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Genetic Approaches to Understanding the Function of Mammalian Oligosaccharides
Summary: John Lowe is interested in defining the functions of glycan molecules expressed at the surface of mammalian cells and studying how these molecules control cell adhesion and signaling processes.
Our goal is to understand the functions of oligosaccharides,
molecules that are displayed on the surfaces of mammalian cells.
Oligosaccharides, or glycans, consist of many different single-sugar
structures linked in complex linear and branching arrays. Their
structures change temporally and spatially during development and
exhibit lineage-specific expression patterns in the adult. These
observations imply that cell surface oligosaccharides may function as
information bearers important to cell-cell interactions. However,
specific functional correlates have been defined for only a few of
these structurally diverse molecules.
Oligosaccharides are constructed by enzymes called
glycosyltransferases. Each linkage between the sugar molecules in an
oligosaccharide is generally constructed by a unique
glycosyltransferase. The enormous number of different oligosaccharides
dictates that many different glycosyltransferases will enter the
construction of these molecules in any particular cell type. Our
efforts to discover oligosaccharide function in mammals now focus on
understanding the mechanistic basis for mutant phenotypes in mice with
intentional genetic alterations in glycosylation. Glycosyltransferase
genes, and genes that control the synthesis of glycosyltransferase
substrates, serve as tools in these efforts.
Some of our work focuses on glycans modified by fucose attached in
α(1,3)anomeric linkage, and by the α(1,3)fucosyltransferases responsible for their synthesis. These studies relate to adhesion between leukocytes and the
endothelial lining of a blood vessel, an initial and required step in
the process used by leukocytes when migrating from the blood to
extravascular sites of inflammation. Leukocyte-endothelial cell
adhesion is mediated by the proteins E-selectin and P-selectin, which
are expressed by endothelial cells during inflammation, and L-selectin,
which is expressed by leukocytes and is required for the
lymphocyte-homing process. E-, P-, and L-selectins bind to ligands
containing sialylated, α(1,3)fucosylated oligosaccharides. Two α(1,3)fucosyltransferase genes (FucT-IV and FucT-VII) are expressed in leukocytes and contribute to selectin
ligand synthesis. We have made and are studying mice deficient in these
genes to define the roles of these enzymes and cognate glycans in
leukocyte-selectin interactions.
In prior years, using mice deficient in FucT-IV, FucT-VII, or both
enzymes, we learned that these two enzymes contribute unequally but
essentially to E- and P-selectin-dependent recruitment of neutrophils
and some subsets of T cells in acute inflammation. This past year, we
completed studies that assign modest and substantial contributions,
respectively, to FucT-IV or FucT-VII in the elaboration of monocyte
selectin ligands, and a corresponding relative rank order contribution
of each to atherosclerotic disease in experimental mice. These
observations suggest that FucT-IV and FucT-VII collaborate to control
monocyte recruitment to atherosclerotic lesions. Confirmation of this
hypothesis is under way, via generation and study of mice in which
expression of these enzymes is restricted to, or deleted from, the
monocyte. (Some of this work was supported by a grant from the National
Institutes of Health to Jonathon Homeister, a senior research fellow in
the laboratory.)
Studies this past year disclose that these two enzymes are also
required for recruitment of eosinophils in a model of allergic disease
(with Takahiro Sato, Tokyo Medical and Dental University) and for the
recruitment of pathogenic T lymphocytes to the brain in autoimmune
encephalomyelitis (with Gabriela Constantin, University of Verona).
These studies suggest that pharmacological inhibitors of these enzymes
might exhibit therapeutic activity in allergy, multiple sclerosis, and
other inflammatory diseases.
This past year, we learned that genetic deficiency of these enzymes
leads to a relative deficit in trafficking of lymphopoietic progenitors
to the thymus. These observations align with our studies identifying
selectin ligands on such progenitors and imply that selectin
ligands—and other cell adhesion receptor-counterreceptor
pairs—cooperatively contribute to progenitor recruitment events
required for thymus-dependent T cell development.
This past year, we examined the roles of a glycan sulfotransferase,
termed L-selectin ligand sulfotransferase (LSST), and core 2 β1,6-N-acetylglucosaminyltransferase-1 (Core2GlcNAcT) in the synthesis of glycans relevant to L-selectin-dependent lymphocyte homing. These studies involved the creation and analysis of mice with genetic deficiencies in LSST and
Core2GlcNAcT. This work indicates that LSST and Core2GlcNAcT cooperate
in elaborating sulfated, fucosylated O-glycans that afford
optimal L-selectin ligand activity for the lymphocyte-homing process
and for the expression of L-selectin ligands that recruit T lymphocytes
to sites of chronic inflammation. (This work, supported in part by
grants from the National Institutes of Health, was a collaboration with
Minoru Fukuda at the Burnham Institute, La Jolla.)
Blood leukocyte numbers are increased in mice with the combined
deficiency of FucT-IV and FucT-VII, in correlation with expansion of a
specific type of leukocyte progenitor in their marrows. Our prior work
implied that increased marrow progenitor number is a direct consequence
of loss of α(1,3)fucosylated glycans on some
marrow cells. Interactions between fucosylated glycans on these cells,
or on their precursors, and molecules that recognize these glycans may
therefore contribute to control of blood leukocyte number. We are
attempting to identify the molecular components of this control and to
understand how they function. (Some of this work is supported by grants
from the National Institutes of Health.)
To understand better the synthesis of GDP-fucose, a substrate
required by all fucosyltransferases, and to help us discover new
functions for fucosylated glycans, we have constructed and study mutant
cell lines with defects in GDP-fucose synthesis. Two cytosolic enzymes,
GMD and FX, are required for constitutive synthesis of GDP-fucose.
Studies with FX or GMD mutant cells disclose that GMD activity is
modulated by fucosylation-dependent changes in its self-association.
Work is in progress to define the molecular basis for these
observations.
We have constructed mice with a deletion of the FX locus. FX-null
mice are deficient in all fucosylated glycans. They exhibit unusual
defects in several organs, including a virtually complete block in the
development of T lymphocytes in the thymus. Deficient T cell
development is rapidly repaired, however, when the mice are fed fucose,
which reconstitutes GDP-fucose synthesis via an FX-independent and
normally inactive pathway. This phenotype is not observed in
FucT-IV/FucT-VII doubly deficient mice, nor in α(1,2)fucosyltransferase-deficient mice we have made, indicating that absence of fucosylated glycans made by other fucosyltransferases accounts for this defect. The fucose-dependent
reversible block to T cell development in FX-null mice is accounted for
by a defect in the T cell progenitors themselves. Studies are in
progress to confirm our localization of this block to a specific class
of lymphoid progenitors in the bone marrow. Work is under way to
identify the glycans and fucosyltransferases that account for this
block. We are also using this system to identify molecules and
mechanisms that operate downstream of this conditional and reversible
block and that direct differentiation of T lymphocyte progenitors in
the marrow. Similar studies are under way to determine the basis for
aberrant development of neutrophil precursors in these mice.
We continue to study a cell surface oligosaccharide, the CT antigen.
CT is synthesized by the N-acetylgalactosaminyltransferase
Galgt2 and is expressed by T lymphocytes and other cells. Our studies
with Galgt2-deficient mice indicate that Galgt2 and Fuc-TVII can
compete for the same glycan substrate to control expression of E- and
P-selectin ligands on some T lymphocytes and on other immune cells.
Work is in progress to define the function of the CT antigen.
Last updated March 01, 2005
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