, Ph.D.,
the team of scientists discovered a new way of synthesizing
glycoproteins, and they report their strategy in the latest
issue of the journal Science.
The strategy, which avoids some of the bottlenecks of previous
methods, involves using a modified form of the bacterium Escherichia
coli to express a glycosylated form of the protein myoglobin.
The E. coli was evolved so that it would insert a glycosylated
amino acid into the sequence of the myoglobins as they were
being produced.
The Tough Task of Making Glycoproteins
Glycoproteins are basically proteins that have been modified
so that one or more carbohydrates (sugars) are attached to
nitrogen or oxygen atoms within the protein's amino acids.
The modifications on glycoproteins are very much a part
of the language of life, and some even call carbohydrates
the third alphabet, behind DNA and proteins. Sugars on proteins
are like the accents on spoken words—they change the
meaning without changing the spelling. If the correct sugars
are not there, the biology is altered.
Some of the most intriguing problems in modern biology and
medicine require scientists and doctors to synthesize proteins
that have been modified with particular sugars attached in
particular places. This presents a sticky problem because
in the human body, the proteins are usually made first and
then modified, and this modification is handled by a number
of intricate mechanisms, not all of which may be reproduced
in the test tube. Producing glycoproteins in the laboratory
has been especially problematic.
Even when it is possible to directly synthesize particular
glycosylated proteins in the test tube, producing them may
be expensive, difficult, time-consuming, and not at all practical.
Some glycosylated proteins are produced in microorganisms
or cultures of eukaryotic cells, like yeast or Chinese hamster
cells—an expensive and sometimes inexact process, which
often involves difficult and expensive purification schemes.
Zhang, Schultz, Wong, and their colleagues have found another
way—making homogeneous pools of glycosylated proteins
in E. coli.
Bacterial cultures like E. coli have been used to
produce proteins cheaply and easily for years, but it has
never been possible to produce glycosylated proteins in them
because bacteria don't normally have the same ability to attach
sugars to proteins as eukaryotic cells do. The Scripps researchers
solved this problem by modifying a form of E. coli
to make homogeneous pools of glycosylated myoglobin protein
with sugars attached at one desired position (see below: The
Basis of the Technology).
Once the protein with the glycosyated amino acid was made
and isolated, the Scripps Research team was able to add additional
sugars to the same site by using a "transfer enzyme," called
glycosyltransferase, which attached the extra sugars.
The use of E. coli to make the myoglobin is a significant
advance because it is a general and versatile method and it
opens up the gates for using bacterial cultures to put other
sugars on other proteins. The method is also scalable, and
should be cheaper than other current technologies.
The Basis of the Technology
The technology that allows this advance is a methodology
that Schultz and his colleagues have developed that exploits
the redundancy of the genetic code of organisms like E.
coli or yeast that allow these cellular factories to mass
produce proteins with unnatural amino acids.
Scientists have for years created proteins with such unnatural
amino acids in the laboratory, but until Schultz and his colleagues
began their work in this field, nobody had ever found a way
to get organisms to add unnatural amino acids into their genetic
code.
When a protein is expressed, an enzyme reads the DNA bases
of a gene (A, G, C, and T), and transcribes them into RNA
(A, G, C, and U). This so-called "messenger RNA" is then translated
by another protein-RNA complex, called the ribosome, into
a protein. The ribosome requires the help of transfer RNA
molecules (tRNA) that have been "loaded" with an amino acid,
and that requires the help of a "loading" enzyme.
Each tRNA recognizes one specific three-base combination,
or "codon," on the mRNA and is loaded with only the one amino
acid that is specific for that codon.
During protein synthesis, the tRNA specific for the next
codon on the mRNA comes in loaded with the right amino acid,
and the ribosome grabs the amino acid and attaches it to the
growing protein chain.
The redundancy of the genetic code comes from the fact that
there are more codons than there are amino acids used. In
fact, there are 4x4x4 = 64 different possible ways to make
a codon—or any three-digit combination of four letters
in the mRNA (UAG, ACG, UTC, etc.). With only 20 amino acids
used by the organisms, not all of the codons are theoretically
necessary.
But nature uses them anyway. Several of the 64 codons are
redundant, coding for the same amino acid, and three of them
are nonsense codons—they don't code for any amino acid
at all. These nonsense codons are useful because normally
when a ribosome that is synthesizing a protein reaches a nonsense
codon, the ribosome dissociates from the mRNA and synthesis
stops. Hence, nonsense codons are also referred to as "stop"
codons. One of these, TAG, played an important role in Schultz's
research.
Schultz and his colleagues knew that if they could provide
their cells with a tRNA molecule that recognizes TAG and also
provide them with a synthetase "loading" enzyme that loaded
the tRNA with a glycosylated form of the amino acid serine,
the scientists would have a way to site-specifically insert
that glycosylated amino acid into any protein they wanted.
They needed to find a functionally "orthogonal" pair—a
tRNA/synthetase pair that react with each other but not with
the normal pairs. They then devised a methodology to evolve
the specificity of the orthogonal synthetase to selectively
accept glycosyl amino acids.
They created a library of cells, each encoding a mutant
synthetase, and they devised a positive selection whereby
only the cells that load the orthogonal tRNA with any amino
acid would survive. Then they designed a negative selection
whereby any cell that recognizes TAG using a tRNA loaded with
a natural amino acid dies. In so doing, they found their orthogonal
synthetase mutants that load the orthogonal tRNA with only
the desired amino acid—which is called N-acetylglucosamine
modified serine.
With this system, a ribosome that was reading an mRNA would
insert the modified serine when it encountered TAG. Furthermore,
any codon in an mRNA that is switched to TAG will encode for
the new amino acid in that place, giving Schultz and his colleagues
a way to site-specifically incorporate glycosylated amino
acids into proteins expressed by the E. coli.
The article, "A New Strategy for the Synthesis of Glycoproteins"
was authored by Zhiwen Zhang, Jeff Gildersleeve, Yu-ying Yang,
Ran Xu, Joseph A. Loo, Sean Uryu, Chi-Huey Wong, and Peter
G. Schultz and will appear in the January 15, 2003 issue of
the journal Science. See: http://www.sciencemag.org.
This work was supported by the Department of Energy, the
National Institutes of Health (NIH), and the Skaggs Institute
for Research. Individual scientists involved in this study
were sponsored through NIH and National Research Service Award
fellowships.
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