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Paulson was working as a postdoctoral fellow when he was
struck by the extraordinary specificity of the carbohydrate
synthetases. As an assistant professor, he began using enzymes
to study the biochemistry of sugars and their synthesis and,
eventually, using sugars as a probe of biological function.
For years he worked for a San Diego biotech company as a senior
executive and researcher. While there, Paulson and others
showed that neutrophils and certain other white blood cells
require a specific sugar structure called sialyl-Lewis X to
traffic normally to sites of inflammation and lymph nodes.
These sugars cause the cells to adhere to and roll on cells
lining the walls of a blood vessel that have produced complementary
binding proteins to 'recruit' the cells to that tissue. Once
slowed down, they stop and squeeze through the cells of the
vessel wall into the surrounding tissue.
In recent years, he has been studying the role of carbohydrates
recognized by another family of carbohydrate-binding proteins,
examining how the binding to carbohydrates modify a cell's
function.
In particular, he is studying CD22, one of the membrane-spanning
"accessory" proteins of the B cell receptor that recognizes
a carbohydrate also expressed on B cells. Previously, he had
cloned a gene for the enzyme that makes the carbohydrate recognized
by CD22. Deletion of this gene in mice with Marth, a collaborator
on this project, resulted in immuno-suppressed B cells, indicating
that the sugar made by the enzyme is a negative regulator
of B cell receptor signaling.
"What we think is happening is that when [CD22] binds the
ligand, it is binding to glycoproteins that sequester it from
the B cell receptor complex," says Paulson. "When the ligand
is not there, it is free to associate and [CD22] exerts its
maximum negative regulatory effect to dampen the immune response."
The mechanism has direct implications for treatment of autoimmune
disease and inflammation, but understanding it fully is hampered
by the difficulty of the research, which spans disciplines
from organic synthesis to pure biochemistry to cell imaging
to whole animal models. Such limitations in the research in
this field are common. This recognition was the impetus for
Paulson and other members of the consortium to consider submitting
the grant application.
The Sweet Smell of Success
The stated purpose of the grant is to define paradigms by
which protein–carbohydrate interactions mediate cell
communication. More specifically, the focus is on the regulation
of glycans displayed on the surfaces of cells and the structure,
function, and regulation of the four major families of carbohydrate-binding
proteins that mediate biological events. "[At the moment],
very little is known about the structure of carbohydrates
on a given cell and about how sugar constellations differ
from cell to cell," says Paulson.
The program includes hypothesis-driven research of the participating
investigators and scientific cores funded by the grant that
provide essential resources for conducting research, a platform
of information about carbohydrate structures and their interaction
with carbohydrate-binding proteins, and an infrastructure
of bioinformatics and databases to facilitate sharing of data
both within the consortium and with the public.
Information generated in the scientific cores will be accessible
within six weeks, and information deposited by participating
investigators will be released as soon as it is made public.
Any investigator with an existing grant in the area of carbohydrates
in cell communication can apply for membership to the consortium.
Membership entitles one to resources produced by the scientific
cores funded by the grant, and to view unpublished data deposited
by other participating investigators under the confines of
a blanket non-disclosure agreement. Investigators, in turn,
commit to providing data back to the consortium.
The Cores
The carbohydrate synthesis and protein expression core will
provide a library of synthetic carbohydrates and other reagents
to be used by the other cores and by investigators. The investigators
should benefit greatly from this resource because one of the
main bottlenecks that has kept the field back up to now has
been lack of commercial availability and the difficulty—particularly
for biologists without extensive organic synthesis training—in
synthesizing these structures.
An analytical core located at UCSD will perform analyses
of carbohydrate structures. This serves a crucial purpose
because, due to the complexity of a branched glycan structure,
systematic sequence analysis relies on highly specialized
methods like high field mass spectrometry and nuclear magnetic
resonance involving stripping sugars off cells and subjecting
them to reliable but tedious separations and individual identifications.
The application of these techniques is still not ready for
high throughput, but "that's something that I'm hoping this
program will have an effect on," says Paulson.
A gene microarray core located at TSRI will produce oligonucleotide
microarrays of human and murine carbohydrate-binding protein
and glycosyltranferase genes.
And a murine genetics core at TSRI and phenotype core at
UCSD will generate up to ten transgenic strains per year with
altered carbohydrate-binding protein and glycosyltranferase
genes that will be phenotyped using all the standard behavioral
and biochemical assays. Once these models are developed, they
will be made publicly available.
A protein-carbohydrate interaction core at the University
of Oklahoma will determine the specificity and affinity of
protein carbohydrate interactions.
Finally, a bioinformatics core located at the Massachusetts
Institute of Technology (M.I.T.) will contain a central database.
The Table is on the Sugar
The database will allow participants to search and link information
to speed up some research activities of the cores and participating
investigators. For instance, for the analytical core, when
the sequencing is linked to the structure database, mapping
the sugars on cell surfaces may become easier, since about
90 percent of carbohydrates that are routinely found on cells
are likely to be common structures that are already identified.
Running a sugar "fingerprint" of cell through a database could
quickly pinpoint new and interesting structures that need
further characterization.
The linking of all types of data is another advantage. Synthesis
data, links to commercially available materials, links to
publications, analysis spectra of carbohydrates, and other
information will eventually be linked to genetic data on the
four major families of carbohydrate-binding proteins from
all major organisms that have been sequenced, database on
the families of glycosyl transferases, and a carbohydrate
structure database developed by Glycominds Ltd., as a contribution
to the project.
Because the database servers are integrated and accessible
through the internet, data entry should be as user-friendly
as data retrieval. Investigators can effortlessly attach tables
and figures to the database using forms and templates that
automatically tag the data appropriately as the database builds
and as the body of knowledge on carbohydrates and their related
binding proteins grows.
While having a complete picture of sugars in the body is
still a long way off, the availability of the database on
the internet should benefit the scientific community as a
whole. The grant will also address the research bottlenecks
in functional glycomics, clearing the road to a more complete
understanding of the paradigms that have evolved to use information
in the glycome to mediate cell communication.
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